**1. Introduction**

Amyloids are large aggregates of misfolded proteins with a highly stable cross β-structure, which are associated with a variety of degenerative illnesses such as Alzheimer's, Parkinson's, and Huntington's diseases [1–3]. Proteins with different functionalities and native structures ranging from α-helical and β-sheet rich to intrinsically unordered are able to form amyloid fibrils *in vitro* with a characteristic cross-β core structure [4–6]. This observation leads to the conclusion that protein fibrillation is a generic property of a polypeptide chain. There are numerous research reports demonstrating that a general fibrillation mechanism involves a partially unfolded protein as the first intermediate state [7, 8]. Steps to follow include the formation of small aggregates and a β-sheet rich nucleus, which generates further protein aggregation and the formation of mature fibrils.

A reduced amount of hydrogen sulfide (H2S) in the brain tissue of patients with Alzheimer's disease has been recently reported [9]. For centuries, people have been interested in H2S for its role as a poisonous chemical. At high concentrations, H2S inhibits cytochrome c and, as a consequence, the electron transport chain [10]. It also binds to hemoglobin forming a sulfhemoglobin complex as detected during sulfhemoglobinemia [11]. More recently, it has been demonstrated that H2S has gasotransmitter functions, similar to CO and NO [12]. For example, a suspended animation-like state in mice has been achieved by administering ppm-levels of H2S at low temperatures. The metabolic rate and body core temperature decrease and fully recover after such exposure, a promising medical benefit that reduces physiological damage after trauma [13]. In the last two decades, significant attention has been paid to understand the physiological role of H2S and its endogenous production. H2S is biosynthesized in mammalian tissue by non-enzymatic reactions and by the enzymatic degradation of cysteine by cystathionine β synthase (CBS), cystathionine γ lyase (CSE), cysteine aminotransferase (CAT), and cysteine lyase (CL) [14]. Consumption of garlic induces non-enzymatic H2S production [15]. Moreover, aged garlic extract has been shown to cause a reduction of *in vivo* Aβ fibrils and soluble amyloid, as well as a decrease in tau conformational changes [16]. This indirect evidence concerning the role of H2S in neurodegenerative diseases has motivated us to investigate the effects of H2S on the formation of amyloid fibrils.

Small molecules can have a significant effect on the formation of amyloid fibrils. There is extensive literature on the inhibitory activity of various small molecules on protein fibrillation [17]. Recently, Arosio and coauthors have reviewed the development of amyloid inhibitors, such as antibodies and chaperones, small molecules (e.g., Congo red and polyphenols), colloidal inhibitors and organic/inorganic nanoparticles, as possible participants in the various states of protein aggregation [17–19]. These states include the inhibition of primary nucleation (monomer-to-oligomer transition), secondary nucleation (oligomer elongation), and postelongation. However, we have not found any published reports on the role of H2S in protein aggregation.

It is well documented that H2S reacts with disulfide bonds, leading one to hypothesize that this reaction could have a significant effect on the mechanism of protein fibrillation. Kumar and co-workers have reported that protecting disulfide bridges with iodoacetamide in an alkaline solution limits the lysozyme fibril growth to 50% [20]. This group has concluded that changing the dynamics of disulfide to *aberrant* disulfide bonds would redirect the process toward the formation of native-like lysozyme aggregates [20]. It has been reported that treating antibodies with H2S has resulted in SS bond modifications, including the formation of trisulfide bonds (SSS) [21]. Surprisingly, no changes in antibody stability and function have been observed. H2S can be incorporated as a sulfane sulfur, a divalent sulfur with six valence electrons, and an oxidation number of zero (S0 ) that only binds to other sulfur atoms to form polysulfides [22]. Several research groups have also reported that the sulfur atom of

**159**

**2. Materials and methodology**

*Inhibition of Protein Fibrillation by Hydrogen Sulfide DOI: http://dx.doi.org/10.5772/intechopen.86221*

H2S can be endogenously incorporated into a large amount of proteins by sulfuration, also known as sulfhydration of cysteines. This leads to the formation of protein persulfides (SSH), which could play an intermediary role in protein SSS formation [23]. Here, we have investigated the effect of H2S on the aggregation of lysozyme, a glycoside hydroxylase responsible for antimicrobial protection in most mammalian species. HEWL is a single chain protein stabilized by four SS bonds in positions cys6-cys127, cys30-cys115, cys64-cys80, and cys76-cys94 [24]. It was found that H2S inhibits the formation of HEWL fibrils. The effect of H2S has been investigated under typical fibrillation conditions such as high temperature and acidic pH using DUVRR and non-resonance Raman spectroscopy, fluorescence, and atomic force microscopy (AFM). We have shown that in the presence of H2S, HEWL forms spherical aggregates of unordered protein under fibrillation conditions. Cytotoxicity tests reveal that these spherical aggregates have no cell toxicity by contrast with typical HEWL fibrils. Our spectroscopic results, buttressed by data that has been published, indicate that H2S reacts with protein disulfide bonds to form trisulfide bridges. This reaction results in significant lysozyme denaturation and the formation of spherical

aggregates of unordered proteins, which prevent protein fibrillation.

However, because myoglobin (Mb) and hemoglobin (Hb) do not have any cysteine chemical bond, we pursued the effect of H2S on the fibril formation of these vital hemeproteins. Mb and Hb are the most studied hemeproteins, because of their biological significance of oxygen binding. The role of Mb and Hb in the body is so important that the minimal unbalance of normal physiology can lead to toxicity and to cascade of reactions generating harmful products. For example, free radical formation in these hemeproteins unavoidably leads to oxidative damage of the heme and amino acids [25]. Also in certain circumstances, Mb and Hb isolated from their cellular environment may crosslink leading to kidney dysfunction, rhabdomyolysis, coma, and subarachnoid brain hemorrhage [26]. Other maladies include heme loss (hemophilia, hemolytic anemia), hemoglobinopathies (thalassemia [α and β], methemoglobinemia, posttranslational alterations), cardiovascular, and renal diseases [27–31]. Likewise, hemoglobin can protagonist sickle cell anemia where a mutation at the β6 position of Hb (β6Glu → Val) results in the polymerization of deoxysickle cell Hb (HbS) and subsequent aggregation into long fibers with amyloid-like structures [32, 33]. Regarding this, it has been shown that Hb under physiological conditions and in the presence of 45% 2,2,2-trifluoethanol (TFE) produces amyloid-like fibril structures. This observation was supported by ThT fluorescence, CD, and FTIR suggesting that Hb β-sheet conformation leads to Hb fibril formation [28]. The mechanism surrounding these fibril events remain almost unknown. Interestingly, our results also show that H2S inhibits the fibril formation in both myoglobin and hemoglobin under physiological conditions and 45% TFE concentration and that increasing concentration of H2S inhibits β-sheet formation and predominates the α-helix structure. The findings demonstrate the same H2S effect on to the fibrillation of Mb monomer and Hb tetramer. Overall, it is very interesting that hydrogen sulfide is able to avoid the formation of fibril derivative in lysozyme and myoglobin and hemoglobin being their structures completely different.

The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO,

USA): 99.7% acetic acid (695092), sodium chloride (NaCl) (S771-3), HEWL (L6876), hemoglobin bovine (H2500), myoglobin from horse skeletal muscle (M0630), 2,2,2-trifluoroethanol 99% (T63002), potassium chloride (P3911), potassium phosphate monobasic (P5655) and potassium phosphate dibasic (1551128),

#### *Inhibition of Protein Fibrillation by Hydrogen Sulfide DOI: http://dx.doi.org/10.5772/intechopen.86221*

*Amyloid Diseases*

**1. Introduction**

Amyloids are large aggregates of misfolded proteins with a highly stable cross β-structure, which are associated with a variety of degenerative illnesses such as Alzheimer's, Parkinson's, and Huntington's diseases [1–3]. Proteins with different functionalities and native structures ranging from α-helical and β-sheet rich to intrinsically unordered are able to form amyloid fibrils *in vitro* with a characteristic cross-β core structure [4–6]. This observation leads to the conclusion that protein fibrillation is a generic property of a polypeptide chain. There are numerous research reports demonstrating that a general fibrillation mechanism involves a partially unfolded protein as the first intermediate state [7, 8]. Steps to follow include the formation of small aggregates and a β-sheet rich nucleus, which gener-

A reduced amount of hydrogen sulfide (H2S) in the brain tissue of patients with Alzheimer's disease has been recently reported [9]. For centuries, people have been interested in H2S for its role as a poisonous chemical. At high concentrations, H2S inhibits cytochrome c and, as a consequence, the electron transport chain [10]. It also binds to hemoglobin forming a sulfhemoglobin complex as detected during sulfhemoglobinemia [11]. More recently, it has been demonstrated that H2S has gasotransmitter functions, similar to CO and NO [12]. For example, a suspended animation-like state in mice has been achieved by administering ppm-levels of H2S at low temperatures. The metabolic rate and body core temperature decrease and fully recover after such exposure, a promising medical benefit that reduces physiological damage after trauma [13]. In the last two decades, significant attention has been paid to understand the physiological role of H2S and its endogenous production. H2S is biosynthesized in mammalian tissue by non-enzymatic reactions and by the enzymatic degradation of cysteine by cystathionine β synthase (CBS), cystathionine γ lyase (CSE), cysteine aminotransferase (CAT), and cysteine lyase (CL) [14]. Consumption of garlic induces non-enzymatic H2S production [15]. Moreover, aged garlic extract has been shown to cause a reduction of *in vivo* Aβ fibrils and soluble amyloid, as well as a decrease in tau conformational changes [16]. This indirect evidence concerning the role of H2S in neurodegenerative diseases has motivated us to investigate the effects of H2S on the formation of amyloid fibrils. Small molecules can have a significant effect on the formation of amyloid fibrils. There is extensive literature on the inhibitory activity of various small molecules on protein fibrillation [17]. Recently, Arosio and coauthors have reviewed the development of amyloid inhibitors, such as antibodies and chaperones, small molecules (e.g., Congo red and polyphenols), colloidal inhibitors and organic/inorganic nanoparticles, as possible participants in the various states of protein aggregation [17–19]. These states include the inhibition of primary nucleation (monomer-to-oligomer transition), secondary nucleation (oligomer elongation), and postelongation. However, we have not found any published reports on the role of H2S in protein aggregation.

It is well documented that H2S reacts with disulfide bonds, leading one to hypothesize that this reaction could have a significant effect on the mechanism of protein fibrillation. Kumar and co-workers have reported that protecting disulfide bridges with iodoacetamide in an alkaline solution limits the lysozyme fibril growth to 50% [20]. This group has concluded that changing the dynamics of disulfide to *aberrant* disulfide bonds would redirect the process toward the formation of native-like lysozyme aggregates [20]. It has been reported that treating antibodies with H2S has resulted in SS bond modifications, including the formation of trisulfide bonds (SSS) [21]. Surprisingly, no changes in antibody stability and function have been observed. H2S can be incorporated as a sulfane sulfur, a divalent sulfur with six valence electrons,

polysulfides [22]. Several research groups have also reported that the sulfur atom of

) that only binds to other sulfur atoms to form

ates further protein aggregation and the formation of mature fibrils.

**158**

and an oxidation number of zero (S0

H2S can be endogenously incorporated into a large amount of proteins by sulfuration, also known as sulfhydration of cysteines. This leads to the formation of protein persulfides (SSH), which could play an intermediary role in protein SSS formation [23].

Here, we have investigated the effect of H2S on the aggregation of lysozyme, a glycoside hydroxylase responsible for antimicrobial protection in most mammalian species. HEWL is a single chain protein stabilized by four SS bonds in positions cys6-cys127, cys30-cys115, cys64-cys80, and cys76-cys94 [24]. It was found that H2S inhibits the formation of HEWL fibrils. The effect of H2S has been investigated under typical fibrillation conditions such as high temperature and acidic pH using DUVRR and non-resonance Raman spectroscopy, fluorescence, and atomic force microscopy (AFM). We have shown that in the presence of H2S, HEWL forms spherical aggregates of unordered protein under fibrillation conditions. Cytotoxicity tests reveal that these spherical aggregates have no cell toxicity by contrast with typical HEWL fibrils. Our spectroscopic results, buttressed by data that has been published, indicate that H2S reacts with protein disulfide bonds to form trisulfide bridges. This reaction results in significant lysozyme denaturation and the formation of spherical aggregates of unordered proteins, which prevent protein fibrillation.

However, because myoglobin (Mb) and hemoglobin (Hb) do not have any cysteine chemical bond, we pursued the effect of H2S on the fibril formation of these vital hemeproteins. Mb and Hb are the most studied hemeproteins, because of their biological significance of oxygen binding. The role of Mb and Hb in the body is so important that the minimal unbalance of normal physiology can lead to toxicity and to cascade of reactions generating harmful products. For example, free radical formation in these hemeproteins unavoidably leads to oxidative damage of the heme and amino acids [25]. Also in certain circumstances, Mb and Hb isolated from their cellular environment may crosslink leading to kidney dysfunction, rhabdomyolysis, coma, and subarachnoid brain hemorrhage [26]. Other maladies include heme loss (hemophilia, hemolytic anemia), hemoglobinopathies (thalassemia [α and β], methemoglobinemia, posttranslational alterations), cardiovascular, and renal diseases [27–31]. Likewise, hemoglobin can protagonist sickle cell anemia where a mutation at the β6 position of Hb (β6Glu → Val) results in the polymerization of deoxysickle cell Hb (HbS) and subsequent aggregation into long fibers with amyloid-like structures [32, 33]. Regarding this, it has been shown that Hb under physiological conditions and in the presence of 45% 2,2,2-trifluoethanol (TFE) produces amyloid-like fibril structures. This observation was supported by ThT fluorescence, CD, and FTIR suggesting that Hb β-sheet conformation leads to Hb fibril formation [28]. The mechanism surrounding these fibril events remain almost unknown. Interestingly, our results also show that H2S inhibits the fibril formation in both myoglobin and hemoglobin under physiological conditions and 45% TFE concentration and that increasing concentration of H2S inhibits β-sheet formation and predominates the α-helix structure. The findings demonstrate the same H2S effect on to the fibrillation of Mb monomer and Hb tetramer. Overall, it is very interesting that hydrogen sulfide is able to avoid the formation of fibril derivative in lysozyme and myoglobin and hemoglobin being their structures completely different.

### **2. Materials and methodology**

The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA): 99.7% acetic acid (695092), sodium chloride (NaCl) (S771-3), HEWL (L6876), hemoglobin bovine (H2500), myoglobin from horse skeletal muscle (M0630), 2,2,2-trifluoroethanol 99% (T63002), potassium chloride (P3911), potassium phosphate monobasic (P5655) and potassium phosphate dibasic (1551128),

sodium sulfide nonahydrate salt (208043), dipropyl disulfide (149225), and trisulfide (6028-61-1).
