**4. Collagen**

Collagens are a large protein family forming a characteristic triple helix of three polypep‐ tide chains giving rise to supramolecular structures in the extracellular matrix: their size, function and tissue distribution vary considerably [25]. So far, 26 genetically distinct collagen types have been described. It is the main fibrous component of skin, bone, tendon and carti‐ lage, accounting in particular for three‐quarters of the dry weight of skin, and representing the most prevalent component of the extracellular matrix. The collagen molecule is a rigid rod‐like structure able to resist stretching. It is composed of three polypeptide left‐handed α chains coiled around each other to form a typical right‐handed rope‐like triple helical rod (approximately, 1.5 nm in diameter, 300 nm in length). The triple helical sequences are com‐ posed of Gly‐X‐Y repeats, X and Y being frequently proline and hydroxy‐proline, respec‐ tively [26]. Stabilization of the triple helix is assured by a lot of structural conditions as the presence of glycine as every third residue, a high content of imino acids with a rigid cyclical structure (proline and hydroxy‐proline) and electrostatic interactions involving lysine and aspartate [27]. Both in the structural and functional properties, a key role is undoubtedly played by water, which provides inter‐chain hydrogen bonds and water bridges and coats triple helix by a cylinder‐like of hydration [28, 29]. Moreover, water critically regulates chain flexibility and assures the water mediated H‐bonds favoring fiber recognition and alignment. Water lacking is a very dangerous process for the protein, damaging the structure in irrevers‐ ible way. Water depletion, caused by tissue maturation, exposition to UV light and/or some pathological processes, such as diabetes, triggers detrimental conformational rearrangements due by nonenzymatic glycation reactions between the protein and the complex carbohydrates present in the matrix, leading to the formation of oxidation products, known as Advanced Glycation End‐products (AGE) [30, 31] having the effect to modify the collagen fibers physi‐ cal properties, inducing an increase in stiffness and breaking load, denaturation temperature, solubility and a decrease in resistance to degradative enzymes [32].

accordance with the approach proposed in the literature to study various hydrated biomate‐ rials [24]. To quantify the integrated absorbance in terms of water surface coverage, we can

where *A*˜ is the integrated absorbance of the component band, *ε* (*νMAX*) is the molar absorp‐ tivity at wave number ν*MAX* corresponding to the peak of the band (L mol−1 cm−1), *c*¯ is the

For water, it is possible to evaluate the molar absorptivity *ε*(ν) at fixed wave number [24]: for example, one can evaluate ε (3600 cm−1) = 1.647 L mol−1 cm−1. By assuming the path length *d* as the thickness of a film of water, the corresponding amount of the water content can be con‐ verted in concentration *c*¯ of water, assumed as only absorbing species. The concentration *c*¯ in each sample prepared at each hydration degree can be evaluated and employed to normalize the amplitude of the subcomponents in the ν(OH) band. The desorption‐adsorption branches of the isotherm curves were therefore obtained for each Gaussian component. As a rule, the desorption experiments were performed by starting from the highest *a*w value (*a*w = 0.97) and accomplished before the adsorption one, to avoid possible damages in the samples produced

Collagens are a large protein family forming a characteristic triple helix of three polypep‐ tide chains giving rise to supramolecular structures in the extracellular matrix: their size, function and tissue distribution vary considerably [25]. So far, 26 genetically distinct collagen types have been described. It is the main fibrous component of skin, bone, tendon and carti‐ lage, accounting in particular for three‐quarters of the dry weight of skin, and representing the most prevalent component of the extracellular matrix. The collagen molecule is a rigid rod‐like structure able to resist stretching. It is composed of three polypeptide left‐handed α chains coiled around each other to form a typical right‐handed rope‐like triple helical rod (approximately, 1.5 nm in diameter, 300 nm in length). The triple helical sequences are com‐ posed of Gly‐X‐Y repeats, X and Y being frequently proline and hydroxy‐proline, respec‐ tively [26]. Stabilization of the triple helix is assured by a lot of structural conditions as the presence of glycine as every third residue, a high content of imino acids with a rigid cyclical structure (proline and hydroxy‐proline) and electrostatic interactions involving lysine and aspartate [27]. Both in the structural and functional properties, a key role is undoubtedly played by water, which provides inter‐chain hydrogen bonds and water bridges and coats triple helix by a cylinder‐like of hydration [28, 29]. Moreover, water critically regulates chain flexibility and assures the water mediated H‐bonds favoring fiber recognition and alignment. Water lacking is a very dangerous process for the protein, damaging the structure in irrevers‐ ible way. Water depletion, caused by tissue maturation, exposition to UV light and/or some pathological processes, such as diabetes, triggers detrimental conformational rearrangements

*A dν* = *ε* (*νMAX* ) *c*¯*d* (3)

or, equivalent mol/L) and *d* is the average

*band*

thickness of the absorbing species film, that is, adsorbed water, in centimeters [9].

assume a modified form of Beer‐Lambert' law

198 Fourier Transforms - High-tech Application and Current Trends

concentration of the absorbing species (mmol/cm<sup>3</sup>

*A*˜ = ∫

by dehydration treatments.

**4. Collagen**

Despite the great amount of studies [33], the explicit relationship between protein and hydra‐ tion water structure is still an open question and the detailed dehydration scheme of collagen and the structural implications are not yet completely elucidated.

### **4.1. Hydration structure of collagen: FTIR spectroscopy and water sorption isotherms**

Complementary FTIR measurements on collagen prepared at very low hydration level (*a*<sup>w</sup> in the range 0.06–0.97) and adsorption isotherm technique have put in evidence the critical hydration level which induces an irreversible conformational change in the protein, respon‐ sible for the first ageing step.

In **Figure 2**, the spectra of collagen extracted from rat tail tendon (type I collagen) are shown during the dehydration run.

In order to study the role of water interacting with collagen, FTIR spectrum was analyzed in the OH stretching region by changing the hydration content of the sample following the method described above (Section 3.2).

**Figure 2.** FTIR spectra of rat tail tendon collagen, recorded at different water activities during dehydration treatment from *a*w = 0.97 to *a*w = 0.06.

OH stretching band (wave number range 4000–3000 cm−1) was analyzed after subtraction of the "dry" spectrum and decomposed into four components (**Figure 3**) whose frequencies were related to different O‐H bond lengths as listed in **Table 2**.

The *ν*(OH) feature composition in Gaussian sub‐bands in such a way can be correlated to the hydrogen bond network around the protein and provides information about the structural modifications induced by changing the hydration level. The four Gaussian components are peaking, for the sample at the maximum hydration, at 3598, 3467, 3295 and 3115 cm−1, follow‐ ing the procedure of the second derivative analysis. They are corresponding to four classes of water molecules bound to the protein, different in vibrational energies and each one char‐ acterized by a single average H‐bond distance (H···OH length): 0.31, 0.29, 0.28 and 0.25 nm, respectively [18, 23].

The sub‐band peaking at the highest wave number region is corresponding to H‐bond dis‐ tances characteristic of vapor‐like state. Similar features, detected for different biological macromolecules [9, 10, 16], were assigned to the non‐H‐bonded or weakly H‐bonded O–H groups. It is reasonable to suppose that in collagen prepared at very low hydration levels, as in our case, they could be originated by the dangling most external water molecules sitting on the outer hydration layer coating the macromolecule. The inability for these molecules to establish active H‐bonds with surrounding water molecules, accounts for the high mobil‐ ity and the large vibrational energy comparable with those of free water molecules in the vapor state. They represent about the 5% of the total amount of water hydrating collagen at the relative humidity settled for the experiment, being the percentages calculated as the

**Figure 3.** Gaussian deconvolution of FTIR spectrum of collagen (*a*w = 0.38) in the ν(OH) region, by means of four component bands, as obtained by means of second derivative method. The sum of the fitted curves is shown as the red line, closely overlapping the experimental data trace, which is shown as a full black line.


**Table 2.** Hydrogen bond distances estimated from the vibrational frequencies following Nakamoto et al. [23].

OH stretching band (wave number range 4000–3000 cm−1) was analyzed after subtraction of the "dry" spectrum and decomposed into four components (**Figure 3**) whose frequencies

The *ν*(OH) feature composition in Gaussian sub‐bands in such a way can be correlated to the hydrogen bond network around the protein and provides information about the structural modifications induced by changing the hydration level. The four Gaussian components are peaking, for the sample at the maximum hydration, at 3598, 3467, 3295 and 3115 cm−1, follow‐ ing the procedure of the second derivative analysis. They are corresponding to four classes of water molecules bound to the protein, different in vibrational energies and each one char‐ acterized by a single average H‐bond distance (H···OH length): 0.31, 0.29, 0.28 and 0.25 nm,

The sub‐band peaking at the highest wave number region is corresponding to H‐bond dis‐ tances characteristic of vapor‐like state. Similar features, detected for different biological macromolecules [9, 10, 16], were assigned to the non‐H‐bonded or weakly H‐bonded O–H groups. It is reasonable to suppose that in collagen prepared at very low hydration levels, as in our case, they could be originated by the dangling most external water molecules sitting on the outer hydration layer coating the macromolecule. The inability for these molecules to establish active H‐bonds with surrounding water molecules, accounts for the high mobil‐ ity and the large vibrational energy comparable with those of free water molecules in the vapor state. They represent about the 5% of the total amount of water hydrating collagen at the relative humidity settled for the experiment, being the percentages calculated as the

**Figure 3.** Gaussian deconvolution of FTIR spectrum of collagen (*a*w = 0.38) in the ν(OH) region, by means of four component bands, as obtained by means of second derivative method. The sum of the fitted curves is shown as the red

line, closely overlapping the experimental data trace, which is shown as a full black line.

were related to different O‐H bond lengths as listed in **Table 2**.

200 Fourier Transforms - High-tech Application and Current Trends

respectively [18, 23].

ratio *A*<sup>i</sup> /*A*tot of the single component area (*A*<sup>i</sup> ) with respect to the total band area (*A*tot). The two component bands peaking at the intermediate wave numbers may be related to H2 O molecules coordinated by two or three, more or less distorted or strained, H bonds. They may be identified with H<sup>2</sup> O molecules deeply located inside the protein helix, acting as water bridges within a single peptide chain and/or interconnecting the different α‐chains in the triple helix. They constitute the 19% and the 31%, respectively, of the total water amount. The corresponding H‐bond distances suggest that they could represent the solvation mol‐ ecules involving C=O groups belonging to glycine (d(C=O···W) = 0.295 nm) and hydroxypro‐ line (Hyp) (d(C=O···W) = 0.284 nm) hydroxyl moieties [29]. The component at 3180 cm−1 may be attributed to water molecules located near to the protein surface and hydrogen bonded to polar and charged groups exposed to the macromolecule surface: they originate some ice‐like tetrahedral structures more or less distorted. The corresponding average H bond length is in fact characteristic for arrangement of H2 O molecules as in small solid water clusters, assum‐ ing the length O···O of hydrogen bonds in ice as 0.276 nm [34]. The broad profile of such a band, extending on a wide wave number range, testifies for the large distribution of water vibrational states in a configuration variety, concurring to the feature. Such a sub‐band repre‐ sents the main hydration fraction at the highest settled humidity, corresponding to the 45% of the total amount of hydration water.

The dehydration treatment at *a*w = 0.06 followed by a subsequent rehydration up to the origi‐ nal relative humidity (*a*w = 0.92) reveals a pronounced hysteresis effect in the ν(OH) band amplitude and a remarkable modification of the profile, as shown in **Figure 4**.

The difference spectrum is peaking at ν ∼3400 cm−1; therefore, it is roughly filling the position of the third component of the overall ν(OH) band. Moreover, it exhibits a shoulder on the low wave number side (ν ∼3000 cm−1). This finding suggests that dehydration treatment causes in collagen the loss of a considerable amount of H2 O molecules truly involved in the internal water bridges, probably coordinated by glycine C=O, and a fraction of molecules coating the polypep‐ tide surface, bound to the Hyp hydroxyls. This water portion once desorbed, only partially can be re‐adsorbed. It accounts for the 21% of the total water amount initially hydrating the sample.

With the aim to better investigate this topic, the peak areas of the two intermediate sub‐bands, peaking at ν = 3467 cm−1 and ν = 3295 cm−1, detected in the spectrum of the sample at the high‐ est hydration level, were plotted as a function of the activity *a*w during the desorption and the subsequent adsorption processes to build the corresponding isotherm curves. **Figure 5** summarizes the isotherm curves related to the two component bands highlighting the large differences occurring in sorption mechanisms of the two related water sets.

**Figure 4.** OH stretching bands measured for collagen at *a*w = 0.97, before (curve a) and after (curve b) dehydration at *a*w = 0.06. Curve c represents the difference between a and b bands and the subtended area (pink dashed area) can be related to the water amount desorbed by the sample during dehydration and no more recovered along rehydration.

**Figure 5.** Desorption and adsorption curves for two sub‐bands (ν = 3467 cm−1 and ν = 3295 cm−1) of OH stretching band measured for collagen, during dehydration (black symbols) and re‐hydration (red symbols) treatments. Blue triangles represent the desorption data obtained by means of the gravimetric experiment. The lines are a guide for the eye.

The band at 3295 cm−1 displays a type II‐like desorption and adsorption behavior [22]. The band amplitude completely recovers at the end of the process; therefore, such component only partially participates to the lacking of recover in amplitude of the total ν(OH) band, the weak hysteresis effect being probably due to the reassessment of the interchain water bridges as a consequence of the perturbation induced by water subtraction. Concerning the band at 3467 cm−1, the mechanism of dehydration is very different than that of rehydration. The desorption branch correlates well with the curve gravimetrically measured by weighing the sample after equilibration at each different humidity level, as shown in **Figure 5** (blue full triangles): the two desorption curves show the same trend. They are as type II curve, where three phases can be distinguished: a starting rapid dehydration step from *a*w = 0.92 to about *a*w = 0.80, a second one in the *a*w range 0.80–0.40, where water desorption occurs more slowly with the decrease in ambient relative humidity and a third phase at low water activities, extending from *a*w = 0.40 to *a*w = 0.06, describing a characteristic "knee" [2]. The first phase may be assigned to the removal of water molecules forming hydrogen‐bonded clusters, the second one to subtraction of H2 O molecules from weakly interacting surface regions and the third branch to the dehydration of strongly bound water molecules by Hyp hydroxyl moi‐ eties exposed at the surface. The result suggests that the whole protein desorption process is mainly concerning the dehydration of the external water layer.

Water uptake exhibits a very different behavior with respect to the dehydration, resulting in a large hysteresis loop, extending over the whole activity range. Adsorption isotherm displays features not conform to any isotherm type in the Brunauer classification [22]. From *a*w = 0.06 to *a*w = 0.40, the amount of water adsorbed is rather negligible. The activity *a*w = 0.40 represents a threshold activity above which a sudden water uptake occurs attaining a saturation hydration value, lower than the moisture content of the freshly prepared sample.

**Figure 4.** OH stretching bands measured for collagen at *a*w = 0.97, before (curve a) and after (curve b) dehydration at *a*w = 0.06. Curve c represents the difference between a and b bands and the subtended area (pink dashed area) can be related to the water amount desorbed by the sample during dehydration and no more recovered along rehydration.

202 Fourier Transforms - High-tech Application and Current Trends

**Figure 5.** Desorption and adsorption curves for two sub‐bands (ν = 3467 cm−1 and ν = 3295 cm−1) of OH stretching band measured for collagen, during dehydration (black symbols) and re‐hydration (red symbols) treatments. Blue triangles represent the desorption data obtained by means of the gravimetric experiment. The lines are a guide for the eye.

Water deprivation and restitution has considerable consequences on the macromolecule struc‐ ture. The changes in the high wave number region of the IR spectrum can be correlated with the secondary structure analysis carried out on Amide I band which was resolved in Gaussian components. Qualitative and quantitative information about the conformational composition of the protein prepared in the different hydration states were obtained. Amide I band in fact was decomposed in sub‐bands whose position and area were related, respectively, to the dif‐ ferent types and to the amount of secondary structures (**Figure 6**). The sum of the areas of the peaks represents the total amount of secondary structure in the protein.

The curve fitting procedure [8, 21] allowed determining the secondary structure composi‐ tion in the freshly prepared sample (*a*w = 0.97), as displayed in **Figure 6A**: β‐turn (1695 cm−1), β‐sheet (1680 cm−1), α‐like helix (1660 cm−1), unordered structure (1644 cm−1), triple helix (1628 cm−1), β‐sheet (1615 cm−1), side chains (1607 cm−1). The multipeak decomposition of the band measured in the sample rehydrated at the original activity, after dehydration at *a*w = 0.06 (**Figure 6B**), revealed the modification occurred in the secondary structure, the most signifi‐ cant change concerning the increase in the broad sub‐band peaking at ν ∼1670 cm−1 largely spanning the high wave number region of the Amide I band and the parallel decrease in the component bands at 1695 and 1660 cm−1. The formation of such structures may be related to the relative increase in β components (antiparallel β‐sheet and aggregated strands) with

**Figure 6.** Amide I band deconvolution for collagen at *a*w = 0.97 before (A) and after (B) dehydration at *a*w = 0.06. The set of Gaussian components can be related to the secondary structure composition of the protein. The full blue lines highlight the component bands related to the antiparallel β‐sheet/aggregated strand motif. The dotted red lines represent the sum of the components.

respect to the α‐helix contribution in the Amide I envelope. Such effect may be explained by assuming the formation of bridges among near collagen molecules laterally associating by forming chain structures side by side interacting, involving Hyp residues [35], spectroscopi‐ cally mimicking β‐sheet structures [36].

These results would be consistent with the physiologic behavior of collagen and may be corre‐ lated to the changes in the structural properties of collagen fiber assembly as a consequence of dehydration. The spectroscopic data are consistent with specific binding of water molecules to collagen chains stabilizing the collagen triple helix by Hyp residues through intramolecu‐ lar water bridges. The difference in interfacial surface water may be the physical reason for the hysteresis phenomena observed in water adsorption isotherms. As a consequence of the removal of water down to water activity *a*w <0.40, the association of the collagen molecules takes place and once water is restituted, the assembled fibers are no more able to re‐adsorb all the water available during the rehydration phase. This arrangement, responsible for the lack‐ ing in the hydration recover of protein, may be considered one of the main causes of collagen maturation and ageing.

### **5. Lysozyme**

Lysozyme is a small globular protein shaped as an approximate ellipsoid of dimension 45 × 30 × 30 Å composed of 129 residues, acting as an enzyme causing lysis of Gram posi‐ tive bacteria by hydrolyzing the β‐(1–4) glycosidic links in the cell wall peptidoglycan [37]. Lysozymes have been isolated from a great variety of sources. Lysozyme from hen egg white (HEWL, MW 14600) is an *α* + *β* protein with 30% helical residues and 13% *β*‐sheet content. As for the majority of protein molecules, lysozyme structure and stability are largely determined by the interaction between the protein and the aqueous surroundings [38–40]. Investigation of the structural changes occurring during the change in the activity of water was important for understanding the mechanism of this interaction. In particular, Careri et al. [41] suggested a connection between the water‐induced onset of enzymatic activity and some water triggered physical properties of the protein system, i.e., heat capacity, diamagnetic susceptibility, IR absorption, percolation threshold for proton conduction, recognizing that the hydration threshold for lysozyme, occurred at 0.22 g of water/g of protein.

### **5.1. Hydration structure of lysozyme: FTIR spectroscopy and water sorption isotherms**

Lysozyme from hen egg white was used without further chemical purification and submitted to FTIR investigation following the experimental method above described (Section 3.2). The solvent architecture around the protein was correlated to the macromolecule conformation by means of two different procedures:

By monitoring secondary structure from the inspection of the changes occurring both in Amide I and Amide III features by changing the protein hydration level. Amide I band analy‐ sis in fact suffers from the extensive overlap of the underlying water H–O–H bending band, lying in close proximity (~1645 cm−1), the line broadening making quantitation arduous. The secondary structure assignments can be reinforced by taking into account Amide III, attrib‐ uted to N–H deformation coupled with the C–N stretching motion and ranging in the wave number region 1220–1340 cm−1. It occurs out of any water absorption region and its behav‐ ior, monitored along secondary structure changes of the protein, may be correlated with the changes suffered by the enzyme structure to provide a consistent picture of the hydrated protein conformational properties [8].

respect to the α‐helix contribution in the Amide I envelope. Such effect may be explained by assuming the formation of bridges among near collagen molecules laterally associating by forming chain structures side by side interacting, involving Hyp residues [35], spectroscopi‐

**Figure 6.** Amide I band deconvolution for collagen at *a*w = 0.97 before (A) and after (B) dehydration at *a*w = 0.06. The set of Gaussian components can be related to the secondary structure composition of the protein. The full blue lines highlight the component bands related to the antiparallel β‐sheet/aggregated strand motif. The dotted red lines represent the sum

These results would be consistent with the physiologic behavior of collagen and may be corre‐ lated to the changes in the structural properties of collagen fiber assembly as a consequence of dehydration. The spectroscopic data are consistent with specific binding of water molecules to collagen chains stabilizing the collagen triple helix by Hyp residues through intramolecu‐ lar water bridges. The difference in interfacial surface water may be the physical reason for the hysteresis phenomena observed in water adsorption isotherms. As a consequence of the removal of water down to water activity *a*w <0.40, the association of the collagen molecules takes place and once water is restituted, the assembled fibers are no more able to re‐adsorb all the water available during the rehydration phase. This arrangement, responsible for the lack‐ ing in the hydration recover of protein, may be considered one of the main causes of collagen

Lysozyme is a small globular protein shaped as an approximate ellipsoid of dimension 45 × 30 × 30 Å composed of 129 residues, acting as an enzyme causing lysis of Gram posi‐ tive bacteria by hydrolyzing the β‐(1–4) glycosidic links in the cell wall peptidoglycan [37]. Lysozymes have been isolated from a great variety of sources. Lysozyme from hen egg white (HEWL, MW 14600) is an *α* + *β* protein with 30% helical residues and 13% *β*‐sheet content. As for the majority of protein molecules, lysozyme structure and stability are largely determined by the interaction between the protein and the aqueous surroundings [38–40]. Investigation of the structural changes occurring during the change in the activity of water was important for understanding the mechanism of this interaction. In particular, Careri et al. [41] suggested a connection between the water‐induced onset of enzymatic activity and some water triggered

cally mimicking β‐sheet structures [36].

204 Fourier Transforms - High-tech Application and Current Trends

maturation and ageing.

**5. Lysozyme**

of the components.

By studying the evolution of the broad *ν*(OH) feature along the protein hydration and dehy‐ dration processes. The OH stretching bands, recorded at each activity value, were decomposed into three Gaussian components, corresponding to the three main water fractions hydrating lysozyme, different in structure and clustering order. By appropriate analysis of the data, sorp‐ tion isotherm curves were built, able to provide information on relationship between the water content and the different protein structural features. The water amount bound to Lysozyme was deduced from the *ν*(OH) band area after subtraction of the spectrum of the "dry" sample.

**Figure 7A** and **B** shows *ν*(OH) bands recorded for the protein prepared at different hydration degree along the dehydration and the rehydration runs.

**Figure 7.** OH stretching bands measured for lysozyme along dehydration (A) and rehydration (B) treatments between *a*w = 0.97 and *a*w = 0.06.

Following the decomposition of the band in Gaussian components, three sub‐bands were detected (**Figure 8**, left side): their positions and the corresponding OH···O distances, mea‐ sured for the sample at the highest hydration degree, are listed in **Table 2**. First component band (I) (**Figure 8A**) peaking at the higher wave number (3539 cm−1) may be attributed to surface water molecules easily adsorbed on the most external hydration layer. They show the dangling OH vibrational frequency typical for free‐like H2 O molecules at the interfaces water‐ air. The sub‐band at ν = 3315 cm−1 (II) (**Figure 8B**) may be related to water molecules directly interacting with protein backbone, particularly engaged in hydrogen bonding with peptide carbonyl moieties (C=O···W). The broadband at ν = 3008 cm−1 (III) (**Figure 8C**), representing the most prevalent component, corresponds to water molecules forming the hydration shells around the protein, interacting with neighboring adsorbed water molecules. The position of the feature is indicative of the intermolecular hydrogen bond distances matching the average

**Figure 8.** Gaussian deconvolution of FTIR spectrum of lysozyme (*a*w = 0.84) in the ν(OH) region, by means of three component bands (dashed areas). For each component band, the isotherm curves were plotted: black symbols represent the desorption data and red symbols represent the adsorption data. The lines are a guide for the eye.

characteristic lengths typical for tetrahedral ice structure but the large bandwidth reveals that it is suffering lengthening and distortions in the distances and orientation.

Following the decomposition of the band in Gaussian components, three sub‐bands were detected (**Figure 8**, left side): their positions and the corresponding OH···O distances, mea‐ sured for the sample at the highest hydration degree, are listed in **Table 2**. First component band (I) (**Figure 8A**) peaking at the higher wave number (3539 cm−1) may be attributed to surface water molecules easily adsorbed on the most external hydration layer. They show the

air. The sub‐band at ν = 3315 cm−1 (II) (**Figure 8B**) may be related to water molecules directly interacting with protein backbone, particularly engaged in hydrogen bonding with peptide carbonyl moieties (C=O···W). The broadband at ν = 3008 cm−1 (III) (**Figure 8C**), representing the most prevalent component, corresponds to water molecules forming the hydration shells around the protein, interacting with neighboring adsorbed water molecules. The position of the feature is indicative of the intermolecular hydrogen bond distances matching the average

**Figure 8.** Gaussian deconvolution of FTIR spectrum of lysozyme (*a*w = 0.84) in the ν(OH) region, by means of three component bands (dashed areas). For each component band, the isotherm curves were plotted: black symbols represent

the desorption data and red symbols represent the adsorption data. The lines are a guide for the eye.

O molecules at the interfaces water‐

dangling OH vibrational frequency typical for free‐like H2

206 Fourier Transforms - High-tech Application and Current Trends

**Figure 8**, right side, displays the isotherm curves obtained by monitoring the areas of the three component bands as a function of the moisture of the sample, both during the sequen‐ tial decrease in the water content of the sample and during the opposite treatment, rehydra‐ tion at RT. Sub‐band I gives rise to a desorption isotherm classified as Type IV, characteristic for the evaporation process of water at the interface water‐air. In the converse process of condensation, the underlying liquid surface acts as to nucleate the adsorption, therefore, the isotherm is quite different, showing the shape of a Type III curve, characteristic for coopera‐ tive interactions. The loop desorption‐adsorption gives rise to a hysteresis because evapora‐ tion and condensation do not take place as exact reverse of each other. The hysteresis effect indicates that between *a*w*=* 0.40 and *a*w*=* 0.80, the amount of water desorbed is greater than that adsorbed, suggesting a change or an assessment of the condensation surface, as a conse‐ quence of dehydration, until the highest moisture degree is achieved. Sub‐band III originates a hysteresis loop as well, although the isotherm curves are not easy to be classified accord‐ ing to the literature [22]. The intermediate component (sub‐band II) reveals an anomalous behavior reflecting an irreversible structural modification of the protein. Water desorption describes a Type IV‐like isotherm but the adsorption branch is a flat line up to *a*w = 0.80, showing a small increase for the highest hydration values attained, but unable to completely recover the original amplitude. It notifies the inability of the macromolecule to re‐adsorb in a reversible manner the water phase originally coordinated by the polypeptide backbone, once desorbed.

The process can be explained by assuming that water subtraction modifies the structural properties of the macromolecule which would tend to narrow down the crevices where water molecules were inserted, by establishing intrachain bonds difficult to reverse. As discussed in the literature, in the absence of water, the protein molecule tends to fill the voids left by water by adopting structures that can continuously fill the space [38]. The hysteresis effect is confirmed by the difference in amplitude between the ν(OH) band of the native sample and the rehydrated one after dehydration, as shown in **Figure 9**. The difference band centered at ν ≈ 3300 cm−1 corresponds to the water fraction desorbed during the dehydration run and no more re‐adsorbed during the subsequent hydration process.

In **Figures 10** and **11**, the spectral windows related to the two polypeptide secondary struc‐ tures, i.e., 1700–1550 cm−1 and 1300–1230 cm−1 corresponding to the Amide I and III ranges are shown for the two samples prepared at the highest hydration degree. The Gaussian decomposition of the native lysozyme Amide I band (**Figure 10**) was carried out in four major components, in agreement with previous studies performed on lysozyme and other kinds of proteins [42, 43]. The band fairly asymmetric and peaking at ~1656 cm−1, is mainly due to the contribution of α‐helical structure (75%), in agreement with the literature [44]. The β‐sheet content is lower and can be measured from the two components around 1618 and 1684 cm−1. The component centered at 1635 cm−1 can be attributed to unordered structures.

**Figure 11** displays Amide III bands for the sample at *a*w = 0.97 and after dehydration down to *a*w = 0.06. The curve fitting analysis for lysozyme in the native hydrated form reveals three

**Figure 9.** OH stretching bands measured for lysozyme at *a*w = 0.97, before (black curve) and after (blue curve) dehydration at *a*w = 0.06. The red curve represents the difference of the two curves and the subtended area (red dashed area) can be related to the water amount desorbed by the sample during dehydration and no more recovered along rehydration.

main sub‐bands peaked at 1338, 1320 and 1300 cm−1, which can be categorized as being α‐helical with a total relative band strength of 40%. The relatively sharp band at 1230 cm−1 is assigned to β sheet structure for 21% and two broadbands at 1260 and 1285 cm−1 indicate the presence of unordered structures for 35%. Dehydration induces modifications of the bands with respect to the spectrum of the native hydrated sample: they can be related to changes in the secondary structure composition.

**Figure 10.** Amide I band deconvolution for lysozyme at *a*w = 0.97. The component bands can be related to the structural contribution of α‐helix (green line), β‐sheet (blue line) and unordered (orange line) structures.

Fourier Transform Infrared Spectroscopy in the Study of Hydrated Biological Macromolecules http://dx.doi.org/10.5772/66576 209

**Figure 11.** Amide III band measured for lysozyme at *a*w = 0.97 (black line) and *a*w = 0.06 (pink line). The deconvolution in Gaussian components is related to the spectrum of the sample at the higher hydration degree. The main components band are as follows: α‐helix at 1338, 1320 and 1300 cm−1, β sheet at 1230 cm−1 and unordered structures at 1260 cm−1 and 1285 cm−1.

In **Figure 12**, the percentage amount of each component band corresponding to each secondary structure element was plotted as a function of the increasing water activity *a*w settled in the dry box.

It appears that dehydration treatment induces small but significant conformational changes involving the increase in the β structure fraction. Interestingly, the plot points out as the α‐helix

main sub‐bands peaked at 1338, 1320 and 1300 cm−1, which can be categorized as being α‐helical with a total relative band strength of 40%. The relatively sharp band at 1230 cm−1 is assigned to β sheet structure for 21% and two broadbands at 1260 and 1285 cm−1 indicate the presence of unordered structures for 35%. Dehydration induces modifications of the bands with respect to the spectrum of the native hydrated sample: they can be related to changes in the secondary

**Figure 10.** Amide I band deconvolution for lysozyme at *a*w = 0.97. The component bands can be related to the structural

contribution of α‐helix (green line), β‐sheet (blue line) and unordered (orange line) structures.

**Figure 9.** OH stretching bands measured for lysozyme at *a*w = 0.97, before (black curve) and after (blue curve) dehydration at *a*w = 0.06. The red curve represents the difference of the two curves and the subtended area (red dashed area) can be related to the water amount desorbed by the sample during dehydration and no more recovered along rehydration.

structure composition.

208 Fourier Transforms - High-tech Application and Current Trends

**Figure 12.** Evolution of the amount of secondary structures in lysozyme, monitored by the deconvolution of Amide III band, along the dehydration treatment, plotted as the relative area peak A<sup>i</sup> /Atot as a function of the activity *a*w. The color code is related to component bands in **Figure 11**.

portion of the protein is heavily affected by water subtraction. The two bands peaking at 1320 and 1300 cm−1, related to this conformational motif, reveal a mutual amplitude interconver‐ sion following a stepped trend, similar but specular, as a function of water content changes. The observed effect could be related to small conformational changes, as a result of hydration changes, involving different fractions of α‐helices which could be affected by mutual conforma‐ tional fluctuations. The observed conformational modification may be due to the change in the orientation of two α domains monitored by the Amide III component bands. Water depriva‐ tion could affect intermolecular distances making different helix structure more or less con‐ sistent with hydrogen bond. Although this result could not be considered as definitive proof, however, it is important in the understanding the dynamical equilibrium of protein structure, crucial for many biological processes involving the enzyme.

The different response to hydration change of the different polypeptide regions at different secondary structure may be interpreted on the light of the crystallographic studies revealing relatively rigid and flexible regions in the macromolecule [45]. The interdomain dynamics modulated by water interaction could be related to the mechanisms involving the active‐site cleft dynamics of the protein needed for the enzymatic activity, whose onset appears over a threshold hydration level and causing the irreversibility of functionality if a critical dehydra‐ tion threshold is exceeded [41].
