**Recent New Characterizations on the Giant Extracellular Hemoglobin of** *Glossoscolex paulistus* **and Some Other Giant Hemoglobins from Different Worms**

Marcel Tabak1, Francisco A.O. Carvalho1, José W.P. Carvalho1, Jose F.R. Bachega2 and Patrícia S. Santiago1,3 *1Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, 2Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, 3Campus Experimental de Registro, UNESP, SP, Brazil* 

### **1. Introduction**

Giant extracellular hemoglobins, also known as erythrocruorins, have been investigated as a model of extreme complexity in oxygen-binding heme proteins [1,2]. They are characterized by a very high molecular mass (*MM*) of several megadalton (MDa), and their oligomeric structure together with the crowded and protected heme environment are two of the main factors responsible for the high redox stability [3,4]. Superoxide dimustase (SOD)-like intrinsic activity, observed for hemoglobins of *Lumbricus terrestris* (HbLt) and of *Arenicola marina* (HbAm), is another important factor [5,6]. These hemoglobins present a highly cooperative oxygen binding and a peculiar behavior associated to their oligomeric dissociation into smaller subunits and possible rearrangement back into the native oligomeric structure [7,8].

Moreover, a strong motivation to study these giant hemoglobins is related to their potential use in medicine as blood substitutes. Studies have been performed in the past for HbLt [9], and are presently underway to test and validate the use of HbAm in this direction [6,10]. They seem to be very promising due to the lack of undesirable immunological reactions in tests with animals, explained by the absence of cell membranes as occurs with human hemoglobin in red blood cells [6,10]. Besides, the resistance to oxidation of extracellular hemoglobins, as noticed by their redox stability, is also an advantage as compared to the use of human hemoglobin in this medical application.

The giant extracellular hemoglobin of *Glossoscolex paulistus* (HbGp) is similar to several homologous proteins described in the literature [11-14]. These extracellular hemoglobins are constituted by a large number of globin-like subunits containing heme groups with *MM* in the range 15–19 kDa. These globin subunits form monomers of 16 kDa (*d*) and disulphide bound hetero-trimers of 51-52 kDa (*abc*), linked by non-heme structures (24–32 kDa), named linkers (*L*) [12,13]. Recent partial characterization of *MM* of HbGp by matrix assisted laser

Recent New Characterizations on the Giant Extracellular Hemoglobin

possible medical application as a blood substitute, are described.

**1.1 Some new extracellular hemoglobins from different worms** 

hemoglobins from different earthworms were studied.

HbRa, is not known from the structural point of view.

research groups in Brazil [27,28].

organic wastes efficiently [32].

of *Glossoscolex paulistus* and Some Other Giant Hemoglobins from Different Worms 339

added, to compare the differences and similarities between these systems: namely hemoglobins of *Rhinodrilus alatus*, *Eisenia andrei* and *Perionyx excavatus*. The focus of this study is the characterization of the different subunits that constitute each protein, aiming to assess in detail the nature of the subunit interactions that maintain the whole extraordinary oligomeric structure. These three new hemoglobins belong to different worm species that have been classified and studied previously only in relation to their biological effects upon the soil condition in their living habitat. Comparing the composition and structures of their subunits could be interesting also in relation to the understanding of the evolutionary changes evolved as a function of their different living environments. Finally, recent results concerning the subunits composition and architecture of HbAm, in connection with its

The giant extracellular hemoglobins found in the Annelids, are characterized by high molecular mass and by an overall hexagonal symmetry [23]. Hemoglobin provides a fascinating example of molecular evolution. In this work four types of extracellular

These species of worms have very specific habitat and are found and live only in some restricted regions. It is not possible to produce some of these earthworm species in large amounts and captivity. *Glossoscolex paulistus* is a worm of the Glossoscolecidae family, very common and endemic in the cities of Piracicaba and Rio Claro, in state of São Paulo, Brazil [26]. The more common use of these earthworms is in the fishing, as baits. However, the extracellular hemoglobin of *Glossoscolex paulistus*, HbGp, has now been studied for more than twenty years, focusing its physical-chemical and structural properties, by several

*Rhinodrilus alatus* is a species endemic and prevalent in sites in the regions of Sete Lagoas and Paraopeba, in the state of Minas Gerais, Brazil [29]. This species was considered to be threatened of extinction, due to its extensive use to achieve natural fertilizer as well as for bait in fishing. This species was well characterized and its anatomy is known [30], especially regarding the circulatory system. *Rhinodrilus alatus* is a giant earthworm, of 56 to 63 cm of length and 11 to 12 mm of diameter [30]. In the period of drought, between April and September, the earthworms of this species enter into spontaneous dormancy, in a camera protected by mucus. *Rhinodrilus alatus* belongs to the same family as the *Glossoscolex paulistus* worm. Differently from HbGp, the extracellular hemoglobin of *Rhinodrilus alatus*,

*Perionyx excavatus* is a worm of the family Megascolecidae and genus Perionyx. *Perionyx excavatus* is an earthworm found commonly over a large area of tropical Asia although it has been transported to Europe and North America [31]. This is an endemic species which lives in organic wastes and high moisture contents, and adequate amounts of suitable organic material are required for populations to become fully established and for them to process

*Perionyx excavatus* had been shown to have efficient biological potential for conversion of organic wastes into high-value useful plant growth media [32]. Differently from the other two species, the *Perionyx excavatus* is produced in earthworm culture and its marketing is

desorption time-of-flight mass spectrometry (MALDI-TOF-MS) confirmed the similarity of its subunits to those of homologous proteins of this class, mentioned above, especially HbLt [15]. This characteristic multi-subunit content confers to the whole protein a double-layered hexagonal oligomeric structure. A common model for the quaternary structure, so-called "bracelet model", has been employed to explain the assembly of this class of proteins into their oligomeric structure.

It is worth of notice that HbGp belongs to the same class of hemoglobins as HbLt, which is one of the most studied hemoglobins in this group. Despite the fact that HbLt has been extensively studied over the past 20 years, the issue of its true *MM* is still not fully understood. HbLt was one of the proteins studied by Theodore Svedberg and collaborators in 1933 [16], and later work by Daniel et al. has argued that the *MM* of HbLt could vary between 3.6 and 4.4 MDa [17]. They propose a model for the whole protein, consisting of twelve equal structures involving a dodecamer, (*abcd*)3, and three linkers *L3*, together with twelve tetramers (*abcd*), in such a way that the protomer corresponding to the 1/12 of the whole oligomer is given by (*abcd*)3*L*3(*abcd*), or alternatively, (*abcd*)4*L*3 [17,18]. On the other hand, Vinogradov et al. [19] have proposed a model for HbLt, where 1/12 of the whole molecule is given by (*abcd*)3 *L*3, so that the difference between the two models is the presence of twelve additional tetramers in the former occupying the central part of the hexagonal bilayer.

Royer et al. describing the crystal structure of HbLt [11,20] has suggested that the Vinogradov model is very consistent with the crystal structure. Besides, very recent preliminary work on the crystal structure of HbGp [21] has also suggested a strong similarity between HbGp and HbLt, both belonging to class 1, where the two hexagonal layers forming the bilayer are rotated to 16 degree one relative to another. It is worthy of mention, that another extracellular hemoglobin from a sea worm, HbAm, has also been investigated by Zal et al. [22,23]. In this case the structure belongs to class 2, where the two hexagonal layers are exactly eclipsed one relative to the other, as demonstrated in recent crystallographic studies at low resolution [24]. Besides, the assembly of the protomer corresponding to 1/12 of the whole HbAm oligomer is different: a dodecamer formed by two monomeric units a1 and a2 together with a trimer (*T*) gives (*a*1)3(*a*2)6*T* associated to an average of 3.5 linkers resulting in a structure of [(*a*1)3(*a*2)6*T*]*L*3.5 [23,25]. This arrangement implies a different number of globin and linker chains for HbAm. In this assembly the contacts between the subunits are quite different as compared to those in HbLt. Moreover, quite recently, the primary sequence of a fourth linker chain for HbLt has been reported, *L<sup>4</sup>* [1]. The unanswered question remains as an open issue regarding the role of four linker chains in a structure that, apparently, only needs three of them. Another interesting question, relevant to the understanding of the overall oligomeric subunit stoichiometry, is the existence of several isoforms for some of the globin chains. This has also been elusive in the description of the crystal structure of the whole assembly at atomic resolution. So, the precise characterization of the several globin and linker subunits that constitute the native extracellular hemoglobins still remains a matter for further research.

In conclusion, this review chapter addresses the issue of the molecular masses of HbGp subunits, as monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF-MS, and their hydrodynamic properties, as monitored by analytical ultracentrifugation (AUC). Also new data from other three giant hemoglobins are

desorption time-of-flight mass spectrometry (MALDI-TOF-MS) confirmed the similarity of its subunits to those of homologous proteins of this class, mentioned above, especially HbLt [15]. This characteristic multi-subunit content confers to the whole protein a double-layered hexagonal oligomeric structure. A common model for the quaternary structure, so-called "bracelet model", has been employed to explain the assembly of this class of proteins into

It is worth of notice that HbGp belongs to the same class of hemoglobins as HbLt, which is one of the most studied hemoglobins in this group. Despite the fact that HbLt has been extensively studied over the past 20 years, the issue of its true *MM* is still not fully understood. HbLt was one of the proteins studied by Theodore Svedberg and collaborators in 1933 [16], and later work by Daniel et al. has argued that the *MM* of HbLt could vary between 3.6 and 4.4 MDa [17]. They propose a model for the whole protein, consisting of twelve equal structures involving a dodecamer, (*abcd*)3, and three linkers *L3*, together with twelve tetramers (*abcd*), in such a way that the protomer corresponding to the 1/12 of the whole oligomer is given by (*abcd*)3*L*3(*abcd*), or alternatively, (*abcd*)4*L*3 [17,18]. On the other hand, Vinogradov et al. [19] have proposed a model for HbLt, where 1/12 of the whole molecule is given by (*abcd*)3 *L*3, so that the difference between the two models is the presence of twelve additional tetramers in the former occupying the central part of the hexagonal

Royer et al. describing the crystal structure of HbLt [11,20] has suggested that the Vinogradov model is very consistent with the crystal structure. Besides, very recent preliminary work on the crystal structure of HbGp [21] has also suggested a strong similarity between HbGp and HbLt, both belonging to class 1, where the two hexagonal layers forming the bilayer are rotated to 16 degree one relative to another. It is worthy of mention, that another extracellular hemoglobin from a sea worm, HbAm, has also been investigated by Zal et al. [22,23]. In this case the structure belongs to class 2, where the two hexagonal layers are exactly eclipsed one relative to the other, as demonstrated in recent crystallographic studies at low resolution [24]. Besides, the assembly of the protomer corresponding to 1/12 of the whole HbAm oligomer is different: a dodecamer formed by two monomeric units a1 and a2 together with a trimer (*T*) gives (*a*1)3(*a*2)6*T* associated to an average of 3.5 linkers resulting in a structure of [(*a*1)3(*a*2)6*T*]*L*3.5 [23,25]. This arrangement implies a different number of globin and linker chains for HbAm. In this assembly the contacts between the subunits are quite different as compared to those in HbLt. Moreover, quite recently, the primary sequence of a fourth linker chain for HbLt has been reported, *L<sup>4</sup>* [1]. The unanswered question remains as an open issue regarding the role of four linker chains in a structure that, apparently, only needs three of them. Another interesting question, relevant to the understanding of the overall oligomeric subunit stoichiometry, is the existence of several isoforms for some of the globin chains. This has also been elusive in the description of the crystal structure of the whole assembly at atomic resolution. So, the precise characterization of the several globin and linker subunits that constitute the native

extracellular hemoglobins still remains a matter for further research.

In conclusion, this review chapter addresses the issue of the molecular masses of HbGp subunits, as monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF-MS, and their hydrodynamic properties, as monitored by analytical ultracentrifugation (AUC). Also new data from other three giant hemoglobins are

their oligomeric structure.

bilayer.

added, to compare the differences and similarities between these systems: namely hemoglobins of *Rhinodrilus alatus*, *Eisenia andrei* and *Perionyx excavatus*. The focus of this study is the characterization of the different subunits that constitute each protein, aiming to assess in detail the nature of the subunit interactions that maintain the whole extraordinary oligomeric structure. These three new hemoglobins belong to different worm species that have been classified and studied previously only in relation to their biological effects upon the soil condition in their living habitat. Comparing the composition and structures of their subunits could be interesting also in relation to the understanding of the evolutionary changes evolved as a function of their different living environments. Finally, recent results concerning the subunits composition and architecture of HbAm, in connection with its possible medical application as a blood substitute, are described.

### **1.1 Some new extracellular hemoglobins from different worms**

The giant extracellular hemoglobins found in the Annelids, are characterized by high molecular mass and by an overall hexagonal symmetry [23]. Hemoglobin provides a fascinating example of molecular evolution. In this work four types of extracellular hemoglobins from different earthworms were studied.

These species of worms have very specific habitat and are found and live only in some restricted regions. It is not possible to produce some of these earthworm species in large amounts and captivity. *Glossoscolex paulistus* is a worm of the Glossoscolecidae family, very common and endemic in the cities of Piracicaba and Rio Claro, in state of São Paulo, Brazil [26]. The more common use of these earthworms is in the fishing, as baits. However, the extracellular hemoglobin of *Glossoscolex paulistus*, HbGp, has now been studied for more than twenty years, focusing its physical-chemical and structural properties, by several research groups in Brazil [27,28].

*Rhinodrilus alatus* is a species endemic and prevalent in sites in the regions of Sete Lagoas and Paraopeba, in the state of Minas Gerais, Brazil [29]. This species was considered to be threatened of extinction, due to its extensive use to achieve natural fertilizer as well as for bait in fishing. This species was well characterized and its anatomy is known [30], especially regarding the circulatory system. *Rhinodrilus alatus* is a giant earthworm, of 56 to 63 cm of length and 11 to 12 mm of diameter [30]. In the period of drought, between April and September, the earthworms of this species enter into spontaneous dormancy, in a camera protected by mucus. *Rhinodrilus alatus* belongs to the same family as the *Glossoscolex paulistus* worm. Differently from HbGp, the extracellular hemoglobin of *Rhinodrilus alatus*, HbRa, is not known from the structural point of view.

*Perionyx excavatus* is a worm of the family Megascolecidae and genus Perionyx. *Perionyx excavatus* is an earthworm found commonly over a large area of tropical Asia although it has been transported to Europe and North America [31]. This is an endemic species which lives in organic wastes and high moisture contents, and adequate amounts of suitable organic material are required for populations to become fully established and for them to process organic wastes efficiently [32].

*Perionyx excavatus* had been shown to have efficient biological potential for conversion of organic wastes into high-value useful plant growth media [32]. Differently from the other two species, the *Perionyx excavatus* is produced in earthworm culture and its marketing is

Recent New Characterizations on the Giant Extracellular Hemoglobin

**2. Experimental methods** 

**3. Results** 

protein structures with atomic resolution [21].

of *Glossoscolex paulistus* and Some Other Giant Hemoglobins from Different Worms 341

In the present work, a review is presented of recent work performed in our laboratory, focusing on the molecular mass *MM* of HbGp, based on electrophoresis, AUC, and MALDI-TOF-MS [3,15]. SDS-PAGE electrophoresis is an extremely useful technique, especially to monitor the subunits contents present in different fractions of the protein obtained from gel exclusion chromatography [26]. Though this technique is not as precise as direct mass spectrometry, it is able to provide informations about the mass characteristic and migration properties of these subunits under the effect of an applied electric field at the same time.

X-Ray crystallography is a powerful structural technique, providing models of complex

Our recent *MM* determination performed on two HbGp samples, at pH 7.0, one in the reduced oxy- form, and the other in the oxidized cyanomet- form, have given values of 3.6 ± 0.1 MDa and 3.7 ± 0.1 MDa, respectively [3]. This value is in excellent agreement with the prediction of the *MM* for the whole molecule, based on the Vinogradov model, and our previous MALDI-TOF-MS analysis [15]: a tetramer *abcd* of 52.1+16.4 = 68.5 kDa is observed, and assuming further an average *MM* for the linkers of 28 kDa, plus 144 heme groups (~0.6 kDa each), a total *MM* of 12×[68.5×3+28×3]+144×0.6 = 3560 kDa is expected for HbGp.

Very recent ultracentrifugation studies regarding the HbGp subunits, obtained from alkaline dissociation at pH 10, provided further information on subunits masses, underlining the difficulties in separating, by simple SEC experiments, the linker chains from the trimer globin subunits due to their similar hydrodynamic properties [37]. In this study, analysis of the equilibrium dissociation of HbGp, at pH 10.0, has shown that the whole protein is constituted by several species. For oxy-HbGp, at pH 10.0, no contribution from un-dissociated whole protein is observed, suggesting that the reduced form is completely dissociated under this alkaline condition. However, for cyanomet-HbGp, 17 % of the protein remains in the native un-dissociated form, implying a higher oligomeric stability of the oxidized cyanomet- form [37]. Results are shown in Tables 1 and 2 and can be summarized as follows: 1) AUC experiments for the monomer *d* alone indicated that this species is quite pure. However, equilibrium is observed involving monomers and dimers of monomers. The dimer contribution is relatively low, around 10%, similar to that observed in previous experiments by MALDI–TOF-MS, increasing at higher protein concentrations. 2) Analysis of oxy-HbGp solution, at pH 10.0, showed the existence of four species in equilibrium, assigned to the following subunits: monomer *d*, dimers of monomers *d2*, trimers *abc* and a fourth species associated to the tetramer *abcd*. The second species (dimers of monomers, *d2*) revealed a significant intensity increase as compared to the pure monomer. This increase in intensity is, possibly, due to a superposition of the contribution from some linker chains, having masses near to the dimers of monomers *d2* one, around 32 kDa. Linker chains are characterized by mass values *MM* in the range of 24-32 kDa, and are, probably, detected as a single peak, corresponding to the peak of the dimers of monomers. The third species, trimers *abc*, is not observed for samples containing the reducing agent, β-mercaptoethanol,

**3.1 Equilibrium of HbGp species obtained from alkaline dissociation at pH 10** 

allowed around the world. For this reason, it is often used in the process of vermicomposting, which is a mesophilic process associated to ingestion, digestion, and absorption of organic waste carried out by earthworms followed by excretion of castings through the worm's metabolic system [32].

Another extracellular hemoglobin that is discussed in this work is collected from the annelid *Eisenia andrei*, HbEa. This is an earthworm of the family Lumbricidae and is known as the red California worm. This earthworm is between 5 to 10 cm in length and 4 to 8 mm in diameter, and it is the most used species in the world for decomposition of organic material [33]. The easy adaptation and reproduction of this species partly justify the *Eisenia andrei* large use around the world.

Some interesting studies suggest that earthworms can be used as biological indicators of soil contamination. Vampré et al. [34] showed that soil contamination by hexachlorobenzene (HCB) can be identified by analysis of soil and earthworm tissue extracts [34]. The earthworm has an important role in the equilibrium of the soil nutrients distribution, in particular, the increase of soil stability, infiltration rates, besides playing an important role in the phosphorus (P) and nitrogen (N) cycles [35]. In this context, interesting work has been developed by Brown et al. regarding the worms population of the state of Paraná, Brazil, aiming to evaluate the soil characteristics of this region [36].

### **1.2 Extracellular worm hemoglobins as potential blood substitutes**

Giant extracellular hemoglobins are easily purified to a homogeneous product avoiding expensive synthetic steps. Besides, it is a product easy to store, the auto-oxidation is quite a slow process and it is less likely to cause immunogenic responses since cell membranes are not present and HbAm, for instance, is not glycosilated [27]. Preliminary experiments have been performed many years ago with the naturally polymerized HbLt. Mice and rats undergoing exchange transfusion with HbLt revealed no apparent behavioral and physical changes [9]. A report on the potential use of HbAm, showing that its structural and functional properties are consistent with the requirements for blood substitutes, has been published recently [6]. These properties should prevent kidney damage, reduce vasoconstrictor effect and oxygen radical formation, often associated with the administration of dissolved mammalian Hb.

Structural analyses of HbAm under simulated human physiological conditions *in vitro*  revealed its dissociation into high molecular weight and functional subunits, while *in vivo*  experiments performed on mice revealed no apparent behavioral or physiopathological changes and absence of allergic response [6]. Recent effort has also been devoted to express some of the globin chains of HbAm considering the possibility of producing larger amounts of material aiming at reconstituting a recombinant hexagonal bilayer hemoglobin (HBL-Hb) by genetic engineering in order to provide a new oxygen carrier for therapeutic applications [10]. Considering the similarity of HbGp and HbLt with HbAm it is possible to conclude that all of them could be potentially useful for blood substitutes. Therefore, our studies aiming to obtain a deeper understanding of the structural and physical-chemical properties of HbGp and the new giant hemoglobins presented in this report can be quite relevant to a better evaluation of their potential biomedical applications.

### **2. Experimental methods**

In the present work, a review is presented of recent work performed in our laboratory, focusing on the molecular mass *MM* of HbGp, based on electrophoresis, AUC, and MALDI-TOF-MS [3,15]. SDS-PAGE electrophoresis is an extremely useful technique, especially to monitor the subunits contents present in different fractions of the protein obtained from gel exclusion chromatography [26]. Though this technique is not as precise as direct mass spectrometry, it is able to provide informations about the mass characteristic and migration properties of these subunits under the effect of an applied electric field at the same time.

X-Ray crystallography is a powerful structural technique, providing models of complex protein structures with atomic resolution [21].

## **3. Results**

340 Stoichiometry and Research – The Importance of Quantity in Biomedicine

allowed around the world. For this reason, it is often used in the process of vermicomposting, which is a mesophilic process associated to ingestion, digestion, and absorption of organic waste carried out by earthworms followed by excretion of castings

Another extracellular hemoglobin that is discussed in this work is collected from the annelid *Eisenia andrei*, HbEa. This is an earthworm of the family Lumbricidae and is known as the red California worm. This earthworm is between 5 to 10 cm in length and 4 to 8 mm in diameter, and it is the most used species in the world for decomposition of organic material [33]. The easy adaptation and reproduction of this species partly justify the *Eisenia andrei*

Some interesting studies suggest that earthworms can be used as biological indicators of soil contamination. Vampré et al. [34] showed that soil contamination by hexachlorobenzene (HCB) can be identified by analysis of soil and earthworm tissue extracts [34]. The earthworm has an important role in the equilibrium of the soil nutrients distribution, in particular, the increase of soil stability, infiltration rates, besides playing an important role in the phosphorus (P) and nitrogen (N) cycles [35]. In this context, interesting work has been developed by Brown et al. regarding the worms population of the state of Paraná, Brazil,

Giant extracellular hemoglobins are easily purified to a homogeneous product avoiding expensive synthetic steps. Besides, it is a product easy to store, the auto-oxidation is quite a slow process and it is less likely to cause immunogenic responses since cell membranes are not present and HbAm, for instance, is not glycosilated [27]. Preliminary experiments have been performed many years ago with the naturally polymerized HbLt. Mice and rats undergoing exchange transfusion with HbLt revealed no apparent behavioral and physical changes [9]. A report on the potential use of HbAm, showing that its structural and functional properties are consistent with the requirements for blood substitutes, has been published recently [6]. These properties should prevent kidney damage, reduce vasoconstrictor effect and oxygen radical formation, often associated with the

Structural analyses of HbAm under simulated human physiological conditions *in vitro*  revealed its dissociation into high molecular weight and functional subunits, while *in vivo*  experiments performed on mice revealed no apparent behavioral or physiopathological changes and absence of allergic response [6]. Recent effort has also been devoted to express some of the globin chains of HbAm considering the possibility of producing larger amounts of material aiming at reconstituting a recombinant hexagonal bilayer hemoglobin (HBL-Hb) by genetic engineering in order to provide a new oxygen carrier for therapeutic applications [10]. Considering the similarity of HbGp and HbLt with HbAm it is possible to conclude that all of them could be potentially useful for blood substitutes. Therefore, our studies aiming to obtain a deeper understanding of the structural and physical-chemical properties of HbGp and the new giant hemoglobins presented in this report can be quite relevant to a

through the worm's metabolic system [32].

aiming to evaluate the soil characteristics of this region [36].

administration of dissolved mammalian Hb.

better evaluation of their potential biomedical applications.

**1.2 Extracellular worm hemoglobins as potential blood substitutes** 

large use around the world.

### **3.1 Equilibrium of HbGp species obtained from alkaline dissociation at pH 10**

Our recent *MM* determination performed on two HbGp samples, at pH 7.0, one in the reduced oxy- form, and the other in the oxidized cyanomet- form, have given values of 3.6 ± 0.1 MDa and 3.7 ± 0.1 MDa, respectively [3]. This value is in excellent agreement with the prediction of the *MM* for the whole molecule, based on the Vinogradov model, and our previous MALDI-TOF-MS analysis [15]: a tetramer *abcd* of 52.1+16.4 = 68.5 kDa is observed, and assuming further an average *MM* for the linkers of 28 kDa, plus 144 heme groups (~0.6 kDa each), a total *MM* of 12×[68.5×3+28×3]+144×0.6 = 3560 kDa is expected for HbGp.

Very recent ultracentrifugation studies regarding the HbGp subunits, obtained from alkaline dissociation at pH 10, provided further information on subunits masses, underlining the difficulties in separating, by simple SEC experiments, the linker chains from the trimer globin subunits due to their similar hydrodynamic properties [37]. In this study, analysis of the equilibrium dissociation of HbGp, at pH 10.0, has shown that the whole protein is constituted by several species. For oxy-HbGp, at pH 10.0, no contribution from un-dissociated whole protein is observed, suggesting that the reduced form is completely dissociated under this alkaline condition. However, for cyanomet-HbGp, 17 % of the protein remains in the native un-dissociated form, implying a higher oligomeric stability of the oxidized cyanomet- form [37]. Results are shown in Tables 1 and 2 and can be summarized as follows: 1) AUC experiments for the monomer *d* alone indicated that this species is quite pure. However, equilibrium is observed involving monomers and dimers of monomers. The dimer contribution is relatively low, around 10%, similar to that observed in previous experiments by MALDI–TOF-MS, increasing at higher protein concentrations. 2) Analysis of oxy-HbGp solution, at pH 10.0, showed the existence of four species in equilibrium, assigned to the following subunits: monomer *d*, dimers of monomers *d2*, trimers *abc* and a fourth species associated to the tetramer *abcd*. The second species (dimers of monomers, *d2*) revealed a significant intensity increase as compared to the pure monomer. This increase in intensity is, possibly, due to a superposition of the contribution from some linker chains, having masses near to the dimers of monomers *d2* one, around 32 kDa. Linker chains are characterized by mass values *MM* in the range of 24-32 kDa, and are, probably, detected as a single peak, corresponding to the peak of the dimers of monomers. The third species, trimers *abc*, is not observed for samples containing the reducing agent, β-mercaptoethanol,

Recent New Characterizations on the Giant Extracellular Hemoglobin

TOF-MS [15] experiments.

**3.2.1 SDS-PAGE electrophoresis data** 

of *Glossoscolex paulistus* and Some Other Giant Hemoglobins from Different Worms 343

As mentioned above, for oxy-HbGp no contribution from un-dissociated protein was observed, implying that the reduced form is fully dissociated at pH 10.0. However, for cyanomet-HbGp, around 17 % of un-dissociated protein remains in its native oligomeric form, consistent with a greater oligomeric stability for this oxidized form, in alkaline medium (Tables 1 and 2). Finally, equilibrium sedimentation data allowed an estimate of the masses *MM* for the four globin chains, monomers *a*, *b*, *c* and *d*. Due to the small difference of masses for monomers *b* and *d* they appear as a single unresolved peak in AUC and MALDI-

**3.2 Initial characterization of the new extracellular hemoglobins from different worms**  In this section new data are presented regarding both electrophoretic and MALDI-TOF-MS measurements of HbGp subunits. Four linker chains are observed for this species by SDS-PAGE, but with quite different concentrations that preclude their detection in the mass spectra experiments. Moreover, multiple chains were detected for the monomeric *c* chain obtained by reduction of the trimer *abc*, as for the isoforms of monomeric subunit *d*, reported earlier [15]. Together with the studies on HbGp, we have recently started to look at three different hemoglobins obtained from different worms, namely HbRa, HbPe, and HbEa. Some preliminary results based on SDS-PAGE, AUC, and MALDI-TOF-MS studies are here reported for the first time, showing a great similarity, but not identity, among the subunit

composition and masses for these extracellular hemoglobins as compared to HbGp.

In Figs.1A and 1B SDS-PAGE gels are shown for the extracellular hemoglobins of HbGp, HbRa, HbPe and HbEa, in the absence and the presence of 2-mercaptoethanol, respectively. The slot (S) corresponds to the standard masses used for calibration. The four hemoglobins in the present study showed a similar, but not identical, band pattern with mass values around 52 ± 2 kDa, associated to the trimer subunit, and also very similar masses for the monomer *d*, around 12 kDa (Fig.1A). The slots II and III correspond to HbGp. The slots IV and V, corresponding to HbRa, showed some differences as compared to HbGp: apparently two bands were observed in the trimer position, while the four linker subunits with different intensities were shifted as compared to HbGp bands. Furthermore, two linkers showed masses similar to those of HbGp and the other two smaller ones. The HbPe bands, shown in slots VI and VII, presented three linker chains, one of them with a higher mass as compared to HbGp and HbRa (mass value around 37 ± 2 kDa). For the HbEa bands presented in slots from VIII to X, also three linker chains were observed, two of them with masses very close to those of HbRa and the third one with a greater one (around 49 kDa). In Fig.1B a SDS-PAGE gel image is shown for the four different hemoglobins, in the presence of the 2-mercaptoethanol. The absence of the band corresponding to the trimer around 52 kDa (see Fig.1A) was observed, as a result of the disulfide bond reduction into the three corresponding monomeric subunits. The linker pattern for HbGp and HbRa were essentially maintained upon addition of the reducing agent (slots II-III and IV-V, respectively). In the slots IV and V it was noticed that the monomeric subunits *a*, *b* and *c* for HbRa were similar to those for HbGp. HbRa had two of these monomers with mass values very close, basically unresolved as a single broad band. HbPe, shown in slots VI and VII, presented one of the trimer monomeric subunits heavier than that for HbGp and HbRa.

since the disulfide bonds are disrupted with the formation of the monomeric species that compose the trimers, monomers *a, b* and *c*, consistent also with the increase in the contribution of the monomeric fraction, observed for this sample (Tables 1 and 2). Calculations of the sedimentation coefficients for the HbLt subunits based on the reported crystal structure [11,20] gave additional useful informations for the interpretation of the obtained AUC data for HbGp: first of all, according to these estimates, the linker chains could appear together either with the monomeric globin chains or with the dimeric globins. Besides, the presence of linker chains associated to globin tetramers would not alter in significant ways the value of s for the pure globin tetramer.


Oxy-HbGp1 –HbGp in the presence of 2-mercaptoethanol. All data obtained at 236 nm. aObserved species 1, 2, 3, 4 and 5 correspond to pure monomer *d,* dimer of monomers *d2,* trimer *(abc)*, tetramer (*abcd*) and tetramer of monomers *d*4 and un-dissociated cyanomet-HbGp, respectively.

Table 1. Sedimentation coefficients for the pure monomer *d*, and for HbGp, dissociated in alkaline medium, under the indicated conditions.


Oxy-HbGp1 – HbGp in the presence of 2-mercaptoethanol. All data obtained at 236 nm. aThe species are the same described in Table 1.

Table 2. Percentage contributions of different species observed in the equilibrium of HbGp, dissociated at alkaline medium, pH 10.0, and of monomer *d*, shown in Table 1.

As mentioned above, for oxy-HbGp no contribution from un-dissociated protein was observed, implying that the reduced form is fully dissociated at pH 10.0. However, for cyanomet-HbGp, around 17 % of un-dissociated protein remains in its native oligomeric form, consistent with a greater oligomeric stability for this oxidized form, in alkaline medium (Tables 1 and 2). Finally, equilibrium sedimentation data allowed an estimate of the masses *MM* for the four globin chains, monomers *a*, *b*, *c* and *d*. Due to the small difference of masses for monomers *b* and *d* they appear as a single unresolved peak in AUC and MALDI-TOF-MS [15] experiments.

### **3.2 Initial characterization of the new extracellular hemoglobins from different worms**

In this section new data are presented regarding both electrophoretic and MALDI-TOF-MS measurements of HbGp subunits. Four linker chains are observed for this species by SDS-PAGE, but with quite different concentrations that preclude their detection in the mass spectra experiments. Moreover, multiple chains were detected for the monomeric *c* chain obtained by reduction of the trimer *abc*, as for the isoforms of monomeric subunit *d*, reported earlier [15]. Together with the studies on HbGp, we have recently started to look at three different hemoglobins obtained from different worms, namely HbRa, HbPe, and HbEa. Some preliminary results based on SDS-PAGE, AUC, and MALDI-TOF-MS studies are here reported for the first time, showing a great similarity, but not identity, among the subunit composition and masses for these extracellular hemoglobins as compared to HbGp.

### **3.2.1 SDS-PAGE electrophoresis data**

342 Stoichiometry and Research – The Importance of Quantity in Biomedicine

since the disulfide bonds are disrupted with the formation of the monomeric species that compose the trimers, monomers *a, b* and *c*, consistent also with the increase in the contribution of the monomeric fraction, observed for this sample (Tables 1 and 2). Calculations of the sedimentation coefficients for the HbLt subunits based on the reported crystal structure [11,20] gave additional useful informations for the interpretation of the obtained AUC data for HbGp: first of all, according to these estimates, the linker chains could appear together either with the monomeric globin chains or with the dimeric globins. Besides, the presence of linker chains associated to globin tetramers would not alter in

> 7.0 2.05 ± 0.05 2.81± 0.03 - 5.7 ± 0.1 10.0 1.86 ±0.06 2.9 ± 0.5 - 4.6 ± 0.9

HbGp 10.0 2.0 ± 0.1 3.06 ± 0.09 3.98 ±0.02 6.6 ± 0.2 58.3 ± 0.5

Oxy-HbGp1 –HbGp in the presence of 2-mercaptoethanol. All data obtained at 236 nm. aObserved species 1, 2, 3, 4 and 5 correspond to pure monomer *d,* dimer of monomers *d2,* trimer *(abc)*, tetramer

Table 1. Sedimentation coefficients for the pure monomer *d*, and for HbGp, dissociated in

Oxy-HbGp 10.0 24 ± 8 29 ± 10 38 ± 3 9 ± 6 - Oxy-HbGp1 10.0 47 ± 4 38 ± 3 - 15 ± 3 -

HbGp 10.0 20 ± 4 20 ± 7 40 ± 3 3 ± 1 17 ± 2

Oxy-HbGp1 – HbGp in the presence of 2-mercaptoethanol. All data obtained at 236 nm. aThe species are

Table 2. Percentage contributions of different species observed in the equilibrium of HbGp,

dissociated at alkaline medium, pH 10.0, and of monomer *d*, shown in Table 1.

Oxy-HbGp 10.0 1.7 ± 0.1 2.67 ± 0.03 4.12±0.06 6.2 ± 0.2 Oxy-HbGp1 10.0 1.64 ± 0.06 2.67 ± 0.02 - 4.6 ± 0.2

(*abcd*) and tetramer of monomers *d*4 and un-dissociated cyanomet-HbGp, respectively.

alkaline medium, under the indicated conditions.

**Samples pH** 

Monomer

Cyanomet-

the same described in Table 1.

**Observed speciesa**

**Observed speciesa**

**% (Area) 1 2 3 4 5** 

7.0 84 ± 8 14 ± 8 - 2 ± 1 - 10.0 89 ± 4 8 ± 4 - 3 ± 1 -

*s0 20,w* **1 2 3 4 5** 

significant ways the value of s for the pure globin tetramer.

**Samples pH** 

Monomer

Cyanomet-

In Figs.1A and 1B SDS-PAGE gels are shown for the extracellular hemoglobins of HbGp, HbRa, HbPe and HbEa, in the absence and the presence of 2-mercaptoethanol, respectively. The slot (S) corresponds to the standard masses used for calibration. The four hemoglobins in the present study showed a similar, but not identical, band pattern with mass values around 52 ± 2 kDa, associated to the trimer subunit, and also very similar masses for the monomer *d*, around 12 kDa (Fig.1A). The slots II and III correspond to HbGp. The slots IV and V, corresponding to HbRa, showed some differences as compared to HbGp: apparently two bands were observed in the trimer position, while the four linker subunits with different intensities were shifted as compared to HbGp bands. Furthermore, two linkers showed masses similar to those of HbGp and the other two smaller ones. The HbPe bands, shown in slots VI and VII, presented three linker chains, one of them with a higher mass as compared to HbGp and HbRa (mass value around 37 ± 2 kDa). For the HbEa bands presented in slots from VIII to X, also three linker chains were observed, two of them with masses very close to those of HbRa and the third one with a greater one (around 49 kDa).

In Fig.1B a SDS-PAGE gel image is shown for the four different hemoglobins, in the presence of the 2-mercaptoethanol. The absence of the band corresponding to the trimer around 52 kDa (see Fig.1A) was observed, as a result of the disulfide bond reduction into the three corresponding monomeric subunits. The linker pattern for HbGp and HbRa were essentially maintained upon addition of the reducing agent (slots II-III and IV-V, respectively). In the slots IV and V it was noticed that the monomeric subunits *a*, *b* and *c* for HbRa were similar to those for HbGp. HbRa had two of these monomers with mass values very close, basically unresolved as a single broad band. HbPe, shown in slots VI and VII, presented one of the trimer monomeric subunits heavier than that for HbGp and HbRa.

Recent New Characterizations on the Giant Extracellular Hemoglobin

**3.2.2 MALDI-TOF-MS data** 

and linkers from 24,000 to 56,000 Da.

of *Glossoscolex paulistus* and Some Other Giant Hemoglobins from Different Worms 345

Fig.2 shows the MALDI-TOF-MS spectrum of a solution of hemoglobin of *Rhinodrilus alatus*, at pH 7.0, using sinapinic acid as matrix in positive ion mode. Fig. 2A shows the peaks obtained for the monomer *d* subunit, the trimer (*abc*) and the linker chains. In agreement with previous studies of hemoglobin of *Glossoscolex paulistus* [15], Fig. 2A shows an intense peak centered around 16.3 kDa, corresponding to the monomer *d*, which is consistent with its relatively easy ionization. Fig.2B shows the expanded region from 15,500 to 18,200 Da, corresponding to the single protonation of the monomer *d*. The contribution of two more intense isoforms, *d1* and *d2*, and another less intense isoform *d3* is evident in Fig.2B.

Fig. 2. (A) MALDI-TOF-MS spectrum of *Rhinodrilus alatus* hemoglobin, HbRa, at pH 7.0. The insert shows the expanded ordinate, corresponding to the intensity in arbitrary units, highlighting the linkers peaks; (B) the expanded region for the mono-protonated monomer *d*+ from 15,500 to 18,300 Da; (C) the expanded region for the mono-protonated trimer *abc*<sup>+</sup>

Moreover, a small contribution around 16,900 ± 30 Da can be associated to *d1* + heme, which is not observed for the other isoforms. In Fig. 2C the expanded region from 24,000 to 56,100 Da is displayed, corresponding to the single protonation of the linker chains *L* and trimer subunits (*abc*). The spectrum clearly shows the existence of three linker chains *L1*, *L2* and *L4*, with 25,770, 26,540 and 26,740 Da, respectively. The remaining linker chain, *L3*, at 32,515 Da,

However, the other two reduced monomeric subunits from the trimer were very similar, as observed for HbGp and HbRa. The linkers pattern for HbPe changed upon disulfide bonds reduction in the presence of the 2-mercaptoethanol. It is not clear if this was only due to partial denaturation of the protein in the presence of the reducing agent, leading to a change in protein migration.

Fig. 1. SDS-PAGE gel electrophoresis for the different extracellular hemoglobins analyzed in this work. The gel concentration was 15% in 25 mmol/L Tris-HCl, 192 mmol/L glycine, pH 8.3, and stained with Comassie Blue R-250. The slot (S) corresponds to the standard masses. (A) Gel electrophoresis in the absence of 2-mercaptoethanol and (B) with the reducing agent. The slots II and III correspond to HbGp, slots IV and V to HbRa, VI and VII to HbPe, and VIII, IX and X to HbEa.

Based on SDS-PAGE, HbEa is the hemoglobin that presents subunits with the highest mass values. All the monomeric subunits obtained from the trimer are heavier as compared to the other hemoglobins studied in this work. Its monomeric subunits have mass values in the range from 12.5 to 18.5 kDa (Fig. 1B). It is also observed one of the linkers with *MM* of 47 ± 2 kDa. These heavier subunits should confer a higher value of *MM* to the whole HbEa as compared to HbGp, HbRa and HbPe. Although, some heavier chains are observed in HbPe and HbEa, it seems that no stoichiometry change occurs for the whole oligomer as compared to HbRa, since the same number of subunits is observed when the disulfide bonds are reduced: namely, four globin monomers and four linkers are detected. This implies that the hemoglobins HbRa, HbPe and HbEa should have a stoichiometry for the whole protein of [(abcd)3L3]12. Nevertheless, for HbGp only two linker chains are observed in the SDS-PAGE, but, probably, each observed band contains two linkers superposed or, alternatively, the amounts of two of the linkers are too low to be observed in the gel.

Interestingly, some differences observed in the migration for the monomeric subunits *d* for all the studied hemoglobins, in the presence of the reducing agent 2-mercaptoethanol, can be associated to the higher unfolding of these subunits, due to the reduction of all disulfide bonds in the hemoglobins. For this reason, when the subunits undergo partial unfolding, their migration through the SDS-PAGE gel becomes more difficult. Being SDS-PAGE electrophoresis alone rather limited, more detailed characterization of the subunits for these hemoglobins by mass spectrometry was performed.

### **3.2.2 MALDI-TOF-MS data**

344 Stoichiometry and Research – The Importance of Quantity in Biomedicine

However, the other two reduced monomeric subunits from the trimer were very similar, as observed for HbGp and HbRa. The linkers pattern for HbPe changed upon disulfide bonds reduction in the presence of the 2-mercaptoethanol. It is not clear if this was only due to partial denaturation of the protein in the presence of the reducing agent, leading to a change

Fig. 1. SDS-PAGE gel electrophoresis for the different extracellular hemoglobins analyzed in this work. The gel concentration was 15% in 25 mmol/L Tris-HCl, 192 mmol/L glycine, pH 8.3, and stained with Comassie Blue R-250. The slot (S) corresponds to the standard masses. (A) Gel electrophoresis in the absence of 2-mercaptoethanol and (B) with the reducing agent. The slots II and III correspond to HbGp, slots IV and V to HbRa, VI and VII to HbPe, and

Based on SDS-PAGE, HbEa is the hemoglobin that presents subunits with the highest mass values. All the monomeric subunits obtained from the trimer are heavier as compared to the other hemoglobins studied in this work. Its monomeric subunits have mass values in the range from 12.5 to 18.5 kDa (Fig. 1B). It is also observed one of the linkers with *MM* of 47 ± 2 kDa. These heavier subunits should confer a higher value of *MM* to the whole HbEa as compared to HbGp, HbRa and HbPe. Although, some heavier chains are observed in HbPe and HbEa, it seems that no stoichiometry change occurs for the whole oligomer as compared to HbRa, since the same number of subunits is observed when the disulfide bonds are reduced: namely, four globin monomers and four linkers are detected. This implies that the hemoglobins HbRa, HbPe and HbEa should have a stoichiometry for the whole protein of [(abcd)3L3]12. Nevertheless, for HbGp only two linker chains are observed in the SDS-PAGE, but, probably, each observed band contains two linkers superposed or,

alternatively, the amounts of two of the linkers are too low to be observed in the gel.

hemoglobins by mass spectrometry was performed.

Interestingly, some differences observed in the migration for the monomeric subunits *d* for all the studied hemoglobins, in the presence of the reducing agent 2-mercaptoethanol, can be associated to the higher unfolding of these subunits, due to the reduction of all disulfide bonds in the hemoglobins. For this reason, when the subunits undergo partial unfolding, their migration through the SDS-PAGE gel becomes more difficult. Being SDS-PAGE electrophoresis alone rather limited, more detailed characterization of the subunits for these

in protein migration.

VIII, IX and X to HbEa.

Fig.2 shows the MALDI-TOF-MS spectrum of a solution of hemoglobin of *Rhinodrilus alatus*, at pH 7.0, using sinapinic acid as matrix in positive ion mode. Fig. 2A shows the peaks obtained for the monomer *d* subunit, the trimer (*abc*) and the linker chains. In agreement with previous studies of hemoglobin of *Glossoscolex paulistus* [15], Fig. 2A shows an intense peak centered around 16.3 kDa, corresponding to the monomer *d*, which is consistent with its relatively easy ionization. Fig.2B shows the expanded region from 15,500 to 18,200 Da, corresponding to the single protonation of the monomer *d*. The contribution of two more intense isoforms, *d1* and *d2*, and another less intense isoform *d3* is evident in Fig.2B.

Fig. 2. (A) MALDI-TOF-MS spectrum of *Rhinodrilus alatus* hemoglobin, HbRa, at pH 7.0. The insert shows the expanded ordinate, corresponding to the intensity in arbitrary units, highlighting the linkers peaks; (B) the expanded region for the mono-protonated monomer *d*+ from 15,500 to 18,300 Da; (C) the expanded region for the mono-protonated trimer *abc*<sup>+</sup> and linkers from 24,000 to 56,000 Da.

Moreover, a small contribution around 16,900 ± 30 Da can be associated to *d1* + heme, which is not observed for the other isoforms. In Fig. 2C the expanded region from 24,000 to 56,100 Da is displayed, corresponding to the single protonation of the linker chains *L* and trimer subunits (*abc*). The spectrum clearly shows the existence of three linker chains *L1*, *L2* and *L4*, with 25,770, 26,540 and 26,740 Da, respectively. The remaining linker chain, *L3*, at 32,515 Da,

Recent New Characterizations on the Giant Extracellular Hemoglobin

of *Glossoscolex paulistus* and Some Other Giant Hemoglobins from Different Worms 347

Fig. 3. MALDI-TOF-MS spectrum of *Eisenia andrei* hemoglobin, HbEa. (A) at pH 7.0, full mass range. (B) Corresponds to the intensity in arbitrary units, highlighting the linkers and triple-protonated dimer of trimer; (C) the expanded region for the mono-protonated trimer

Our results indicate that the HbEa presents two significant differences when compared with the HbGp: one of the monomeric chains *a*, *b* or *c*, that constitute the trimer *abc*, has a molecular mass higher than the corresponding HbGp one, due to the mass difference of 2 kDa observed in the trimer of the HbEa (Table 3). The second is the presence of one

The data for hemoglobin of *Perionyx excavatus* are shown in Table 3. In this case the molecular mass of monomer *d* is analogous to that observed for the other hemoglobins. Moreover, it was noticed that a small proportion of the monomer *d* remained associated to heme with a mass of 16,844 ± 20 Da. In general, the subunits of the HbPe are very similar to those of HbGp, since no significant differences in the molecular masses are observed. Very recent studies of HbGp, in the presence of 2-mercaptoethanol [38], have shown that the monomer *c* chain presents also four isoforms with *MM* in the range of 17.3-17.6 kDa. This is, probably, why the trimer *T* is also characterized by two isoforms (Table 3). Further experiments in the future, in the presence of 2-mercaptoethanol will be necessary to elucidate more clearly the masses differences of the globin subunits forming the trimer *abc*,

All AUC experiments were performed in 100 mmol/L Tris-HCl containing 50 mmol/L NaCl, at pH 7.0 or 5.0, and at 20 oC. The protein concentrations were in the range 100 to 300

*abc*+ and tetramer *abcd*+ from 48,000 to 75,000 Da.

especially regarding the new hemoglobins.

**3.2.3 AUC data** 

additional linker chain (*L4*) in the oligomeric structure of HbEa.

can be superposed with the dimers of monomer *2d*. Finally, an intense contribution at 51,470 Da is observed, associated to the trimer (*abc*)+, while the less intense species at 67,690 Da is due to the contribution of the tetramer (*abcd*)+ (Table 3).

Our results for HbRa suggest that the molecular masses of its subunits are very similar to those for HbGp, but the presence of two isoforms for the trimer subunit, as noticed for HbGp [15], is not observed in this hemoglobin. This might indicate that the monomers *a*, *b* and *c* do not have isoforms.

In Table 3, the results of analysis of MALDI-TOF-MS data for hemoglobin of *Eisenia andrei* are shown. In Fig. 3A the peaks obtained for the mono- and di-protonated monomer *d* subunit are significantly more intense as compared to the trimer (*abc*) and the linker chains ones. The molecular mass of the monomer *d* subunit is very similar to those for the HbGp and HbRa hemoglobins, but the peak corresponding to *d1* bound to the heme is not observed. In Fig.3B the three linker chains *L1*, *L2* and *L4*, with masses of 25,110, 26,470 and 27,445 Da, respectively, and another linker *L3* with a more intense peak at 32,775 Da, are shown. Two additional contributions at 53,270 ± 80 and 69,590 ± 100 Da are also shown in Fig. 3C, which can be assigned to the trimer (*abc*) and tetramer (*abcd*), respectively. Differently from the HbRa and HbGp, the hemoglobin of *Eisenia andrei* has only one predominant monomeric isoform *d1* with molecular mass at 16,344 ± 24 Da. The remaining monomeric isoforms have low intensities.


\*The value of mass corresponds very closely to the dimer of the monomer isoform *d*<sup>2</sup>

Table 3. Molecular masses in (Da) of extracellular hemoglobin subunits, obtained from MALDI-TOF-MS, and for different worms.

Fig. 3. MALDI-TOF-MS spectrum of *Eisenia andrei* hemoglobin, HbEa. (A) at pH 7.0, full mass range. (B) Corresponds to the intensity in arbitrary units, highlighting the linkers and triple-protonated dimer of trimer; (C) the expanded region for the mono-protonated trimer *abc*+ and tetramer *abcd*+ from 48,000 to 75,000 Da.

Our results indicate that the HbEa presents two significant differences when compared with the HbGp: one of the monomeric chains *a*, *b* or *c*, that constitute the trimer *abc*, has a molecular mass higher than the corresponding HbGp one, due to the mass difference of 2 kDa observed in the trimer of the HbEa (Table 3). The second is the presence of one additional linker chain (*L4*) in the oligomeric structure of HbEa.

The data for hemoglobin of *Perionyx excavatus* are shown in Table 3. In this case the molecular mass of monomer *d* is analogous to that observed for the other hemoglobins. Moreover, it was noticed that a small proportion of the monomer *d* remained associated to heme with a mass of 16,844 ± 20 Da. In general, the subunits of the HbPe are very similar to those of HbGp, since no significant differences in the molecular masses are observed. Very recent studies of HbGp, in the presence of 2-mercaptoethanol [38], have shown that the monomer *c* chain presents also four isoforms with *MM* in the range of 17.3-17.6 kDa. This is, probably, why the trimer *T* is also characterized by two isoforms (Table 3). Further experiments in the future, in the presence of 2-mercaptoethanol will be necessary to elucidate more clearly the masses differences of the globin subunits forming the trimer *abc*, especially regarding the new hemoglobins.

### **3.2.3 AUC data**

346 Stoichiometry and Research – The Importance of Quantity in Biomedicine

can be superposed with the dimers of monomer *2d*. Finally, an intense contribution at 51,470 Da is observed, associated to the trimer (*abc*)+, while the less intense species at 67,690 Da is

Our results for HbRa suggest that the molecular masses of its subunits are very similar to those for HbGp, but the presence of two isoforms for the trimer subunit, as noticed for HbGp [15], is not observed in this hemoglobin. This might indicate that the monomers *a*, *b*

In Table 3, the results of analysis of MALDI-TOF-MS data for hemoglobin of *Eisenia andrei* are shown. In Fig. 3A the peaks obtained for the mono- and di-protonated monomer *d* subunit are significantly more intense as compared to the trimer (*abc*) and the linker chains ones. The molecular mass of the monomer *d* subunit is very similar to those for the HbGp and HbRa hemoglobins, but the peak corresponding to *d1* bound to the heme is not observed. In Fig.3B the three linker chains *L1*, *L2* and *L4*, with masses of 25,110, 26,470 and 27,445 Da, respectively, and another linker *L3* with a more intense peak at 32,775 Da, are shown. Two additional contributions at 53,270 ± 80 and 69,590 ± 100 Da are also shown in Fig. 3C, which can be assigned to the trimer (*abc*) and tetramer (*abcd*), respectively. Differently from the HbRa and HbGp, the hemoglobin of *Eisenia andrei* has only one predominant monomeric isoform *d1* with molecular mass at 16,344 ± 24 Da. The remaining

Mass of the extracellular hemoglobins in (Da)

*d1* 16,370 16,272 ± 16 16,344 ± 24 16,220 ± 19 *d2* 16,415 ± 10 16,490 ± 20 16,495 ± 12 16,438 ± 20 *d3* 16,650 ± 40 16,710 ± 25 16,560 16,656 ± 25

Subunits HbGp HbRa HbEa HbPe

*d4* 16,850 ± 40 - 16,790 *d1 + heme* 16,900 ± 30 16,844 ± 20 *L1* 25,780 ± 30 25,770 25,110 25,670 *L2* 26,750 ± 80 26,540 26,470 27,145 *L4* - 26,740 27,445 - *L3 or2d*\* 32,870 32,515 32,775 32,410 *T1 (abc)* 51,200 51,470 53,270 ± 80 51,240 *T2 (abc)* 51,985 - - *abcd* 68,400 67,690 69,590 ± 100 67,620

\*The value of mass corresponds very closely to the dimer of the monomer isoform *d*<sup>2</sup>

Table 3. Molecular masses in (Da) of extracellular hemoglobin subunits, obtained from

due to the contribution of the tetramer (*abcd*)+ (Table 3).

and *c* do not have isoforms.

monomeric isoforms have low intensities.

MALDI-TOF-MS, and for different worms.

All AUC experiments were performed in 100 mmol/L Tris-HCl containing 50 mmol/L NaCl, at pH 7.0 or 5.0, and at 20 oC. The protein concentrations were in the range 100 to 300

Recent New Characterizations on the Giant Extracellular Hemoglobin

1HbGp at pH 5.0. 2HbGp at pH 7.0.

worms, at 20.0 ± 0.1 °C and pH 7.0.

average mass of 326/12 =27 kDa.

of *Glossoscolex paulistus* and Some Other Giant Hemoglobins from Different Worms 349

*Glossoscolex paulistus* (HbGp)1 58.6 ± 0.4 3600 ± 80 *Glossoscolex paulistus* (HbGp)2 58.1 ± 0.4 3500 ± 100 *Rhinodrilus alatus* (HbRa) 59.1 ± 0.1 3500 ± 70 *Eisenia andrei* (HbEa) 64.6 ± 0.4 3850 ± 50 *Perionyx excavatus* (HbPe) 58.9 ± 0.2 3500 ± 50

Table 4. AUC hydrodynamic data analysis, for extracellular hemoglobins from different

Fig. 5. Continuous molecular mass distribution of extracellular hemoglobins. The c(M) fittings for hemoglobins, at a concentration of 300 μg/mL, in 100 mmol/L Tris–HCl containing 50 mmol/L NaCl are shown. The insert shows the enhanced plot in the *MM* range from 0 to 400 kDa. The *MM* for each fitted curve was determined as the maximum of

the peaks of c(M) curves. Absorbance was monitored at 415 nm.

In Fig.5 the c(M) distributions are shown for the four types of studied extracellular hemoglobins, at a protein concentration of 300 µg/mL. It is noticed that HbEa has a higher *MM* than the other three hemoglobins one (Table 4). This 330 kDa difference can be attributed to two factors: 1) the difference of the subunits masses constituting the oligomeric structure, and 2) a different subunit stoichiometry for the HbEa oligomer. Thus, the inclusion of another linker chain in the structure of HbEa, giving a model like [(*abcd*)3*L*4]12, could contribute with a 326 kDa increase in the total mass of the molecule, consistent with the results observed for HbEa in the c(M) values. This fourth linker chain would have an

**Hemoglobins** ( ) <sup>0</sup> *<sup>s</sup>*20,*<sup>w</sup> <sup>S</sup> MM* by c(*M*) **(kDa)**

µg/mL, and the speed rotor was 20,000 rpm. The SEDFIT software was used in the analysis of sedimentation velocity (SV) data. The V*bar* parameter used in the fits was the standard value of 0.733 mL/g and *f/f0* was allowed to float being a regularization parameter.

Fig.4 shows sedimentation coefficient distributions c(S), at 200 µg/mL, for the four extracellular hemoglobins. Our results indicate that three of the four hemoglobins present a good homogeneity with a single species in solution. However, for the extracellular hemoglobin of *Eisenia andrei* two different populations are observed, the first one around 10 S can be associated to the dodecamer subunits while the other species at 64 S corresponds to the whole protein. Although these proteins are quite stable in these conditions [3], the observed dissociation is, probably, due to the purification process used for this hemoglobin.

Fig. 4. Continuous sedimentation coefficient distribution of extracellular hemoglobins. The c(S) fittings for hemoglobins, at a concentration of 200 μg/mL, in 100 mmol/L Tris–HCl containing 50 mmol/L NaCl, are shown. The *s,20w* for each fitted curve was determined as the maximum of the peaks of c(S) curves. Absorbance was monitored at 415 nm.

The work of Krebs et al [39] on the properties of the dodecamer subunit, (*abcd*)3, of HbLt showed that the dodecamer obtained from urea dissociation of the whole protein consisted of an equilibrium of three species with sedimentation coefficients of 8.5 - 9.4 S, 3.6 - 4.4 S and 1.9 S. According to our AUC data for HbGp, the dodecamer is associated to the 8.5 - 9.4 S species. The other two species correspond to trimers and monomers, in order of decreasing *s*, implying that urea dissociation in HbLt [39] produces an equilibrium of three species.

The *s* values were corrected to standard conditions (water and 20 oC), and the *s0 20,w* values, corresponding to *s20,w* at 0 mg/mL protein, were obtained by linear regression extrapolation. The *s020,w*, values for the oxy-HbGp at pH 5.0 and 7.0 were 58.6 ± 0.4 and 58.1 ± 0.4 S, respectively, as shown in Table 4. These results suggest that HbGp is very stable in both pH values, and does not undergo oligomeric dissociation at pH 5.0. For the other two hemoglobins, HbRa (*Rhinodrilus alatus*) and HbPe (*Perionyx excavatus*), similar results were observed as for HbGp [3], with *s020,w* around of 59 S. The mass values are very similar, within the experimental error, with the exception of HbEa that has a higher mass (Table 4).


1HbGp at pH 5.0. 2HbGp at pH 7.0.

348 Stoichiometry and Research – The Importance of Quantity in Biomedicine

µg/mL, and the speed rotor was 20,000 rpm. The SEDFIT software was used in the analysis of sedimentation velocity (SV) data. The V*bar* parameter used in the fits was the standard

Fig.4 shows sedimentation coefficient distributions c(S), at 200 µg/mL, for the four extracellular hemoglobins. Our results indicate that three of the four hemoglobins present a good homogeneity with a single species in solution. However, for the extracellular hemoglobin of *Eisenia andrei* two different populations are observed, the first one around 10 S can be associated to the dodecamer subunits while the other species at 64 S corresponds to the whole protein. Although these proteins are quite stable in these conditions [3], the observed dissociation is, probably, due to the purification process used for this hemoglobin.

value of 0.733 mL/g and *f/f0* was allowed to float being a regularization parameter.

Fig. 4. Continuous sedimentation coefficient distribution of extracellular hemoglobins. The c(S) fittings for hemoglobins, at a concentration of 200 μg/mL, in 100 mmol/L Tris–HCl containing 50 mmol/L NaCl, are shown. The *s,20w* for each fitted curve was determined as

The work of Krebs et al [39] on the properties of the dodecamer subunit, (*abcd*)3, of HbLt showed that the dodecamer obtained from urea dissociation of the whole protein consisted of an equilibrium of three species with sedimentation coefficients of 8.5 - 9.4 S, 3.6 - 4.4 S and 1.9 S. According to our AUC data for HbGp, the dodecamer is associated to the 8.5 - 9.4 S species. The other two species correspond to trimers and monomers, in order of decreasing *s*, implying that urea dissociation in HbLt [39] produces an equilibrium of three species.

The *s* values were corrected to standard conditions (water and 20 oC), and the *s020,w* values, corresponding to *s20,w* at 0 mg/mL protein, were obtained by linear regression extrapolation. The *s020,w*, values for the oxy-HbGp at pH 5.0 and 7.0 were 58.6 ± 0.4 and 58.1 ± 0.4 S, respectively, as shown in Table 4. These results suggest that HbGp is very stable in both pH values, and does not undergo oligomeric dissociation at pH 5.0. For the other two hemoglobins, HbRa (*Rhinodrilus alatus*) and HbPe (*Perionyx excavatus*), similar results were

the experimental error, with the exception of HbEa that has a higher mass (Table 4).

*20,w* around of 59 S. The mass values are very similar, within

the maximum of the peaks of c(S) curves. Absorbance was monitored at 415 nm.

observed as for HbGp [3], with *s0*

Table 4. AUC hydrodynamic data analysis, for extracellular hemoglobins from different worms, at 20.0 ± 0.1 °C and pH 7.0.

In Fig.5 the c(M) distributions are shown for the four types of studied extracellular hemoglobins, at a protein concentration of 300 µg/mL. It is noticed that HbEa has a higher *MM* than the other three hemoglobins one (Table 4). This 330 kDa difference can be attributed to two factors: 1) the difference of the subunits masses constituting the oligomeric structure, and 2) a different subunit stoichiometry for the HbEa oligomer. Thus, the inclusion of another linker chain in the structure of HbEa, giving a model like [(*abcd*)3*L*4]12, could contribute with a 326 kDa increase in the total mass of the molecule, consistent with the results observed for HbEa in the c(M) values. This fourth linker chain would have an average mass of 326/12 =27 kDa.

Fig. 5. Continuous molecular mass distribution of extracellular hemoglobins. The c(M) fittings for hemoglobins, at a concentration of 300 μg/mL, in 100 mmol/L Tris–HCl containing 50 mmol/L NaCl are shown. The insert shows the enhanced plot in the *MM* range from 0 to 400 kDa. The *MM* for each fitted curve was determined as the maximum of the peaks of c(M) curves. Absorbance was monitored at 415 nm.

Recent New Characterizations on the Giant Extracellular Hemoglobin

associations between subunits *d* and *a* in the whole oligomer.

of *Glossoscolex paulistus* and Some Other Giant Hemoglobins from Different Worms 351

subunit *d*, which leads us to conclude that such an arrangement is a consequence of

Fig. 6. Schematic representation of the hierarchy in the oligomeric assembly of HbGp into the native structure as obtained from crystallographic data analysis. The whole HbGp structure is composed by twelve protomers, each of them containing a dodecameric structure of globins and three linker subunits, (*abcd*)3*L*3. The dodecameric structure is composed by four types of globin chains: *a*, *b, c* and *d*. The four globin chains are associated

In order to evaluate the potential usefulness of such a giant protein such as HbGp for biomedical applications, a detailed knowledge of its constituent subunits is very relevant. In the present review chapter, recent results for HbGp were described, based on the use of SDS-PAGE, MALDI-TOF-MS and AUC. Preliminary characterization of three new extracellular hemoglobins of different worm species was also described for the first time. The electrophoresis analysis of these hemoglobins shows that they have a subunits structure very similar to that described for HbGp. Moreover, our present results based on AUC data show that two of the new hemoglobins have a total *MM* similar to that of HbGp, namely 3.6 MDa, while the fourth one has a higher *MM* of 3.8 MDa. It appears clear from our data that the globin chains are a total of four with a trimer of 52 - 53 kDa and a monomer of 16 kDa as a common feature for all these hemoglobins. The highest *MM* hemoglobin, HbEa, has heavier linker chains as well as one of the globin monomers. MALDI-TOF-MS analysis for HbGp showed that the monomer *c* presents four isoforms and that the trimer, *abc,* is characterized by two isoforms, *T1* and *T2*. In the final part of this review some crystal structures are also reported, which might be helpful in understanding the oligomeric structure of this class of proteins. Our present studies for HbGp are consistent with literature reports on several other extracellular hemoglobins, such as the HbLt and HbAm. Future more detailed characterization of the new hemoglobins will be important to assess all the subunit masses to model their oligomeric structure. We believe our results represent a nice contribution and an important and necessary step in the complete characterization of

as a hetero-tetramer (where *a*, *b*, and *c* form a trimer, linked by disulfide bonds).

**4. Conclusions** 

HbGp oligomeric structure.

In the c(M) distribution curve for the hemoglobin of *Eisenia andrei* an additional species of 210 kDa is observed, which is assigned to the dodecamer (*abcd*)3. This mass value is in agreement with the literature reports of 205 kDa [19]. Moreover, the peak at 3.8 MDa corresponding to the whole native protein is broadened, which is, probably, due to a superposition of two contributions: from species lacking the dodecamer and from the whole protein.

The *MM* of three of the hemoglobins shown in Table 4 is around 3.6 MDa, indicating that these proteins have the same stoichiometry and subunits with quite similar mass. Moreover, these *MM* values strongly suggest that the Vinogradov's model [(*abcd*)3*L*3]12 [19] is adequate for the hemoglobins of *Glossoscolex paulistus*, HbGp, *Rhinodrilus alatus,* HbRa, and *Perionyx excavatus,* HbPe. However, our results indicate that for the hemoglobin of *Eisenia andrei* this model is not appropriate, since a significant mass difference is observed corresponding to 12 additional linker chains (with an average mass of 27 kDa).

### **3.3 Structural model and stoichiometry**

The first crystallization experiments with giant hemoglobins are dated from 1840, when crystals from HbLt were obtained. Indeed, HbLt was the first protein crystallized ever [40]. Nevertheless, it took more than one hundred and sixty years to obtain a complete crystallographic structure [20]. The crystal structure of HbLt, at 3.5 Å of resolution, elucidated how the hierarchical levels of an erythrocruorin is made, and represented a fundamental step to better understand these structures. Recently, our group, reported the preliminary structure of HbGp, at 3.15 Å of resolution [21]. The HbGp and HbLt share an estimated identity for the monomer subunit *d* of 54 % [41], with the HbGp structure showing the same hierarchical levels as HbLt (Fig. 6), and the same global association, into type 1. It was the second time that an entire type 1 erythrocrurin has been reported. As in HbLt, the electron density map of the HbGp reveals important details, such as the conserved calcium biding sites, inter/intra chains disulfide bonds and heme group positions. Very recently, crystals from the giant hemoglobins from HbEa, and HbPe were also obtained in our group and the preliminary structure for HbEa has been solved, with a resolution of 4.7 Å, which is enough to conclude that, like HbGp and HbLt, HbEa also belongs to type 1 array. Further experiments are needed to improve the resolution and find evidences on the electron density map where HbEa shows an accumulation of mass [42].

Another very recent relevant advance in the structural analysis was the characterization of the isolated subunit *d* from HbGp. In the biological unit, subunits type *d* form an interface with subunits type *a*, involving the heme groups from both chains, and at the same time an interface with two other subunits *d*, forming a trimeric array, as shown in Fig. 6. We showed that once isolated, the subunit *d* shows, as a first level of aggregation, a dimer *d-d*, with an interface involving heme groups, similar to the one observed between *a*-*d* in the biological unit (where *a* and *d* shares 27% of identity). In the next level, the crystal lattice shows a stable dimer of dimers *d-d. T*his tetrameric array is unrelated with the hetero tetramer (*abcd*) in the biological unit [43]. The existence of these oligomeric structures has also been confirmed by AUC, which shows that the pure monomer *d* solution contains also three species, monomer, dimer and tetramer, and increasing the protein concentration the contribution of the monomeric species reduces from 90 to 80 % [44]. On the other hand, the trimer formed by the subunits *d* in the biological unit has not been observed for the isolated subunit *d*, which leads us to conclude that such an arrangement is a consequence of associations between subunits *d* and *a* in the whole oligomer.

Fig. 6. Schematic representation of the hierarchy in the oligomeric assembly of HbGp into the native structure as obtained from crystallographic data analysis. The whole HbGp structure is composed by twelve protomers, each of them containing a dodecameric structure of globins and three linker subunits, (*abcd*)3*L*3. The dodecameric structure is composed by four types of globin chains: *a*, *b, c* and *d*. The four globin chains are associated as a hetero-tetramer (where *a*, *b*, and *c* form a trimer, linked by disulfide bonds).

## **4. Conclusions**

350 Stoichiometry and Research – The Importance of Quantity in Biomedicine

In the c(M) distribution curve for the hemoglobin of *Eisenia andrei* an additional species of 210 kDa is observed, which is assigned to the dodecamer (*abcd*)3. This mass value is in agreement with the literature reports of 205 kDa [19]. Moreover, the peak at 3.8 MDa corresponding to the whole native protein is broadened, which is, probably, due to a superposition of two contributions: from species lacking the dodecamer and from the whole

The *MM* of three of the hemoglobins shown in Table 4 is around 3.6 MDa, indicating that these proteins have the same stoichiometry and subunits with quite similar mass. Moreover, these *MM* values strongly suggest that the Vinogradov's model [(*abcd*)3*L*3]12 [19] is adequate for the hemoglobins of *Glossoscolex paulistus*, HbGp, *Rhinodrilus alatus,* HbRa, and *Perionyx excavatus,* HbPe. However, our results indicate that for the hemoglobin of *Eisenia andrei* this model is not appropriate, since a significant mass difference is observed corresponding to 12

The first crystallization experiments with giant hemoglobins are dated from 1840, when crystals from HbLt were obtained. Indeed, HbLt was the first protein crystallized ever [40]. Nevertheless, it took more than one hundred and sixty years to obtain a complete crystallographic structure [20]. The crystal structure of HbLt, at 3.5 Å of resolution, elucidated how the hierarchical levels of an erythrocruorin is made, and represented a fundamental step to better understand these structures. Recently, our group, reported the preliminary structure of HbGp, at 3.15 Å of resolution [21]. The HbGp and HbLt share an estimated identity for the monomer subunit *d* of 54 % [41], with the HbGp structure showing the same hierarchical levels as HbLt (Fig. 6), and the same global association, into type 1. It was the second time that an entire type 1 erythrocrurin has been reported. As in HbLt, the electron density map of the HbGp reveals important details, such as the conserved calcium biding sites, inter/intra chains disulfide bonds and heme group positions. Very recently, crystals from the giant hemoglobins from HbEa, and HbPe were also obtained in our group and the preliminary structure for HbEa has been solved, with a resolution of 4.7 Å, which is enough to conclude that, like HbGp and HbLt, HbEa also belongs to type 1 array. Further experiments are needed to improve the resolution and find evidences on the

Another very recent relevant advance in the structural analysis was the characterization of the isolated subunit *d* from HbGp. In the biological unit, subunits type *d* form an interface with subunits type *a*, involving the heme groups from both chains, and at the same time an interface with two other subunits *d*, forming a trimeric array, as shown in Fig. 6. We showed that once isolated, the subunit *d* shows, as a first level of aggregation, a dimer *d-d*, with an interface involving heme groups, similar to the one observed between *a*-*d* in the biological unit (where *a* and *d* shares 27% of identity). In the next level, the crystal lattice shows a stable dimer of dimers *d-d. T*his tetrameric array is unrelated with the hetero tetramer (*abcd*) in the biological unit [43]. The existence of these oligomeric structures has also been confirmed by AUC, which shows that the pure monomer *d* solution contains also three species, monomer, dimer and tetramer, and increasing the protein concentration the contribution of the monomeric species reduces from 90 to 80 % [44]. On the other hand, the trimer formed by the subunits *d* in the biological unit has not been observed for the isolated

additional linker chains (with an average mass of 27 kDa).

electron density map where HbEa shows an accumulation of mass [42].

**3.3 Structural model and stoichiometry** 

protein.

In order to evaluate the potential usefulness of such a giant protein such as HbGp for biomedical applications, a detailed knowledge of its constituent subunits is very relevant. In the present review chapter, recent results for HbGp were described, based on the use of SDS-PAGE, MALDI-TOF-MS and AUC. Preliminary characterization of three new extracellular hemoglobins of different worm species was also described for the first time. The electrophoresis analysis of these hemoglobins shows that they have a subunits structure very similar to that described for HbGp. Moreover, our present results based on AUC data show that two of the new hemoglobins have a total *MM* similar to that of HbGp, namely 3.6 MDa, while the fourth one has a higher *MM* of 3.8 MDa. It appears clear from our data that the globin chains are a total of four with a trimer of 52 - 53 kDa and a monomer of 16 kDa as a common feature for all these hemoglobins. The highest *MM* hemoglobin, HbEa, has heavier linker chains as well as one of the globin monomers. MALDI-TOF-MS analysis for HbGp showed that the monomer *c* presents four isoforms and that the trimer, *abc,* is characterized by two isoforms, *T1* and *T2*. In the final part of this review some crystal structures are also reported, which might be helpful in understanding the oligomeric structure of this class of proteins. Our present studies for HbGp are consistent with literature reports on several other extracellular hemoglobins, such as the HbLt and HbAm. Future more detailed characterization of the new hemoglobins will be important to assess all the subunit masses to model their oligomeric structure. We believe our results represent a nice contribution and an important and necessary step in the complete characterization of HbGp oligomeric structure.

Recent New Characterizations on the Giant Extracellular Hemoglobin

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### **5. Acknowledgments**

Thanks are due to the Brazilian agencies FAPESP, CNPq, and CAPES for partial financial support. P.S. Santiago is grateful to FAPESP and CNPq for postdoctoral grants. F. A. O. Carvalho is the recipient of a PhD. grant from FAPESP (2009/17261-6). J. W. P. Carvalho is the recipient of a PhD. grant from FAPESP (2010/09719-0). M. Tabak is grateful to CNPq for a research grant. Thanks are also due to Mr. Ézer Biazin for efficient support in the sample preparations and size exclusion chromatography experiments. The authors are indebted to Prof. Paolo di Mascio from Instituto de Química, Universidade de São Paulo, São Paulo, for making available the MALDI-TOF-MS instrumentation and to Mrs. Izaura N. Toma for excellent technical support in the experiments. The authors are greatful to Prof. Richard Garrat and Mr. Fernando Maluf, from the Instituto de Física de São Carlos, University of São Paulo, São Carlos, Brazil, respectively, for constant interest on the crystal structure analysis and support with purification of HbEa and HbPe. Thanks are also due to Mr. Afrânio Augusto Guimarães, from Minhobox (www.minhobox.com.br) for making available *Eisenia andrei* and *Peryonix excavatus* worms.

### **6. References**


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[23] Zal, F; Green, N; Lallier, F.H; Vinogradov S.N; Toulmond, A. (1997). Quaternary

[24] Royer, W.E, Omartian, M.N, Knapp, J.E. (2007). Low resolution structure

[25] Rousselot, M; Le Guen, D; Chabasse, C; Zal, F. (2006). Novel dissociation mechanism of

[26] Santiago, P.S; Moura, F; Moreira, L.M; Domingues, M.M; Santos, N.C; Tabak, M. (2008).

[28] Bispo, J.A.C; Santos, J.L.R; Landini, G.F; Goncalves, J.M; Bonafe, C.F.S. (2007). PH

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**16** 

Mehdi Cherif

*Montreal, Quebec* 

*Canada* 

*McGill University, Department of Biology,* 

**Biological Stoichiometry: The Elements** 

All organisms in their complexities of shapes, structures and functions use the same building blocks, elements, assembled and cemented together by energy. But only a subset of the elements available on Earth is used by the organisms in their biomass (Figure 1). Furthermore, the elements most commonly used by living beings (e.g., H, C, N, O, P, S, Na, K, Ca, Fe) are essential components of all organisms biomass. This reveals on one hand the common origin of all species and on the other hand the very specific requirements of the various life processes, to which only a subset of the elements are adapted (see Fraústo da Silva & Williams 2001 for a thorough discussion of the adequacy of elements to their

Fig. 1. The distribution of elements essential for life in the periodic table (from Fraústo da

Organisms need to get their elements from the environment. To do so, they engage in a variety of interactions with their physical environment and with the other species surrounding them. This fundamental need for elements and energy is thus the fulcrum of many ecological interactions. Furthermore, the sum of all element and energy consumption

**1. Introduction** 

biological functions).

Silva & Williams 2001).

**at the Heart of Biological Interactions** 


## **Biological Stoichiometry: The Elements at the Heart of Biological Interactions**

Mehdi Cherif

*McGill University, Department of Biology, Montreal, Quebec Canada* 

### **1. Introduction**

356 Stoichiometry and Research – The Importance of Quantity in Biomedicine

[43] Bachega JFR, Horjales ER, Tabak M, Garratt RC. (2011). Preliminary Crystallographic

Structure of Subunit D from the Giant Hemoglobin of *Glossoscolex paulistus*. *XL Reunião Anualda Sociedade Brasileira de Bioquímica e Biologia Molecular - SBBq*. No. M-

[42] Bachega, J.F.R et al, work in progress.

[44] Bachega, J.F.R et al., manuscript in preparation.

54, pp. 76

All organisms in their complexities of shapes, structures and functions use the same building blocks, elements, assembled and cemented together by energy. But only a subset of the elements available on Earth is used by the organisms in their biomass (Figure 1). Furthermore, the elements most commonly used by living beings (e.g., H, C, N, O, P, S, Na, K, Ca, Fe) are essential components of all organisms biomass. This reveals on one hand the common origin of all species and on the other hand the very specific requirements of the various life processes, to which only a subset of the elements are adapted (see Fraústo da Silva & Williams 2001 for a thorough discussion of the adequacy of elements to their biological functions).

Fig. 1. The distribution of elements essential for life in the periodic table (from Fraústo da Silva & Williams 2001).

Organisms need to get their elements from the environment. To do so, they engage in a variety of interactions with their physical environment and with the other species surrounding them. This fundamental need for elements and energy is thus the fulcrum of many ecological interactions. Furthermore, the sum of all element and energy consumption

Biological Stoichiometry: The Elements at the Heart of Biological Interactions 359

promising approach. It views organisms as a single molecule, made of a combination of the various essential elements (C, N, P, Fe…). Accordingly, it treats ecological interactions as chemical reactions, during which elements are exchanged between a consumer, its resource and the environment. It generally assumes that the organisms stoichiometry is constant, i.e., that their elemental composition is homeostatic. But this assumption is not essential to the theory. More essential is the mass conservation principle, which constrains the ecologists to

This theory has led to major advances in our understanding of ecological interactions across biological scales. Among them, there are: the realization that the growth of higher, complex organisms can be limited by the availability of one specific element in their food (Urabe & Watanabe 1992); the uncovering of indirect effects from plants on their supply of mineral nutrients through herbivores, because of mismatches between their elemental compositions (Sterner 1990); the exposure of a causal relationship between the elemental compositions of organisms and their growth rates (Elser *et al.* 2003a). Surprising insights from the theory also extend to other fields of biology, such as reproductive biology (Bertram *et al.* 2006), human cancer (Elser *et al.* 2007), evolution (Souza *et al.* 2008) and genomics (Acquisti *et al.* 2009). The earliest contributions of ecological stoichiometry to biology are covered in the 4th section. The latest contributions are covered in the 5th section. These advances led to the coinage of a new term, "Biological Stoichiometry". This term is meant to emphasize the potential of the theory to link processes across all the scales of biology, from molecules to the biosphere. The last section of this chapter will briefly evaluate how far the theory has gone in this unifying endeavour and what are the challenges ahead of biological stoichiometry, before it can claim to realistically portray some of the important interrelations between molecular and

Justus von Liebig was probably the first influential scientist to apply chemistry to study plant and animal physiologies in a systematic way. It is probably his vision, that there was no distinction between chemical reactions within and outside organisms, that led him to investigate the elemental compositions of organisms and the effects of this composition on

He came to realize that plant nutrition could be entirely satisfied by inorganic compounds, as long as they contained all the elements that made up plant biomass. Liebig's law of the minimum (Figure 2) emerged from this work and has become a central law of ecology. This law was the first example of an application of the principle of conservation of matter to the biological realm, albeit restricted to plants. For society at large, Liebig's work led to the invention and large-scale application of inorganic fertilizers, in other words, what was later

Liebig's work on plant mineral nutrition started a long tradition of research on plant growth limitation by elements. It also oriented researchers towards the pursuit of the original source of mineral nutrients in soils. Quickly, it was understood that microbes (bacteria and fungi) were the main providers of mineral nutrients to plants through their decomposition of organic matter in soils. This organic matter itself originates from dead parts of plants (shed leaves, fallen twigs and trunks, dead roots…) or from animal waste.

biotic processes such as plant growth and decomposition (Playfair & Liebig 1843).

track the fate of all the important elements exchanged in an ecological interaction.

ecosystemic processes.

known as the "Green Revolution".

**2. The long road to ecological stoichiometry** 

rates in an ecosystem represents a major driver of the biogeochemical cycles of elements. On geological timescales, organism-driven cycling of elements has effected great changes at a biosphere level, which fed back on life itself (Lovelock & Margulis 1974; Mcelroy 1983).

One would thus expect that the elemental nutrition of organisms would have been an early concern of ecologists and biogeochemists. Oddly enough, this was not the case, despite the fact that, very early on, Alfred James Lotka, a founding figure of ecology, called the attention of ecologists towards the crucial role of elements in shaping what he called the "drama of life", i.e., the sum of all ecological interactions (Lotka 1925). His call has been mostly ignored during the following six decades. Instead, ecologists focused almost exclusively on energy fluxes between organisms. Apart from some specialized fields of ecology, and until recently, most ecological approaches ignored the potential role of elements in shaping ecological interactions, emphasizing instead the importance of energy acquisition. The reasons are many, some conceptual, others practical. Energy in ecosystems is generally fixed from light in newly-formed organic matter by photosynthetic plants (hence the term primary producers used to describe photosynthetic organisms). It is then transmitted to those organisms that consume plant production, herbivores and decomposers. Energy then moves up the food web, thanks to predators eating other consumers. Once organic matter is consumed, the energy is either used for growth, excreted in molecules that cannot be used by plants, or dissipated as heat. Hence, there is no recycling of energy back to plants. The fate of all energy entering ecosystems through plants is either to be rapidly degraded into heat or buried into organic matter recalcitrant to decomposition. Energy flow within ecosystems is thus relatively simple. Consequently, its study led to straightforward, successful theories and concepts, such as Eltonian pyramids (Elton 1927), ecological efficiencies and food webs (Lindeman 1942).

On the other hand, the nutrients flow in ecosystems is a cycle. For a given element, autotrophic organisms are those that incorporate in their biomass the element in its mineral form from the environment. Generally, they are also primary producers, but many heterotrophic microorganisms show this ability, too. Mineral elements in ecosystems come partly from external outputs, but the bulk comes from the decomposition of carcasses and wastes from the organisms themselves. On very long timescales, because the Earth is virtually a closed system, most available elements should cycle at least once through living organisms, short of those spurted from the depths of the planet by volcanoes. This cycling adds a level of complexity that can hardly translate into simple, general laws, as is the case for energy (Loreau 2010). Faced with this complexity, few theoreticians attempted to look for generalities about the flows of matter in food webs and ecosystems, despite Lotka's (1925) longstanding call (DeAngelis 1992).

Admittedly, there have been some fields in which approaches based on elemental composition were applied. Moreover, there have been some sporadic attempts at taking into account the repercussions of the organisms elemental composition on their interactions or on the availability of nutrients in ecosystems. The next section is dedicated to a presentation of these fields that considered the role of elements in biology, but did not serve in their time as stepping stones towards a comprehensive theory of the role of elements in biological interactions. The field of ecology had to wait until the early nineties for such a theory, called "Ecological Stoichiometry", to emerge. It is a recent, exciting theory, presented in the third section, which tackles the role of elements in ecological interactions with a novel and

rates in an ecosystem represents a major driver of the biogeochemical cycles of elements. On geological timescales, organism-driven cycling of elements has effected great changes at a biosphere level, which fed back on life itself (Lovelock & Margulis 1974; Mcelroy 1983).

One would thus expect that the elemental nutrition of organisms would have been an early concern of ecologists and biogeochemists. Oddly enough, this was not the case, despite the fact that, very early on, Alfred James Lotka, a founding figure of ecology, called the attention of ecologists towards the crucial role of elements in shaping what he called the "drama of life", i.e., the sum of all ecological interactions (Lotka 1925). His call has been mostly ignored during the following six decades. Instead, ecologists focused almost exclusively on energy fluxes between organisms. Apart from some specialized fields of ecology, and until recently, most ecological approaches ignored the potential role of elements in shaping ecological interactions, emphasizing instead the importance of energy acquisition. The reasons are many, some conceptual, others practical. Energy in ecosystems is generally fixed from light in newly-formed organic matter by photosynthetic plants (hence the term primary producers used to describe photosynthetic organisms). It is then transmitted to those organisms that consume plant production, herbivores and decomposers. Energy then moves up the food web, thanks to predators eating other consumers. Once organic matter is consumed, the energy is either used for growth, excreted in molecules that cannot be used by plants, or dissipated as heat. Hence, there is no recycling of energy back to plants. The fate of all energy entering ecosystems through plants is either to be rapidly degraded into heat or buried into organic matter recalcitrant to decomposition. Energy flow within ecosystems is thus relatively simple. Consequently, its study led to straightforward, successful theories and concepts, such as Eltonian pyramids

On the other hand, the nutrients flow in ecosystems is a cycle. For a given element, autotrophic organisms are those that incorporate in their biomass the element in its mineral form from the environment. Generally, they are also primary producers, but many heterotrophic microorganisms show this ability, too. Mineral elements in ecosystems come partly from external outputs, but the bulk comes from the decomposition of carcasses and wastes from the organisms themselves. On very long timescales, because the Earth is virtually a closed system, most available elements should cycle at least once through living organisms, short of those spurted from the depths of the planet by volcanoes. This cycling adds a level of complexity that can hardly translate into simple, general laws, as is the case for energy (Loreau 2010). Faced with this complexity, few theoreticians attempted to look for generalities about the flows of matter in food webs and ecosystems, despite Lotka's

Admittedly, there have been some fields in which approaches based on elemental composition were applied. Moreover, there have been some sporadic attempts at taking into account the repercussions of the organisms elemental composition on their interactions or on the availability of nutrients in ecosystems. The next section is dedicated to a presentation of these fields that considered the role of elements in biology, but did not serve in their time as stepping stones towards a comprehensive theory of the role of elements in biological interactions. The field of ecology had to wait until the early nineties for such a theory, called "Ecological Stoichiometry", to emerge. It is a recent, exciting theory, presented in the third section, which tackles the role of elements in ecological interactions with a novel and

(Elton 1927), ecological efficiencies and food webs (Lindeman 1942).

(1925) longstanding call (DeAngelis 1992).

promising approach. It views organisms as a single molecule, made of a combination of the various essential elements (C, N, P, Fe…). Accordingly, it treats ecological interactions as chemical reactions, during which elements are exchanged between a consumer, its resource and the environment. It generally assumes that the organisms stoichiometry is constant, i.e., that their elemental composition is homeostatic. But this assumption is not essential to the theory. More essential is the mass conservation principle, which constrains the ecologists to track the fate of all the important elements exchanged in an ecological interaction.

This theory has led to major advances in our understanding of ecological interactions across biological scales. Among them, there are: the realization that the growth of higher, complex organisms can be limited by the availability of one specific element in their food (Urabe & Watanabe 1992); the uncovering of indirect effects from plants on their supply of mineral nutrients through herbivores, because of mismatches between their elemental compositions (Sterner 1990); the exposure of a causal relationship between the elemental compositions of organisms and their growth rates (Elser *et al.* 2003a). Surprising insights from the theory also extend to other fields of biology, such as reproductive biology (Bertram *et al.* 2006), human cancer (Elser *et al.* 2007), evolution (Souza *et al.* 2008) and genomics (Acquisti *et al.* 2009). The earliest contributions of ecological stoichiometry to biology are covered in the 4th section. The latest contributions are covered in the 5th section. These advances led to the coinage of a new term, "Biological Stoichiometry". This term is meant to emphasize the potential of the theory to link processes across all the scales of biology, from molecules to the biosphere. The last section of this chapter will briefly evaluate how far the theory has gone in this unifying endeavour and what are the challenges ahead of biological stoichiometry, before it can claim to realistically portray some of the important interrelations between molecular and ecosystemic processes.

### **2. The long road to ecological stoichiometry**

Justus von Liebig was probably the first influential scientist to apply chemistry to study plant and animal physiologies in a systematic way. It is probably his vision, that there was no distinction between chemical reactions within and outside organisms, that led him to investigate the elemental compositions of organisms and the effects of this composition on biotic processes such as plant growth and decomposition (Playfair & Liebig 1843).

He came to realize that plant nutrition could be entirely satisfied by inorganic compounds, as long as they contained all the elements that made up plant biomass. Liebig's law of the minimum (Figure 2) emerged from this work and has become a central law of ecology. This law was the first example of an application of the principle of conservation of matter to the biological realm, albeit restricted to plants. For society at large, Liebig's work led to the invention and large-scale application of inorganic fertilizers, in other words, what was later known as the "Green Revolution".

Liebig's work on plant mineral nutrition started a long tradition of research on plant growth limitation by elements. It also oriented researchers towards the pursuit of the original source of mineral nutrients in soils. Quickly, it was understood that microbes (bacteria and fungi) were the main providers of mineral nutrients to plants through their decomposition of organic matter in soils. This organic matter itself originates from dead parts of plants (shed leaves, fallen twigs and trunks, dead roots…) or from animal waste.

Biological Stoichiometry: The Elements at the Heart of Biological Interactions 361

aquatic and terrestrial ecosystems established that most bacterial production is generally consumed by heterotrophic predators also called microbivores (mainly protists, such as amoebae, ciliates and flagellates and, in soils, nematodes). The elements locked in the biomass of the ingested microbial decomposers are then mineralized as catabolic byproducts, or because microbivores themselves fall prey to other predators higher up the food web, closing what was called the "microbial loop" (Caron 1994; Clarholm 1994;

Fig. 3. Mineralization (A) and immobilization (B) of mineral nitrogen by a microbial decomposer, depending on whether organic matter is too rich or too poor in nitrogen,

Following the breakthrough of the microbial loop concept, a change in the paradigm of decomposition occurred. Now, the whole detrital food web, not only microbial decomposers, was seen as contributing to the mineralization of the elements essential to plant growth. In this new model, the elemental composition of microbial decomposers plays a central role. On one hand, it determines the extent of immobilization occurring. On the other hand, it also affects the excretion of elements by microbivores, since elements in excess of microbivores needs are excreted (Nakano 1994). Unfortunately, this increasing awareness of the roles of the elemental compositions of microbes, organic matter and microbivores within the communities of researchers interested in soils, did not spread to other fields of

respectively, in comparison to the decomposer nitrogen requirement.

ecology, at least until the emergence of the ecological stoichiometry theory.

figure 4).

To a lesser degree, Liebig's work also attracted attention to the role of animals as resuppliers of elements to plants. Overall, it helped entrench a prevalent view of ecosystems where plants are at the centre, bringing inorganic nutrients into the world of organic matter, and consumers are dissipators of energy and resuppliers of inorganic nutrients to plants, with decomposers taking the largest stack. The abiotic components of ecosystems considered are mainly those that affect mineral uptake by plants and the decomposition of organic matter into minerals (e.g., atmospheric deposition of minerals, leaching, temperature, light conditions…). This model came to reinforce the energy-based food chain model formalized by Lindeman (1942). The effect of elements on the growth and reproduction of animals and microbes was seldom considered, although Liebig himself invoked the possibility that the availability of elements in an animal's diet could limit its growth (Playfair & Liebig 1843). Elements were not yet seen as a factor able to affect the food webs structure and dynamics.

Fig. 2. Liebig's Law of the Minimum and the barrel analogy.

Later developments, however, made it harder to ignore the importance of the elemental needs of some consumers, both for their growth and for their recycling of nutrients. For example, it was known since a long time that microbial decomposers could, in some circumstances, take up inorganic elements instead of mineralize them (Waksman 1917). This uptake of mineral nutrients by heterotrophic microbes was called immobilization. It was quickly understood that the main controlling factor for microbial decomposition or immobilization of nutrients was the mineral content of the microbial biomass in relation to the content of organic matter (Figure 3).

The ecological consequences of the microbial decomposers making up shortages of essential elements in their resource by tapping into the stocks of inorganic nutrients were worked out later on (Bratbak & Thingstad 1985; Harte & Kinzig 1993; Daufresne & Loreau 2001a; Cherif & Loreau 2007). Nutrient-limited primary producers tend to generate carbon-rich organic matter, promoting microbial immobilization. This leads to a paradox, with nutrient-limited plants driving themselves towards stronger nutrient limitation by promoting immobilization. How, then, do limiting nutrients cycle back to plants and support continuous primary productivity if they are locked into the biomass of microbial decomposers? The solution to this paradox was found when food web studies in both

To a lesser degree, Liebig's work also attracted attention to the role of animals as resuppliers of elements to plants. Overall, it helped entrench a prevalent view of ecosystems where plants are at the centre, bringing inorganic nutrients into the world of organic matter, and consumers are dissipators of energy and resuppliers of inorganic nutrients to plants, with decomposers taking the largest stack. The abiotic components of ecosystems considered are mainly those that affect mineral uptake by plants and the decomposition of organic matter into minerals (e.g., atmospheric deposition of minerals, leaching, temperature, light conditions…). This model came to reinforce the energy-based food chain model formalized by Lindeman (1942). The effect of elements on the growth and reproduction of animals and microbes was seldom considered, although Liebig himself invoked the possibility that the availability of elements in an animal's diet could limit its growth (Playfair & Liebig 1843). Elements were not yet seen as a factor able to

Later developments, however, made it harder to ignore the importance of the elemental needs of some consumers, both for their growth and for their recycling of nutrients. For example, it was known since a long time that microbial decomposers could, in some circumstances, take up inorganic elements instead of mineralize them (Waksman 1917). This uptake of mineral nutrients by heterotrophic microbes was called immobilization. It was quickly understood that the main controlling factor for microbial decomposition or immobilization of nutrients was the mineral content of the microbial biomass in relation to

The ecological consequences of the microbial decomposers making up shortages of essential elements in their resource by tapping into the stocks of inorganic nutrients were worked out later on (Bratbak & Thingstad 1985; Harte & Kinzig 1993; Daufresne & Loreau 2001a; Cherif & Loreau 2007). Nutrient-limited primary producers tend to generate carbon-rich organic matter, promoting microbial immobilization. This leads to a paradox, with nutrient-limited plants driving themselves towards stronger nutrient limitation by promoting immobilization. How, then, do limiting nutrients cycle back to plants and support continuous primary productivity if they are locked into the biomass of microbial decomposers? The solution to this paradox was found when food web studies in both

affect the food webs structure and dynamics.

Fig. 2. Liebig's Law of the Minimum and the barrel analogy.

the content of organic matter (Figure 3).

aquatic and terrestrial ecosystems established that most bacterial production is generally consumed by heterotrophic predators also called microbivores (mainly protists, such as amoebae, ciliates and flagellates and, in soils, nematodes). The elements locked in the biomass of the ingested microbial decomposers are then mineralized as catabolic byproducts, or because microbivores themselves fall prey to other predators higher up the food web, closing what was called the "microbial loop" (Caron 1994; Clarholm 1994; figure 4).

Fig. 3. Mineralization (A) and immobilization (B) of mineral nitrogen by a microbial decomposer, depending on whether organic matter is too rich or too poor in nitrogen, respectively, in comparison to the decomposer nitrogen requirement.

Following the breakthrough of the microbial loop concept, a change in the paradigm of decomposition occurred. Now, the whole detrital food web, not only microbial decomposers, was seen as contributing to the mineralization of the elements essential to plant growth. In this new model, the elemental composition of microbial decomposers plays a central role. On one hand, it determines the extent of immobilization occurring. On the other hand, it also affects the excretion of elements by microbivores, since elements in excess of microbivores needs are excreted (Nakano 1994). Unfortunately, this increasing awareness of the roles of the elemental compositions of microbes, organic matter and microbivores within the communities of researchers interested in soils, did not spread to other fields of ecology, at least until the emergence of the ecological stoichiometry theory.

Biological Stoichiometry: The Elements at the Heart of Biological Interactions 363

Furthermore, Redfield proposed that the phytoplankton balanced its consumption of nitrogen relative to phosphorus by fixing the atmospheric nitrogen when P was in excess, so that their ratio became equal to that of their chemical composition (the now famous N:P Redfield ratio equal to 16). This is equivalent to assuming that it was the phosphorus that, on geological timescales, limited the growth of phytoplankton, while nitrogen was simply adjusted by the biological activity of the nitrogen-fixing phytoplankton (Redfield 1958).

This stoichiometric model of the oceanic biogeochemical cycle has prompted work on the elemental stoichiometry of recycling in oceans (e.g., Berner). Moreover, it encouraged much speculation on the genesis and maintenance of the ocean and the atmosphere chemical properties (Walker 1974; Griffith *et al.* 1977; Lovelock & Watson 1982). Curiously, this did not lead to any serious attempt to generalize these results to other ecosystems, other elements beyond nitrogen, phosphorus and carbon, or the roles of other trophic levels

On the other hand, the assertion that phosphorus should be the element limiting the growth of phytoplankton contradicted accumulated empirical evidence showing that the oceanic phytoplankton was primarily limited by nitrogen (Smith 1984). This contradiction spurred several decades of studies on the growth and chemical composition of phytoplankton grown in the presence of several potentially limiting factors, especially nitrogen and phosphorus (Droop 1974; Rhee 1978; Goldman *et al.* 1979; Tett *et al.* 1985). These experiments showed that both the chemical composition and type of growth-limiting element vary among phytoplankton species. Although constant at the scale of ocean basins, the Redfield ratio is thus probably the result of several processes rather than a fixed property of the

The accumulation of knowledge on the diversity of elemental requirements, limitations and chemical compositions among phytoplankton species led to believe that it was possible to explain the "paradox of the plankton" proposed by George Evelyn Hutchinson (1961). Hutchinson wondered how the phytoplankton could harbour so many species (in the order of several tens) in the relatively homogeneous environment of surface waters, despite the low number of resources shared by these species. This seemed to contradict the principle of competitive exclusion, which predicts that there cannot be more species than limiting

Based on earlier work (Macarthur & Levins 1964; Rapport 1971; Leon & Tumpson 1975), David Tilman developed a theory that could predict the outcome of resource competition between several species of phytoplankton according to their elemental requirements, their chemical compositions and external supplies of elements in the ecosystem (Tilman 1980). Later, expanding his theory to integrate spatiotemporal fluctuations in external supplies, he demonstrated that these variations allowed the coexistence of more species than resources (Tilman *et al.* 1982). He thus provided what he considered as a definitive response to Hutchinson's "paradox of the plankton". Furthermore, seeking to explain possible sources of variation in the supplies of elements, he advanced the hypothesis that "If some nutrients (e.g., phosphorus) are regenerated more rapidly than others, zooplankton may significantly affect phytoplankton community structure by changing nutrient supply rates and ratios" (Tilman *et al.* 1982). Only a few steps remained until the

phytoplankton (Falkowski 2000; Geider & La Roche 2002; Klausmeier *et al.* 2004).

beyond the phytoplankton.

resources at equilibrium (Hardin 1960).

ecological stoichiometry theory.

Fig. 4. The microbial loop in soils. Microbial decomposers (fungi and bacteria) immobilize the inorganic elements (here N). They are consumed by microbivores (here nematodes) who release the immobilized N as a by-product of catabolism and are consumed by other consumers (here mites).

The closest to an early stoichiometric thinking, anterior to the ecological stoichiometry theory, appeared in oceanography, a discipline interested into patterns at a larger spatial scale than most other disciplines, but which turned its attention towards the phytoplankton, among the smallest organisms on the planet. Biomass elemental composition was part of oceanographic thinking since at least the 40s, thanks to the work of Alfred Clarence Redfield (1934). Redfield compared the contents of carbon, nitrogen and phosphorus in the phytoplankton of open ocean areas to dissolved nutrient concentrations in surface water and regenerated in the deep ocean (Table 1). The similarity between these ratios led him to state that the plankton chemical composition determined, on geological time, the chemical composition of the ocean (Redfield 1958).


Table 1. Atomic ratios of elements in components of the oceanic biochemical cycle (Adapted from Redfield 1958)

Fig. 4. The microbial loop in soils. Microbial decomposers (fungi and bacteria) immobilize the inorganic elements (here N). They are consumed by microbivores (here nematodes) who

The closest to an early stoichiometric thinking, anterior to the ecological stoichiometry theory, appeared in oceanography, a discipline interested into patterns at a larger spatial scale than most other disciplines, but which turned its attention towards the phytoplankton, among the smallest organisms on the planet. Biomass elemental composition was part of oceanographic thinking since at least the 40s, thanks to the work of Alfred Clarence Redfield (1934). Redfield compared the contents of carbon, nitrogen and phosphorus in the phytoplankton of open ocean areas to dissolved nutrient concentrations in surface water and regenerated in the deep ocean (Table 1). The similarity between these ratios led him to state that the plankton chemical composition determined, on geological time, the chemical

Table 1. Atomic ratios of elements in components of the oceanic biochemical cycle (Adapted

release the immobilized N as a by-product of catabolism and are consumed by other

consumers (here mites).

from Redfield 1958)

composition of the ocean (Redfield 1958).

Furthermore, Redfield proposed that the phytoplankton balanced its consumption of nitrogen relative to phosphorus by fixing the atmospheric nitrogen when P was in excess, so that their ratio became equal to that of their chemical composition (the now famous N:P Redfield ratio equal to 16). This is equivalent to assuming that it was the phosphorus that, on geological timescales, limited the growth of phytoplankton, while nitrogen was simply adjusted by the biological activity of the nitrogen-fixing phytoplankton (Redfield 1958).

This stoichiometric model of the oceanic biogeochemical cycle has prompted work on the elemental stoichiometry of recycling in oceans (e.g., Berner). Moreover, it encouraged much speculation on the genesis and maintenance of the ocean and the atmosphere chemical properties (Walker 1974; Griffith *et al.* 1977; Lovelock & Watson 1982). Curiously, this did not lead to any serious attempt to generalize these results to other ecosystems, other elements beyond nitrogen, phosphorus and carbon, or the roles of other trophic levels beyond the phytoplankton.

On the other hand, the assertion that phosphorus should be the element limiting the growth of phytoplankton contradicted accumulated empirical evidence showing that the oceanic phytoplankton was primarily limited by nitrogen (Smith 1984). This contradiction spurred several decades of studies on the growth and chemical composition of phytoplankton grown in the presence of several potentially limiting factors, especially nitrogen and phosphorus (Droop 1974; Rhee 1978; Goldman *et al.* 1979; Tett *et al.* 1985). These experiments showed that both the chemical composition and type of growth-limiting element vary among phytoplankton species. Although constant at the scale of ocean basins, the Redfield ratio is thus probably the result of several processes rather than a fixed property of the phytoplankton (Falkowski 2000; Geider & La Roche 2002; Klausmeier *et al.* 2004).

The accumulation of knowledge on the diversity of elemental requirements, limitations and chemical compositions among phytoplankton species led to believe that it was possible to explain the "paradox of the plankton" proposed by George Evelyn Hutchinson (1961). Hutchinson wondered how the phytoplankton could harbour so many species (in the order of several tens) in the relatively homogeneous environment of surface waters, despite the low number of resources shared by these species. This seemed to contradict the principle of competitive exclusion, which predicts that there cannot be more species than limiting resources at equilibrium (Hardin 1960).

Based on earlier work (Macarthur & Levins 1964; Rapport 1971; Leon & Tumpson 1975), David Tilman developed a theory that could predict the outcome of resource competition between several species of phytoplankton according to their elemental requirements, their chemical compositions and external supplies of elements in the ecosystem (Tilman 1980). Later, expanding his theory to integrate spatiotemporal fluctuations in external supplies, he demonstrated that these variations allowed the coexistence of more species than resources (Tilman *et al.* 1982). He thus provided what he considered as a definitive response to Hutchinson's "paradox of the plankton". Furthermore, seeking to explain possible sources of variation in the supplies of elements, he advanced the hypothesis that "If some nutrients (e.g., phosphorus) are regenerated more rapidly than others, zooplankton may significantly affect phytoplankton community structure by changing nutrient supply rates and ratios" (Tilman *et al.* 1982). Only a few steps remained until the ecological stoichiometry theory.

Biological Stoichiometry: The Elements at the Heart of Biological Interactions 365

1991; Urabe 1993). Daufresne and Loreau (2001b) provided an elegant explanation of these discrepancies by showing theoretically that the effect of herbivores on the plants limitation depends not only on their chemical composition, but also on the physiological response of plants to herbivory. Indeed, the decrease in plant biomass caused by herbivory results in increased elemental requirements of plants to compensate for the loss. These additional requirements can vary between elements and between phytoplankton species. The nature of the limiting element promoted by herbivores is determined by the result of these

physiological adjustments and the differential recycling of elements by herbivores.

Fig. 5. The stoichiometric hypothesis of consumer-driven elemental limitation of

Getting rid of this excess, they enrich the environment in N and thus drive the phytoplankton towards P limitation. N-rich copepods (lower panel) drive the

though, that no comprehensive review of the topic has appeared since then.

phytoplankton towards N limitation by a similar mechanism.

phytoplankton. P-rich Daphnia (upper panel) ingest N in excess from the phytoplankton.

Efforts were also undertaken to broaden the stoichiometric approach beyond the sphere of pelagic freshwater organisms and towards other ecosystems. Several studies have therefore turned to other habitats: marine (Elser & Hassett 1994), arctic (Dobberfuhl & Elser 2000), terrestrial (Elser *et al.* 2000), benthic (Frost *et al.* 2002), arid (Schade *et al.* 2003) and even fossil ecosystems (Elser *et al.* 2006). Other organisms and trophic levels were also investigated: bacteria (Chrzanowski & Kyle 1996; Makino *et al.* 2003), protists (Grover & Chrzanowski 2006), vertebrates (Vanni *et al.* 2002), and insects (Woods *et al.* 2002; Markow *et al.* 2006).

Most of the advances in ecological stoichiometry, from its birth to the early years of this century are well summarized in the book "Ecological stoichiometry: The biology of elements from molecules to the biosphere" published in 2002 by Sterner and Elser. It is worth noting

### **3. Ecological stoichiometry: An attempt at a systematic approach**

In 1988, Elser and colleagues observed a correlation between the type of element limiting the growth of the phytoplankton and the species composition of the zooplankton community of experimental lakes in Michigan (Elser *et al.* 1988). When the zooplankton community was dominated by copepods, the phytoplankton was limited by nitrogen, whereas it was limited by phosphorus when dominated by cladocerans such as Daphnia. They already knew that the zooplankton recycled nitrogen and phosphorus with different efficiencies (Lehman 1984). They could not explain, though, the different effects of copepods and cladocerans, especially since many differences between the two types of zooplankton could intervene: copepods are mainly small and select the species they consume, while cladocerans are generally larger (up to several millimetres) and consume very effectively all the species captured in their filters. An explanation was soon offered by R. W. Sterner (1990) when he showed theoretically that zooplankton species with different chemical compositions should recycle elements with different efficiencies, provided that their chemical composition is kept constant over time. Andersen and Hessen (1991) then found that copepods and cladocerans were widely dissimilar in their chemical composition; copepods are rich in nitrogen while Daphnia are richer in phosphorus. Moreover, they found little variation in their compositions over time, despite variations in the composition of their resources.

All these related observations and hypotheses merged together to give the first fullystoichiometric description of a trophic interaction: " Herbivore species with a high ratio of N:P in their tissues should resupply nutrients at a relatively low N:P ratio compared to herbivore species with low body N:P" (Sterner *et al.* 1992). The increased availability of the element in excess of the herbivores needs should drive the phytoplankton to a limitation by the same factor that limits the growth of herbivores (N for herbivores with a high ratio of N: P and P for those with a low N: P) (Figure 5).

Surprisingly, the main criticisms of this hypothesis concerned a point that is not essential to the mechanism that underlies it, but derives from subsequent works (Hessen 1992; Urabe & Watanabe 1992). The possibility of a direct limitation of the growth of the zooplankton by mineral elements, either N or P, raised many objections from researchers thinking that resource limitation of growth was possible only by biochemical substances such as amino acids or fatty acids (Brett 1993, Müller-Navarra 1995). Links between growth rate and diet elemental content were seen as simple correlations. Much research was then devoted to prove or disprove the negative effects of nitrogen and phosphorus-deficient phytoplankton on the growth of the zooplankton (Sterner *et al.* 1993; Anderson & Hessen 1995; Urabe *et al.* 1997; Urabe & Sterner 2001).

Regeneration of nitrogen and phosphorus by the zooplankton with a ratio reflecting the difference between its chemical composition and that of its resources is a part of the hypothesis that was largely confirmed afterwards (Sterner & Smith 1993; Urabe 1993; Balseiro *et al.* 1997; Elser & Urabe 1999). On the other hand, the alteration of phytoplankton limitation that is supposed to result from this differential recycling of elements by zooplankton was, if not less studied, rarely highlighted. Some studies showed indeed a strong effect of herbivore chemical composition and recycling on the phytoplankton growth limitation (Carpenter *et al.* 1993; Rothhaupt 1997; MacKay & Elser 1998). Other results did not concur with the predictions from the stoichiometric hypothesis (Moegenburg & Vanni

In 1988, Elser and colleagues observed a correlation between the type of element limiting the growth of the phytoplankton and the species composition of the zooplankton community of experimental lakes in Michigan (Elser *et al.* 1988). When the zooplankton community was dominated by copepods, the phytoplankton was limited by nitrogen, whereas it was limited by phosphorus when dominated by cladocerans such as Daphnia. They already knew that the zooplankton recycled nitrogen and phosphorus with different efficiencies (Lehman 1984). They could not explain, though, the different effects of copepods and cladocerans, especially since many differences between the two types of zooplankton could intervene: copepods are mainly small and select the species they consume, while cladocerans are generally larger (up to several millimetres) and consume very effectively all the species captured in their filters. An explanation was soon offered by R. W. Sterner (1990) when he showed theoretically that zooplankton species with different chemical compositions should recycle elements with different efficiencies, provided that their chemical composition is kept constant over time. Andersen and Hessen (1991) then found that copepods and cladocerans were widely dissimilar in their chemical composition; copepods are rich in nitrogen while Daphnia are richer in phosphorus. Moreover, they found little variation in their

**3. Ecological stoichiometry: An attempt at a systematic approach** 

compositions over time, despite variations in the composition of their resources.

P and P for those with a low N: P) (Figure 5).

1997; Urabe & Sterner 2001).

All these related observations and hypotheses merged together to give the first fullystoichiometric description of a trophic interaction: " Herbivore species with a high ratio of N:P in their tissues should resupply nutrients at a relatively low N:P ratio compared to herbivore species with low body N:P" (Sterner *et al.* 1992). The increased availability of the element in excess of the herbivores needs should drive the phytoplankton to a limitation by the same factor that limits the growth of herbivores (N for herbivores with a high ratio of N:

Surprisingly, the main criticisms of this hypothesis concerned a point that is not essential to the mechanism that underlies it, but derives from subsequent works (Hessen 1992; Urabe & Watanabe 1992). The possibility of a direct limitation of the growth of the zooplankton by mineral elements, either N or P, raised many objections from researchers thinking that resource limitation of growth was possible only by biochemical substances such as amino acids or fatty acids (Brett 1993, Müller-Navarra 1995). Links between growth rate and diet elemental content were seen as simple correlations. Much research was then devoted to prove or disprove the negative effects of nitrogen and phosphorus-deficient phytoplankton on the growth of the zooplankton (Sterner *et al.* 1993; Anderson & Hessen 1995; Urabe *et al.*

Regeneration of nitrogen and phosphorus by the zooplankton with a ratio reflecting the difference between its chemical composition and that of its resources is a part of the hypothesis that was largely confirmed afterwards (Sterner & Smith 1993; Urabe 1993; Balseiro *et al.* 1997; Elser & Urabe 1999). On the other hand, the alteration of phytoplankton limitation that is supposed to result from this differential recycling of elements by zooplankton was, if not less studied, rarely highlighted. Some studies showed indeed a strong effect of herbivore chemical composition and recycling on the phytoplankton growth limitation (Carpenter *et al.* 1993; Rothhaupt 1997; MacKay & Elser 1998). Other results did not concur with the predictions from the stoichiometric hypothesis (Moegenburg & Vanni 1991; Urabe 1993). Daufresne and Loreau (2001b) provided an elegant explanation of these discrepancies by showing theoretically that the effect of herbivores on the plants limitation depends not only on their chemical composition, but also on the physiological response of plants to herbivory. Indeed, the decrease in plant biomass caused by herbivory results in increased elemental requirements of plants to compensate for the loss. These additional requirements can vary between elements and between phytoplankton species. The nature of the limiting element promoted by herbivores is determined by the result of these physiological adjustments and the differential recycling of elements by herbivores.

Fig. 5. The stoichiometric hypothesis of consumer-driven elemental limitation of phytoplankton. P-rich Daphnia (upper panel) ingest N in excess from the phytoplankton. Getting rid of this excess, they enrich the environment in N and thus drive the phytoplankton towards P limitation. N-rich copepods (lower panel) drive the phytoplankton towards N limitation by a similar mechanism.

Efforts were also undertaken to broaden the stoichiometric approach beyond the sphere of pelagic freshwater organisms and towards other ecosystems. Several studies have therefore turned to other habitats: marine (Elser & Hassett 1994), arctic (Dobberfuhl & Elser 2000), terrestrial (Elser *et al.* 2000), benthic (Frost *et al.* 2002), arid (Schade *et al.* 2003) and even fossil ecosystems (Elser *et al.* 2006). Other organisms and trophic levels were also investigated: bacteria (Chrzanowski & Kyle 1996; Makino *et al.* 2003), protists (Grover & Chrzanowski 2006), vertebrates (Vanni *et al.* 2002), and insects (Woods *et al.* 2002; Markow *et al.* 2006).

Most of the advances in ecological stoichiometry, from its birth to the early years of this century are well summarized in the book "Ecological stoichiometry: The biology of elements from molecules to the biosphere" published in 2002 by Sterner and Elser. It is worth noting though, that no comprehensive review of the topic has appeared since then.

Biological Stoichiometry: The Elements at the Heart of Biological Interactions 367

proportions of proteins and nucleic acids also differ in their N:P ratios (Sterner & Elser 2002). The next step was to look at the distribution of these molecules within cell components and organelles. Through cell fractionation techniques and calculations, it quickly became clear that ribosomal RNA (rRNA) generally represent the major stock of nucleic acids and P within cells (Elser *et al.* 1996). Proteins and associated N atoms are more evenly distributed within cells, although mitochondria emerge as a particularly N-rich organelles (Sterner & Elser 2002). These observations led to one of the first and major unexpected predictions to stem from the ecological stoichiometry theory, namely the "growth rate hypothesis". This hypothesis is simply a causal link made between the observation that rRNA generally represent the major stock of P in cells and organisms on one side, and the observation that the need for ribosomes increases with growth rate on the other side (Elser *et al.* 1996). From there came the hypothesis that there should be a direct relation between the growth rate of organisms and their P content. Since proteins and nucleic acids have similar N contents, replacing one with another should not alter the N content. So, there should be also a direct relation between growth rate and the N:P ratio. The hypothesis was put to test, using data collections and experiments. Many studies showed results that are congruent with the growth rate hypothesis in various taxa (Elser *et al.* 2003a; Karpinets *et al.* 2006). However, it was found to be of limited validity across other taxa, because of species-specific differences in the percentage of P linked to RNA (Matzek & Vitousek 2009). Moreover, maintenance costs for high P levels in biomass can also impair the relation between P content and growth rate (Urabe & Shimizu 2008). Finally, P content was often decoupled from growth rate when organisms were grown under a limiting factor other than P, probably because they can store P that is in excess of their needs (Elser *et al.*

Curiously, although there is a strong association between mitochondria and cell N content, there was no hypothesis set forth to predict potential relations between cell N content and physiological correlates of mitochondrial cellular density. More generally, the role of N content has been understudied in biological stoichiometry, probably reflecting the fact that: i) N content is less variable than P content ii) P-rich Daphnia are model organisms in many fields of ecology and genetics and iii) P is thought to be the predominant limiting factor in

In summary, there are three main factors that determine the elemental composition of organisms: i) the relative amounts of important biomolecules in cells, such as proteins and RNA; ii) the type and amount of structural material in the organism; and iii) the reserves of elements that are supplied in excess of the organismal requirement. Each of these three factors is related to ecologically relevant life history traits, thus building a bridge between the stoichiometry and the ecology of organisms. For example, P content in P limited organisms, is related to RNA content which itself is a function of growth rate (the Growth Rate Hypothesis). Organisms with more structural material, tending to be larger than related species with fewer structures, show lower specific metabolic rates (Brown *et al.* 2004). Species storing non-limiting resources fare better than similar organisms without storage when the resource becomes limiting, but at the expenses of a higher maximum growth rate (Sommer 1985). Hence, progress in understanding the underlying biological determinants of the organisms stoichiometry is relevant, not only because it advances our knowledge of the physiology of organisms, but also because it sheds light on the ecology of organisms as

freshwater ecosystems and the main agent of anthropogenic eutrophication.

2003a; Matzek & Vitousek 2009).

### **4. Understanding stoichiometry: The biological bases of the economy of elements in organisms**

Different organisms vary widely in their elemental composition. This variation is what makes the study of stoichiometry relevant for developing a better understanding of ecological interactions. On a different level, these elemental differences also lead to questions about the underlying biological causes for these differences. Sterner and Elser's synthetic book (2002) represents the first attempt at an in-depth investigation of the origins of the differences among the elemental stoichiometries of organisms.

A first obvious source of stoichiometric diversity is the various extents to which organisms invest in structural materials that often present distinct stoichiometric signatures. Wellknown examples are the investment of molluscs in calcium carbonate shells, which should increase their calcium content, the hydroxyapatite-based bones of vertebrates, the siliceous frustules of diatoms and the carbon-rich, nitrogen-poor woody stems of many terrestrial plants. A precursor to the ecological stoichiometry theory, William A. Reiners (1986) assumed a fixed composition for the protoplasm of all organisms (the cellular components, without any structural material) and assigned all variations in elemental composition to variations in the proportions of different structural material. Since then, it became obvious that protoplasms can differ in their elemental compositions, such that even relatively taxonomically close species can show different stoichiometries (Andersen & Hessen 1991). The basic building blocks of organic matter (amino-acids, lipids, carbohydrates, nucleotides…) and the polymers assembled from them (proteins, nucleotides…) show obvious stoichiometric differences (Figure 6).

Fig. 6. Nitrogen and phosphorus composition of important biomolecules. Percentage nitrogen and percentage phosphorus are given in terms of weight. Dotted lines depict standard values of atomic (molar) N:P ratios, the most commonly used units reported in ecological stoichiometry studies (reproduced from Elser *et al.* 1996)

Among these biomolecules, proteins and nucleic acids stand out, being very abundant and having contrasting elemental compositions. Proteins are rich in N and nucleic acids are rich in P. It was postulated and then shown that otherwise close organisms that differ in their

Different organisms vary widely in their elemental composition. This variation is what makes the study of stoichiometry relevant for developing a better understanding of ecological interactions. On a different level, these elemental differences also lead to questions about the underlying biological causes for these differences. Sterner and Elser's synthetic book (2002) represents the first attempt at an in-depth investigation of the origins

A first obvious source of stoichiometric diversity is the various extents to which organisms invest in structural materials that often present distinct stoichiometric signatures. Wellknown examples are the investment of molluscs in calcium carbonate shells, which should increase their calcium content, the hydroxyapatite-based bones of vertebrates, the siliceous frustules of diatoms and the carbon-rich, nitrogen-poor woody stems of many terrestrial plants. A precursor to the ecological stoichiometry theory, William A. Reiners (1986) assumed a fixed composition for the protoplasm of all organisms (the cellular components, without any structural material) and assigned all variations in elemental composition to variations in the proportions of different structural material. Since then, it became obvious that protoplasms can differ in their elemental compositions, such that even relatively taxonomically close species can show different stoichiometries (Andersen & Hessen 1991). The basic building blocks of organic matter (amino-acids, lipids, carbohydrates, nucleotides…) and the polymers assembled from them (proteins, nucleotides…) show obvious stoichiometric differences

Fig. 6. Nitrogen and phosphorus composition of important biomolecules. Percentage nitrogen and percentage phosphorus are given in terms of weight. Dotted lines depict standard values of atomic (molar) N:P ratios, the most commonly used units reported in

Among these biomolecules, proteins and nucleic acids stand out, being very abundant and having contrasting elemental compositions. Proteins are rich in N and nucleic acids are rich in P. It was postulated and then shown that otherwise close organisms that differ in their

ecological stoichiometry studies (reproduced from Elser *et al.* 1996)

**4. Understanding stoichiometry: The biological bases of the economy of** 

of the differences among the elemental stoichiometries of organisms.

**elements in organisms** 

(Figure 6).

proportions of proteins and nucleic acids also differ in their N:P ratios (Sterner & Elser 2002). The next step was to look at the distribution of these molecules within cell components and organelles. Through cell fractionation techniques and calculations, it quickly became clear that ribosomal RNA (rRNA) generally represent the major stock of nucleic acids and P within cells (Elser *et al.* 1996). Proteins and associated N atoms are more evenly distributed within cells, although mitochondria emerge as a particularly N-rich organelles (Sterner & Elser 2002). These observations led to one of the first and major unexpected predictions to stem from the ecological stoichiometry theory, namely the "growth rate hypothesis". This hypothesis is simply a causal link made between the observation that rRNA generally represent the major stock of P in cells and organisms on one side, and the observation that the need for ribosomes increases with growth rate on the other side (Elser *et al.* 1996). From there came the hypothesis that there should be a direct relation between the growth rate of organisms and their P content. Since proteins and nucleic acids have similar N contents, replacing one with another should not alter the N content. So, there should be also a direct relation between growth rate and the N:P ratio. The hypothesis was put to test, using data collections and experiments. Many studies showed results that are congruent with the growth rate hypothesis in various taxa (Elser *et al.* 2003a; Karpinets *et al.* 2006). However, it was found to be of limited validity across other taxa, because of species-specific differences in the percentage of P linked to RNA (Matzek & Vitousek 2009). Moreover, maintenance costs for high P levels in biomass can also impair the relation between P content and growth rate (Urabe & Shimizu 2008). Finally, P content was often decoupled from growth rate when organisms were grown under a limiting factor other than P, probably because they can store P that is in excess of their needs (Elser *et al.* 2003a; Matzek & Vitousek 2009).

Curiously, although there is a strong association between mitochondria and cell N content, there was no hypothesis set forth to predict potential relations between cell N content and physiological correlates of mitochondrial cellular density. More generally, the role of N content has been understudied in biological stoichiometry, probably reflecting the fact that: i) N content is less variable than P content ii) P-rich Daphnia are model organisms in many fields of ecology and genetics and iii) P is thought to be the predominant limiting factor in freshwater ecosystems and the main agent of anthropogenic eutrophication.

In summary, there are three main factors that determine the elemental composition of organisms: i) the relative amounts of important biomolecules in cells, such as proteins and RNA; ii) the type and amount of structural material in the organism; and iii) the reserves of elements that are supplied in excess of the organismal requirement. Each of these three factors is related to ecologically relevant life history traits, thus building a bridge between the stoichiometry and the ecology of organisms. For example, P content in P limited organisms, is related to RNA content which itself is a function of growth rate (the Growth Rate Hypothesis). Organisms with more structural material, tending to be larger than related species with fewer structures, show lower specific metabolic rates (Brown *et al.* 2004). Species storing non-limiting resources fare better than similar organisms without storage when the resource becomes limiting, but at the expenses of a higher maximum growth rate (Sommer 1985). Hence, progress in understanding the underlying biological determinants of the organisms stoichiometry is relevant, not only because it advances our knowledge of the physiology of organisms, but also because it sheds light on the ecology of organisms as

Biological Stoichiometry: The Elements at the Heart of Biological Interactions 369

turnover, assimilation costs, maintenance costs, and respiration. These endeavours to understand the regulation of organismal stoichiometry by including the metabolism of biomolecules lead to connections between biological stoichiometry and nutritional sciences

Fig. 7. Relation between the gross growth efficiencies of two unspecified elements (X and Y) as imposed by the constraint of a homeostatic regulation of the stoichiometry of a consumer. A) Mass balance equations for consumption. B) Definition of the gross growth efficiency. C) Stoichiometric constraint imposed on the GGEs for X and Y, as a consequence of the constancy of the X:Y ratio of consumers. D) A simple model that assumes a maximal GGE for the limiting element leads to the GGE of the non-limiting element to be a function of the maximal GGE,

Physiological adaptation is the short-term response of consumers to imbalanced resources. Analyses at the levels of genomes and transcriptomes, showed that transcriptional changes are among the mechanisms used by consumers to achieve stoichiometric homeostasis. For example, transcriptional changes in the expression of phosphate transporters, activation of alternative metabolic pathways, changes in allocations to roots (for plants), are triggered under P deficiency (Jeyasingh & Weider 2007). Recent studies uncovered long-term evolutionary responses to element deprivations: N-deprived organisms react by substituting N-rich amino-acids by amino-acids with N-poor residues in their expressed proteins or by expressing shorter mRNAs (Grzymski & Dussaq 2011). They also show RNAs with more Npoor nucleotides (Elser *et al.* 2011). As for DNA, since GC and AT pairs have almost the same N content, N limitation seems to have less effect. These advances on the links between elemental limitation and cell polymer composition lead to the coining of a new term:

and the X:Y ratios of the consumer and its resource (adapted from Sterner 1990).

"stoichiogenomics" (Elser *et al.* 2011).

(Raubenheimer *et al.* 2009).

affected by their stoichiometry. The previous section described how Ecological Stoichiometry opened up the way to link the organisms stoichiometry to the biogeochemical cycles within ecosystems. This section shows how Biological Stoichiometry represents a link from molecules to the ecology of organisms. Since Biological Stoichiometry encompasses Ecological Stoichiometry, then it is a theory that has the potential to link molecules to ecosystems, as claimed by its main proponents in their reference work (Sterner & Elser 2002). Nevertheless, the theory just set the tools (the elemental composition regulation in organisms) and the method (balancing the multi-elemental exchanges between organisms) to reach such integration across the levels of biology, without offering a-priori predictions about how processes at different biological levels articulate. These predictions need to be worked out by mixing stoichiometric reasoning with observations. Such was the case for the Growth Rate Hypothesis, which stemmed from the observations that a majority of P in cells is associated with rRNA, itself associated with growth rate, and concluded that fast-growing organisms should be P-rich and thus more sensitive to P deficiencies in their food. The next section is meant to present some of the most recent advances towards this integrative goal.

### **5. Recent advances in biological stoichiometry**

Following Sterner and Elser's 2002 book, researchers in the field of biological stoichiometry pursued two main directions. On one hand, there was an urge to have a more accurate knowledge of the mechanisms by which organisms regulate their elemental composition, in order to more realistically link their stoichiometric properties to their impacts on the cycles of elements. On the other hand, other researchers carried on with the process of extending the use of the stoichiometric approach to other fields in biology, always with the objective of integrating seemingly separate fields.

Consumers that strive to keep their elemental composition constant, in the face of stoichiometrically imbalanced resources, need to alter their gross growth efficiencies for elements in a controlled way (Figure 7). These gross growth efficiencies, in turn, determine the differential recycling of elements by consumers (Sterner 1990).

How consumers regulate their gross growth efficiencies was, and still is, largely unknown. In the absence of empirical data, in early stoichiometric models, the gross growth efficiency of the element limiting the consumer growth was set to a maximum value, ensuring maximal growth, while the gross growth efficiencies of the other elements were adjusted to yield a constant stoichiometry for the consumer (Figure 7). But theoretically, there is an infinite number of possible alternative strategies (Andersen 1997). Recent experiments also show that net assimilation of the limiting element is not necessarily at its maximal efficiency (DeMott *et al.* 1998) and that non-limiting elements too have impacts on growth (Boersma & Elser 2006; Zehnder & Hunter 2009). Moreover, it was shown that the quantity of resources, besides their stoichiometric compositions, affects the gross growth efficiencies of elements (Anderson & Hessen 2005). Furthermore, models that included a description of the distribution of elements among the various biomolecules in cells have shown that it is not even always possible to set one element as the growth- limiting factor (Anderson *et al.* 2004). It thus became clear that more physiologically grounded models of the use of elements by organisms were needed. Anderson et al's now classical model (2005) strikes a good balance between physiological realism and simplicity, including processes such as biomass

affected by their stoichiometry. The previous section described how Ecological Stoichiometry opened up the way to link the organisms stoichiometry to the biogeochemical cycles within ecosystems. This section shows how Biological Stoichiometry represents a link from molecules to the ecology of organisms. Since Biological Stoichiometry encompasses Ecological Stoichiometry, then it is a theory that has the potential to link molecules to ecosystems, as claimed by its main proponents in their reference work (Sterner & Elser 2002). Nevertheless, the theory just set the tools (the elemental composition regulation in organisms) and the method (balancing the multi-elemental exchanges between organisms) to reach such integration across the levels of biology, without offering a-priori predictions about how processes at different biological levels articulate. These predictions need to be worked out by mixing stoichiometric reasoning with observations. Such was the case for the Growth Rate Hypothesis, which stemmed from the observations that a majority of P in cells is associated with rRNA, itself associated with growth rate, and concluded that fast-growing organisms should be P-rich and thus more sensitive to P deficiencies in their food. The next section is meant to present some of the most recent advances towards this integrative goal.

Following Sterner and Elser's 2002 book, researchers in the field of biological stoichiometry pursued two main directions. On one hand, there was an urge to have a more accurate knowledge of the mechanisms by which organisms regulate their elemental composition, in order to more realistically link their stoichiometric properties to their impacts on the cycles of elements. On the other hand, other researchers carried on with the process of extending the use of the stoichiometric approach to other fields in biology, always with the objective of

Consumers that strive to keep their elemental composition constant, in the face of stoichiometrically imbalanced resources, need to alter their gross growth efficiencies for elements in a controlled way (Figure 7). These gross growth efficiencies, in turn, determine

How consumers regulate their gross growth efficiencies was, and still is, largely unknown. In the absence of empirical data, in early stoichiometric models, the gross growth efficiency of the element limiting the consumer growth was set to a maximum value, ensuring maximal growth, while the gross growth efficiencies of the other elements were adjusted to yield a constant stoichiometry for the consumer (Figure 7). But theoretically, there is an infinite number of possible alternative strategies (Andersen 1997). Recent experiments also show that net assimilation of the limiting element is not necessarily at its maximal efficiency (DeMott *et al.* 1998) and that non-limiting elements too have impacts on growth (Boersma & Elser 2006; Zehnder & Hunter 2009). Moreover, it was shown that the quantity of resources, besides their stoichiometric compositions, affects the gross growth efficiencies of elements (Anderson & Hessen 2005). Furthermore, models that included a description of the distribution of elements among the various biomolecules in cells have shown that it is not even always possible to set one element as the growth- limiting factor (Anderson *et al.* 2004). It thus became clear that more physiologically grounded models of the use of elements by organisms were needed. Anderson et al's now classical model (2005) strikes a good balance between physiological realism and simplicity, including processes such as biomass

**5. Recent advances in biological stoichiometry** 

the differential recycling of elements by consumers (Sterner 1990).

integrating seemingly separate fields.

turnover, assimilation costs, maintenance costs, and respiration. These endeavours to understand the regulation of organismal stoichiometry by including the metabolism of biomolecules lead to connections between biological stoichiometry and nutritional sciences (Raubenheimer *et al.* 2009).


Fig. 7. Relation between the gross growth efficiencies of two unspecified elements (X and Y) as imposed by the constraint of a homeostatic regulation of the stoichiometry of a consumer. A) Mass balance equations for consumption. B) Definition of the gross growth efficiency. C) Stoichiometric constraint imposed on the GGEs for X and Y, as a consequence of the constancy of the X:Y ratio of consumers. D) A simple model that assumes a maximal GGE for the limiting element leads to the GGE of the non-limiting element to be a function of the maximal GGE, and the X:Y ratios of the consumer and its resource (adapted from Sterner 1990).

Physiological adaptation is the short-term response of consumers to imbalanced resources. Analyses at the levels of genomes and transcriptomes, showed that transcriptional changes are among the mechanisms used by consumers to achieve stoichiometric homeostasis. For example, transcriptional changes in the expression of phosphate transporters, activation of alternative metabolic pathways, changes in allocations to roots (for plants), are triggered under P deficiency (Jeyasingh & Weider 2007). Recent studies uncovered long-term evolutionary responses to element deprivations: N-deprived organisms react by substituting N-rich amino-acids by amino-acids with N-poor residues in their expressed proteins or by expressing shorter mRNAs (Grzymski & Dussaq 2011). They also show RNAs with more Npoor nucleotides (Elser *et al.* 2011). As for DNA, since GC and AT pairs have almost the same N content, N limitation seems to have less effect. These advances on the links between elemental limitation and cell polymer composition lead to the coining of a new term: "stoichiogenomics" (Elser *et al.* 2011).

Biological Stoichiometry: The Elements at the Heart of Biological Interactions 371

complexities need to be fully incorporated into the theory of Biological Stoichiometry before it really can link molecules to ecosystems. This is obviously a long journey but the road is

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Away from these ventures into the heart of the cellular machinery, other researchers looked for new insights from applying stoichiometric approaches to yet untouched fields. For example, models and experiments had shown that primary producers with a very low nutrient content could drive grazers to extinction and prevent their invasion of a habitat where they are absent (Andersen *et al.* 2004). This barrier to herbivore establishment led to speculations that the lack of grazers before the early Cambrian explosion and their "sudden" appearance afterwards could be linked to a change in the P supply at the scale of the globe (Elser *et al.* 2006). The same authors tested the hypothesis using modern stromatolites, which are very similar to the fossilized Cambrian stromatolites.

Another unexpected fallout from the application of the stoichiometric approach is the uncovering of stoichiometry-related effects on the relation between sexes and on the process of sexual selection. Sexes often differ in their investment in sex-related characters and organs. This difference is likely to be reflected in their elemental needs and use of their resources (Morehouse *et al.* 2010). A well-studied example is the positive correlation found between the phosphorus body content in male crickets and their song performance. Another area in which the stoichiometric approach proved fruitful hits closer to our everyday concerns: our health. An ingenious application of the growth rate hypothesis to cancer, gave rise to the hypothesis that fast-growing cancerous tumours should be richer in P. Thus, they should enter into competition with their neighbouring healthy cells for P and possibly see their growth be P-limited (Elser *et al.* 2007). This hypothesis brought forth by ecologists, that sees tumours as an ecosystem in itself, attracted favourable attention from the medical world (Baudouin-Cornu 2008; Pienta *et al.* 2008) and has potential medical applications ((Elser *et al.* 2003b). Because pathogens share the same requirements for elements as their hosts, they too can be affected by shortages in some key elements, such as iron (Smith 2007). Competition between host cells and pathogens for elements as well as external supplies of elements become an important parameter of the outcome of infections (Hall *et al.* 2009). Hence, the stoichiometry of diseases opens the door for more rational medical treatments through nutrition (Cotter *et al.* 2011).

### **6. Future needs and challenges ahead**

Biological stoichiometry proved to be astonishingly successful in bringing new insights in seemingly disconnected fields of biology, such as molecular evolution, palaeontology and parasitology. But these advances have not yet allowed us to draw a complete picture of the elements role in structuring biological entities from molecules to ecosystems. Huge gaps remain between levels: for example, the stoichiometric theory currently attributes a unique elemental composition to all individuals within one population. But it is known that stoichiometry varies with ontogeny (E.g., Main *et al.* 1997). Hence, the stoichiometry of a population probably differs from the stoichiometry of its individual components and might vary through time, a fact rarely considered (Nakazawa 2011).

Life is complex: different organisms might show different strategies to regulate their elemental composition (Mitra & Flynn 2005, 2007); food webs are diverse, both horizontally (i.e., within the same trophic level) and vertically (i.e., across trophic levels). Predictions that apply to one trophic level may prove erroneous if they neglect other species from the same (Danger *et al.* 2008) or from different trophic levels (Cherif & Loreau 2009). These additional complexities need to be fully incorporated into the theory of Biological Stoichiometry before it really can link molecules to ecosystems. This is obviously a long journey but the road is already drawn on the map!

### **7. References**

370 Stoichiometry and Research – The Importance of Quantity in Biomedicine

Away from these ventures into the heart of the cellular machinery, other researchers looked for new insights from applying stoichiometric approaches to yet untouched fields. For example, models and experiments had shown that primary producers with a very low nutrient content could drive grazers to extinction and prevent their invasion of a habitat where they are absent (Andersen *et al.* 2004). This barrier to herbivore establishment led to speculations that the lack of grazers before the early Cambrian explosion and their "sudden" appearance afterwards could be linked to a change in the P supply at the scale of the globe (Elser *et al.* 2006). The same authors tested the hypothesis using modern

Another unexpected fallout from the application of the stoichiometric approach is the uncovering of stoichiometry-related effects on the relation between sexes and on the process of sexual selection. Sexes often differ in their investment in sex-related characters and organs. This difference is likely to be reflected in their elemental needs and use of their resources (Morehouse *et al.* 2010). A well-studied example is the positive correlation found between the phosphorus body content in male crickets and their song performance. Another area in which the stoichiometric approach proved fruitful hits closer to our everyday concerns: our health. An ingenious application of the growth rate hypothesis to cancer, gave rise to the hypothesis that fast-growing cancerous tumours should be richer in P. Thus, they should enter into competition with their neighbouring healthy cells for P and possibly see their growth be P-limited (Elser *et al.* 2007). This hypothesis brought forth by ecologists, that sees tumours as an ecosystem in itself, attracted favourable attention from the medical world (Baudouin-Cornu 2008; Pienta *et al.* 2008) and has potential medical applications ((Elser *et al.* 2003b). Because pathogens share the same requirements for elements as their hosts, they too can be affected by shortages in some key elements, such as iron (Smith 2007). Competition between host cells and pathogens for elements as well as external supplies of elements become an important parameter of the outcome of infections (Hall *et al.* 2009). Hence, the stoichiometry of diseases opens the door for more rational medical treatments

Biological stoichiometry proved to be astonishingly successful in bringing new insights in seemingly disconnected fields of biology, such as molecular evolution, palaeontology and parasitology. But these advances have not yet allowed us to draw a complete picture of the elements role in structuring biological entities from molecules to ecosystems. Huge gaps remain between levels: for example, the stoichiometric theory currently attributes a unique elemental composition to all individuals within one population. But it is known that stoichiometry varies with ontogeny (E.g., Main *et al.* 1997). Hence, the stoichiometry of a population probably differs from the stoichiometry of its individual components and might

Life is complex: different organisms might show different strategies to regulate their elemental composition (Mitra & Flynn 2005, 2007); food webs are diverse, both horizontally (i.e., within the same trophic level) and vertically (i.e., across trophic levels). Predictions that apply to one trophic level may prove erroneous if they neglect other species from the same (Danger *et al.* 2008) or from different trophic levels (Cherif & Loreau 2009). These additional

stromatolites, which are very similar to the fossilized Cambrian stromatolites.

through nutrition (Cotter *et al.* 2011).

**6. Future needs and challenges ahead** 

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## *Edited by Alessio Innocenti*

The aim of this book is to provide an overview of the importance of stoichiometry in the biomedical field. It proposes a collection of selected research articles and reviews which provide up-to-date information related to stoichiometry at various levels. The first section deals with host-guest chemistry, focusing on selected calixarenes, cyclodextrins and crown ethers derivatives. In the second and third sections the book presents some issues concerning stoichiometry of metal complexes and lipids and polymers architecture. The fourth section aims to clarify the role of stoichiometry in the determination of protein interactions, while in the fifth section some selected experimental techniques applied to specific systems are introduced. The last section of the book is an attempt at showing some interesting connections between biomedicine and the environment, introducing the concept of biological stoichiometry. On this basis, the present volume would definitely be an ideal source of scientific information to researchers and scientists involved in biomedicine, biochemistry and other areas involving stoichiometry evaluation.

Stoichiometry and Research - The Importance of Quantity in Biomedicine

Stoichiometry and Research

The Importance of Quantity in Biomedicine

*Edited by Alessio Innocenti*

Photo by borzaya / iStock