**2.6 Tryptophan and ThT fluorescence**

Fluorescence spectra were measured in a JobinYvon Fluoromax-3 spectrofluorometer (JobinYvon, Edison, NJ). Intrinsic tryptophan fluorescence of

**161**

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

ment under a pH = 7.4 and T = 37°.

contributions [28, 40, 41].

curvature radius.

**3. Results**

**2.8 Atomic force microscope**

**2.7 Circular dichroism spectroscopy**

25 mg/mL HEWL was measured in a 10-μm path length cell without dilutions. The UV absorption was <0.05 at an excitation wavelength of 295.5 nm. The excitation and emission slits were 0.5 and 5 nm, respectively. Three spectral accumulations were taken, and the spectra were averaged for each sample. HEWL fibrils formed after 90 minutes of incubation were also characterized using intrinsic tryptophan fluorescence. Fibrils were washed in acetic acid solution twice in a procedure which included sonication for 10 minutes, centrifugation for 4 minutes at 13,000 rpm, the removal of supernatant liquid and re-suspension in an acetic acid solution. Fluorescence dye thioflavin T (ThT) is one of the most used probes for identification and analysis in the formation of amyloid fibrils both *in vivo* and *in vitro*. Once ThT binds to β-sheet-rich amyloid fibril structures, there is a characteristic blue shift in the emission spectrum from 510 to 480 nm [37–39]. In the ThT fluorescence assay, aliquots of 25 mg/mL HEWL were diluted in a molar ratio of 1:10 (HEWL:thioflavin T (ThT) dye) to a final concentration of 2.5 mM ThT. The excitation and emission wavelengths were 450 and 480 nm, respectively. The excitation and emission slits were 5 nm. Three recorded spectra were averaged for each measurement. ThT fluorescence intensity of Mb and Hb was monitored during fibrillation experiment: λex = 440 nm and λem = 480 nm. The final protein sample and ThT concentration were 10 and 10 μM, respectively [28]. Fluorescence was measured using Biotek Synergy 4 (multi-mode microplate reader) with a 96 well-plate in a continuous acquisition (kinetics mode), every 5 minutes for 20 hours

with gently orbital shake. We examined the time-dependence of Mb and Hb amyloid-like structures by monitoring thioflavin T fluorescence (480 nm) enhance-

Far-UV circular dichroism (CD) measurements of Hb and Mb samples were performed on a Jasco-815 spectropolarimeter. Each spectrum was the average of six scans. Three replicates for each time point of the kinetic experiment were analyzed.

Aliquots of HEWL incubated at 60°C, pH 2.0, 100 mM NaCl were cooled to room temperature and deposited on freshly cleaved mica. After a few minutes of exposure, the mica surface was rinsed with MQ water and dried. AFM images were collected using the SmartSPM 1000 system (AIST-NT, Novato, CA). Images were acquired in the tapping mode using silicon cantilevers with a 10–25 nm tip

To form fibrils, HEWL was incubated at 60°C in 20% acetic acid (pH 2.0) and 100 mM NaCl, from here on referred to as control conditions. The morphology of lysozyme aggregates formed in the course of incubation under the fibrillogenic

**3.1 Aggregation and structural rearrangements of lysozyme**

Spectra were acquired at 0.5-nm intervals with a 4-s integration time and a bandwidth of 1.0 nm. Mb and Hb were analyzed at a final concentration of 0.5 mg/mL. All measurements were performed under nitrogen flow. The results were expressed as the mean residue ellipticity. Data were corrected for buffer

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

*Amyloid Diseases*

fide (6028-61-1).

**2.1 HEWL solution preparation**

to evaluate H2S effect [28, 34].

the range of 400–1800 cm<sup>−</sup><sup>1</sup>

**2.4 TCEP test for trisulfides**

was used for data processing.

**2.6 Tryptophan and ThT fluorescence**

**2.3 Non-resonance Raman experiments**

**2.2 Myoglobin and hemoglobin fibril preparation**

sodium sulfide nonahydrate salt (208043), dipropyl disulfide (149225), and trisul-

Lysozyme was dissolved (25 mg/mL) in 20% acetic acid and 100 mM NaCl at pH 2.0 and incubated at 60°C to form fibrils under initial (control) conditions. To study the effect of H2S, sodium sulfide nonahydrate salt (12 mM) was added to the control solution in a molar ratio of 1:5 (HEWL:H2S), prior to the temperature elevation.

Myoglobin and hemoglobin were dissolved (15 and 20 mg/mL, respectively) in 20 mM phosphate buffer at pH 7.4; samples of Mb and Hb solutions were mixed with 45% of 2,2,2-trifluoroethanol and incubated at 37°C for 24 hours to form the fibrils (control conditions). Another batch of samples was prepared with sodium sulfide nonahydrate salt (60–300 μM, 12 mM) under the same control conditions,

Powder samples of native and aggregated HEWL were prepared by drying the solutions under nitrogen at room temperature, which removed the acetic acid. Raman spectra (785-nm excitation) of HEWL powder samples and pure dipropyl di- and trisulfide liquids were recorded using a Renishaw inVia confocal Raman spectrometer equipped with a research grade Leica microscope and 50× objective (numerical aperture, 0.55). Five accumulations of 30 s each were collected for each sample in

power of approximately 11.5 mW was used to avoid sample photo-degradation.

drying the corresponding solutions under a nitrogen flow.

**2.5 Deep UV resonance Raman spectroscopy (DUVRR)**

A reaction with tris(2-carboxyethyl)phosphine (TCEP) reducing agent was used as a test for trisulfides [35]. Hen egg white lysozyme (HEWL) in native and aggregated form was incubated at pH 2.0 and room temperature for 90 minutes in the presence of TCEP. The reaction products were analyzed using normal Raman spectroscopy. Powder samples of HEWL-aggregates incubated at different concentrations of TCEP were prepared for non-resonance Raman spectroscopic analysis by

DUVRR spectra (199.7 nm excitation) of 25 mg/mL HEWL were collected using

a home built instrument equipped with a CCD camera (Roper Scientific, Inc.) cooled in liquid nitrogen [36]. A spinning quartz NMR tube with a magnetic stirrer was used for sampling. Each spectrum recorded an average of 20 accumulations with 30 s acquisition time. GRAMS/AI 7.0 software (Thermo Galactic, Salem, NH)

Fluorescence spectra were measured in a JobinYvon Fluoromax-3 spectrofluorometer (JobinYvon, Edison, NJ). Intrinsic tryptophan fluorescence of

. Wire 4.0 software was used for data collection. A laser

**160**

25 mg/mL HEWL was measured in a 10-μm path length cell without dilutions. The UV absorption was <0.05 at an excitation wavelength of 295.5 nm. The excitation and emission slits were 0.5 and 5 nm, respectively. Three spectral accumulations were taken, and the spectra were averaged for each sample. HEWL fibrils formed after 90 minutes of incubation were also characterized using intrinsic tryptophan fluorescence. Fibrils were washed in acetic acid solution twice in a procedure which included sonication for 10 minutes, centrifugation for 4 minutes at 13,000 rpm, the removal of supernatant liquid and re-suspension in an acetic acid solution. Fluorescence dye thioflavin T (ThT) is one of the most used probes for identification and analysis in the formation of amyloid fibrils both *in vivo* and *in vitro*. Once ThT binds to β-sheet-rich amyloid fibril structures, there is a characteristic blue shift in the emission spectrum from 510 to 480 nm [37–39]. In the ThT fluorescence assay, aliquots of 25 mg/mL HEWL were diluted in a molar ratio of 1:10 (HEWL:thioflavin T (ThT) dye) to a final concentration of 2.5 mM ThT. The excitation and emission wavelengths were 450 and 480 nm, respectively. The excitation and emission slits were 5 nm. Three recorded spectra were averaged for each measurement. ThT fluorescence intensity of Mb and Hb was monitored during fibrillation experiment: λex = 440 nm and λem = 480 nm. The final protein sample and ThT concentration were 10 and 10 μM, respectively [28]. Fluorescence was measured using Biotek Synergy 4 (multi-mode microplate reader) with a 96 well-plate in a continuous acquisition (kinetics mode), every 5 minutes for 20 hours with gently orbital shake. We examined the time-dependence of Mb and Hb amyloid-like structures by monitoring thioflavin T fluorescence (480 nm) enhancement under a pH = 7.4 and T = 37°.

#### **2.7 Circular dichroism spectroscopy**

Far-UV circular dichroism (CD) measurements of Hb and Mb samples were performed on a Jasco-815 spectropolarimeter. Each spectrum was the average of six scans. Three replicates for each time point of the kinetic experiment were analyzed. Spectra were acquired at 0.5-nm intervals with a 4-s integration time and a bandwidth of 1.0 nm. Mb and Hb were analyzed at a final concentration of 0.5 mg/mL. All measurements were performed under nitrogen flow. The results were expressed as the mean residue ellipticity. Data were corrected for buffer contributions [28, 40, 41].

#### **2.8 Atomic force microscope**

Aliquots of HEWL incubated at 60°C, pH 2.0, 100 mM NaCl were cooled to room temperature and deposited on freshly cleaved mica. After a few minutes of exposure, the mica surface was rinsed with MQ water and dried. AFM images were collected using the SmartSPM 1000 system (AIST-NT, Novato, CA). Images were acquired in the tapping mode using silicon cantilevers with a 10–25 nm tip curvature radius.

### **3. Results**

#### **3.1 Aggregation and structural rearrangements of lysozyme**

To form fibrils, HEWL was incubated at 60°C in 20% acetic acid (pH 2.0) and 100 mM NaCl, from here on referred to as control conditions. The morphology of lysozyme aggregates formed in the course of incubation under the fibrillogenic

conditions and in the presence of H2S was characterized by AFM. The presence of typical long rode-like fibrils was evident after 90 minutes of incubation under control conditions (**Figure 1A**). However, incubation of HEWL in the presence of H2S resulted in the formation of spherical aggregates instead of fibrils, as evident from AFM images (**Figure 1B**).

ThT fluorescence is used often to monitor the formation of amyloid fibrils. ThT fluorescence intensity increased dramatically after 70 minutes of incubation of lysozyme under control conditions, indicating the formation of amyloid fibrils (**Figure 1C**). However, no increase in ThT fluorescence intensity was observed for the HEWL solution incubated with H2S within 48 hours. We investigated changes in the lysozyme secondary structure during incubation with and without H2S using deep UV resonance Raman (DUVRR) spectroscopy. DUVRR has been used to study structural rearrangements of HEWL at all stages of fibrillation [36, 45, 46]. The DUVRR spectrum of HEWL excited at 199.7 nm was mainly composed of the amide chromosphere and the aromatic amino acid (Phe and Tyr) contributions [47]. A noticeable increase in the intensity and sharpness of the Am I band (approximately 1672 cm<sup>−</sup><sup>1</sup> ) indicated the appearance of β-sheets due to the formation of fibrils [36, 48–50]. The DUVRR spectrum of fibrillated lysozyme under control conditions confirmed the formation of β-sheets. The spectrum of HEWL after 30 minutes of incubation under control conditions (**Figure 1D**, red) is similar to that reported previously for HEWL fibrils [36]. However, the DUVRR spectrum of lysozyme

#### **Figure 1.**

*Lysozyme forms β-sheet-rich fibrils under fibrillogenic control conditions and spherical aggregates of unordered protein under fibrillogenic conditions with H2S incubation. AFM images of (A) HEWL fibrils formed after incubation of the control solution for 90 minutes and (B) HEWL aggregates formed after incubation of the solution in the presence of H2S for 48 hours; scale bars are 1 μm. (C) Aggregation kinetics (ThT fluorescence) of HEWL incubated under control conditions (red) and in the presence of H2S (black). (D) DUVRR spectra of native HEWL (blue), HEWL fibrils (red), and HEWL spherical aggregates (black) formed in the presence of H2S; all spectra were normalized using the aromatic amino acid Raman band (approximately 1600 cm<sup>−</sup><sup>1</sup> ) for comparison. The amide I vibrational mode (Am I) is dominated by C*〓*O stretching, with minor contributions from C▬N stretching and N▬H bending [42]. Amide II (Am II) and amide III (Am III) bands involve significant C▬N stretching, N▬H bending, and C▬C stretching [43]. The Cα▬H bending vibrational mode involves Cα▬H symmetric bending and C▬Cα stretching [44].*

**163**

490 cm<sup>−</sup><sup>1</sup>

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

case, the Am I band (approximately 1670 cm<sup>−</sup><sup>1</sup>

mately 1250 cm<sup>−</sup><sup>1</sup>

**rearrangement**

control fibrillation conditions.

the range of 505–550 cm<sup>−</sup><sup>1</sup>

A strong 507 cm<sup>−</sup><sup>1</sup>

523 cm<sup>−</sup><sup>1</sup>

**3.3 Rearrangement of disulfide bonds**

decreased, and a new peak appeared at 490 cm<sup>−</sup><sup>1</sup>

investigating the effect of H2S concentrations. The 1003 cm<sup>−</sup><sup>1</sup>

incubated in the presence of H2S confirmed the lack of β-sheet formation. In this

change (**Figure 1D**, black). Instead, the Am I band shifted slightly to a higher frequency, signifying the formation of an unordered protein [36, 42]. This was further

of α-helix melting [51]. A significant change in Raman bands for Am III (approxi-

transition of α-helix to unordered protein. Therefore, AFM, ThT fluorescence, and DUVRR spectroscopy indicated the formation of unordered spherical aggregates of

Tryptophan (Trp) fluorescence is an efficient intrinsic marker of local environments, which is often used for monitoring tertiary structural changes in proteins [47]. Native lysozyme at neutral pH shows a maximum Trp emission at 340 nm [52]. At pH 2.0 (20% acetic acid), the Trp fluorescence peak shifts to 345 nm, indicating a partial denaturation of lysozyme. A further minor shift to 347 nm due to HEWL fibril formation under control conditions was observed (**Figure 2B** and **C**). To confirm that the intrinsic Trp fluorescence is dominated by the signal from HEWL fibrils, the solutions (after incubation for 40 and 90 minutes) were sonicated, centrifuged, and re-suspended in 20% acetic acid to remove possible monomeric and oligomeric forms of the protein (**Figure 2B**). A significant shift of the Trp emission maximum, from 345 to 357 nm, was observed after 90 minutes of lysozyme incubation in the presence of H2S (**Figure 2A** and **C**), with no further changes for at least 48 hours. This significant red shift is consistent with the previously reported maximum emission at 352 nm for fully denatured lysozyme in 6 M guanidinium-HCl at pH 7.0 [53]. Therefore, we conclude that incubation of lysozyme in the presence of H2S results in a stronger denaturation than that which occurs during

Non-resonance Raman spectroscopy of proteins offers a unique opportunity for characterizing the conformation of disulfide bridges [49]. The SS symmetric stretching vibrational mode is typically represented as a strong Raman band in

change significantly in the SS signature region with incubation time (**Figure 3A**).

tion of the fourth SS bond of lysozyme [49, 55]. The amplitudes of these peaks

in the presence of 12 mM H2S, indicating significant rearrangements of SS bonds (**Figure 3A**). The concentration of 12 mM H2S corresponded to a 5:1 (H2S:HEWL) molar ratio, chosen so that a sufficient number of H2S molecules could react with all four lysozyme SS bonds assuming a 1:1 stoichiometric ratio. We are currently

to phenylalanine was used to normalize Raman spectra in **Figure 3A** (region not shown). **Figure 3C** shows synchronous kinetic change in the area of the 507 and

were observed during 48 hours of additional incubation in the presence of H2S

bands with incubation time up to 90 minutes [56]. No further changes

peak can be attributed to the gauche-gauche-trans (g-g-t) configura-

gauche-gauche-gauche (g-g-g) configuration of three SS bonds, and a small

[49, 54]. The Raman spectrum of HEWL was found to

as a result of HEWL incubation

peak corresponding

peak in the Raman spectrum of native HEWL represents the

) and Am II (approximately 1555 cm<sup>−</sup><sup>1</sup>

**3.2 Intrinsic tryptophan fluorescence marker of the tertiary structural** 

supported by the increase in Cα▬H band intensity at 1390 cm<sup>−</sup><sup>1</sup>

HEWL by contrast with β-sheet-rich fibrils in the presence of H2S.

) did not show a significant intensity

) was consistent with the

that was indicative

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

*Amyloid Diseases*

1672 cm<sup>−</sup><sup>1</sup>

from AFM images (**Figure 1B**).

conditions and in the presence of H2S was characterized by AFM. The presence of typical long rode-like fibrils was evident after 90 minutes of incubation under control conditions (**Figure 1A**). However, incubation of HEWL in the presence of H2S resulted in the formation of spherical aggregates instead of fibrils, as evident

ThT fluorescence is used often to monitor the formation of amyloid fibrils. ThT fluorescence intensity increased dramatically after 70 minutes of incubation of lysozyme under control conditions, indicating the formation of amyloid fibrils (**Figure 1C**). However, no increase in ThT fluorescence intensity was observed for the HEWL solution incubated with H2S within 48 hours. We investigated changes in the lysozyme secondary structure during incubation with and without H2S using deep UV resonance Raman (DUVRR) spectroscopy. DUVRR has been used to study structural rearrangements of HEWL at all stages of fibrillation [36, 45, 46]. The DUVRR spectrum of HEWL excited at 199.7 nm was mainly composed of the amide chromosphere and the aromatic amino acid (Phe and Tyr) contributions [47]. A noticeable increase in the intensity and sharpness of the Am I band (approximately

) indicated the appearance of β-sheets due to the formation of fibrils [36, 48–50]. The DUVRR spectrum of fibrillated lysozyme under control conditions confirmed the formation of β-sheets. The spectrum of HEWL after 30 minutes of incubation under control conditions (**Figure 1D**, red) is similar to that reported previously for HEWL fibrils [36]. However, the DUVRR spectrum of lysozyme

*Lysozyme forms β-sheet-rich fibrils under fibrillogenic control conditions and spherical aggregates of unordered protein under fibrillogenic conditions with H2S incubation. AFM images of (A) HEWL fibrils formed after incubation of the control solution for 90 minutes and (B) HEWL aggregates formed after incubation of the solution in the presence of H2S for 48 hours; scale bars are 1 μm. (C) Aggregation kinetics (ThT fluorescence) of HEWL incubated under control conditions (red) and in the presence of H2S (black). (D) DUVRR spectra of native HEWL (blue), HEWL fibrils (red), and HEWL spherical aggregates (black) formed in the presence of H2S; all spectra were normalized using the aromatic amino acid Raman band (approximately 1600 cm<sup>−</sup><sup>1</sup>*

*comparison. The amide I vibrational mode (Am I) is dominated by C*〓*O stretching, with minor contributions from C▬N stretching and N▬H bending [42]. Amide II (Am II) and amide III (Am III) bands involve significant C▬N stretching, N▬H bending, and C▬C stretching [43]. The Cα▬H bending vibrational mode* 

*involves Cα▬H symmetric bending and C▬Cα stretching [44].*

*) for* 

**162**

**Figure 1.**

incubated in the presence of H2S confirmed the lack of β-sheet formation. In this case, the Am I band (approximately 1670 cm<sup>−</sup><sup>1</sup> ) did not show a significant intensity change (**Figure 1D**, black). Instead, the Am I band shifted slightly to a higher frequency, signifying the formation of an unordered protein [36, 42]. This was further supported by the increase in Cα▬H band intensity at 1390 cm<sup>−</sup><sup>1</sup> that was indicative of α-helix melting [51]. A significant change in Raman bands for Am III (approximately 1250 cm<sup>−</sup><sup>1</sup> ) and Am II (approximately 1555 cm<sup>−</sup><sup>1</sup> ) was consistent with the transition of α-helix to unordered protein. Therefore, AFM, ThT fluorescence, and DUVRR spectroscopy indicated the formation of unordered spherical aggregates of HEWL by contrast with β-sheet-rich fibrils in the presence of H2S.

## **3.2 Intrinsic tryptophan fluorescence marker of the tertiary structural rearrangement**

Tryptophan (Trp) fluorescence is an efficient intrinsic marker of local environments, which is often used for monitoring tertiary structural changes in proteins [47]. Native lysozyme at neutral pH shows a maximum Trp emission at 340 nm [52]. At pH 2.0 (20% acetic acid), the Trp fluorescence peak shifts to 345 nm, indicating a partial denaturation of lysozyme. A further minor shift to 347 nm due to HEWL fibril formation under control conditions was observed (**Figure 2B** and **C**). To confirm that the intrinsic Trp fluorescence is dominated by the signal from HEWL fibrils, the solutions (after incubation for 40 and 90 minutes) were sonicated, centrifuged, and re-suspended in 20% acetic acid to remove possible monomeric and oligomeric forms of the protein (**Figure 2B**). A significant shift of the Trp emission maximum, from 345 to 357 nm, was observed after 90 minutes of lysozyme incubation in the presence of H2S (**Figure 2A** and **C**), with no further changes for at least 48 hours. This significant red shift is consistent with the previously reported maximum emission at 352 nm for fully denatured lysozyme in 6 M guanidinium-HCl at pH 7.0 [53]. Therefore, we conclude that incubation of lysozyme in the presence of H2S results in a stronger denaturation than that which occurs during control fibrillation conditions.

### **3.3 Rearrangement of disulfide bonds**

Non-resonance Raman spectroscopy of proteins offers a unique opportunity for characterizing the conformation of disulfide bridges [49]. The SS symmetric stretching vibrational mode is typically represented as a strong Raman band in the range of 505–550 cm<sup>−</sup><sup>1</sup> [49, 54]. The Raman spectrum of HEWL was found to change significantly in the SS signature region with incubation time (**Figure 3A**). A strong 507 cm<sup>−</sup><sup>1</sup> peak in the Raman spectrum of native HEWL represents the gauche-gauche-gauche (g-g-g) configuration of three SS bonds, and a small 523 cm<sup>−</sup><sup>1</sup> peak can be attributed to the gauche-gauche-trans (g-g-t) configuration of the fourth SS bond of lysozyme [49, 55]. The amplitudes of these peaks decreased, and a new peak appeared at 490 cm<sup>−</sup><sup>1</sup> as a result of HEWL incubation in the presence of 12 mM H2S, indicating significant rearrangements of SS bonds (**Figure 3A**). The concentration of 12 mM H2S corresponded to a 5:1 (H2S:HEWL) molar ratio, chosen so that a sufficient number of H2S molecules could react with all four lysozyme SS bonds assuming a 1:1 stoichiometric ratio. We are currently investigating the effect of H2S concentrations. The 1003 cm<sup>−</sup><sup>1</sup> peak corresponding to phenylalanine was used to normalize Raman spectra in **Figure 3A** (region not shown). **Figure 3C** shows synchronous kinetic change in the area of the 507 and 490 cm<sup>−</sup><sup>1</sup> bands with incubation time up to 90 minutes [56]. No further changes were observed during 48 hours of additional incubation in the presence of H2S

#### **Figure 2.**

*Time dependent Trp fluorescence changes of lysozyme (A) incubated in the presence of H2S for 0 minutes (dashes ---), 10 minutes (dots …), 90 minutes (long dashes ---), 48 hours (solid line \_\_\_); (B) incubated with control solution for 0 minutes (solid line \_\_\_), 40 minutes (long dashes ---), and 90 minutes (dots …); (C) Trp maximum emission wavelength of HEWL incubated with H2S (A circles) and fibrillation under control conditions (B squares).*

(data not shown). As discussed in detail below, dipropyl-trisulfide (DPTS) Raman spectrum contains a 485 cm<sup>−</sup><sup>1</sup> band (**Figure 3D**) characteristic to the trisulfide moiety that motivated us to investigate the possibility of assigning 490 cm<sup>−</sup><sup>1</sup> band in HEWL aggregate Raman spectrum to the SSS group. The non-resonance Raman spectroscopy of HEWL fibrillation under control conditions indicate that the 507 cm<sup>−</sup><sup>1</sup> peak does not change significantly during fibril formation (**Figure 3B**). Therefore, in the absence of H2S, HEWL SS bands remain intact and the g-g-g conformation dominates, in agreement with our previous report [57].

#### **3.4 Reduction of trisulfide bridges by TCEP**

To test the hypothesis about the formation of trisulfide groups, we investigated the reaction of HEWL aggregates with TCEP reduction agent by normal Raman spectroscopy. TCEP reaction with SS groups is well known to result in oxidation of TCEP and formation of TCEP(O) and R-SH groups [58]. More recently, Cumnock et al. reported that TCEP reacted preferentially with SSS moieties in the presence of SS bridges until the majority of SSS groups were consumed according to Eqs. (1) and (2) [35]. SS bridges and thiophosphine TCEP(S) species are main products of TCEP-SSS reaction [35]. **Figure 4** shows Raman spectra of HEWL aggregates after incubation with different concentrations of TCEP (0.5, 1, 2.5, and 10 mM). The amount of aggregated HEWL molecules in these samples was kept about 3.0 mM.

**165**

**Figure 3.**

*523 cm<sup>−</sup><sup>1</sup>*

SSS band at 490 cm<sup>−</sup><sup>1</sup>

*area of the 507 and 490 cm<sup>−</sup><sup>1</sup>*

*formation (490 cm<sup>−</sup><sup>1</sup>*

*(red dots, …).*

ment with an increase in 2575 cm<sup>−</sup><sup>1</sup>

490 and 507 cm<sup>−</sup><sup>1</sup>

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

*TCEP* + *R* − *SSS* − *R TCEP*(*S*) + *R* − *SS* − *R* (1)

*Evolution of lysozyme disulfide bonds in the presence of H2S probed by normal Raman spectroscopy. Raman spectra of HEWL incubated (A) in the presence of H2S and (B) under control conditions, where 507 and* 

*the presence of H2S. (D) The difference spectrum between normal Raman spectra of HEWL aggregated in the presence of H2S acquired at 90- and 0-minute incubation [shown in (A), gray solid line]. The latter spectrum is represented by the expected spectral change demonstrating the disulfide-to-trisulfide transition symbolized by the inverted Raman spectrum of dipropyl disulfide (black dots and dash, -.-.-) and dipropyl trisulfide* 

*) and the decrease in the amount of RSSR (507 cm<sup>−</sup><sup>1</sup>*

 *bands correspond to g-g-g and g-g-t SS configurations, respectively. Synchronous kinetic change in the* 

 *bands is assigned to the newly formed RSSSR group. (C) The kinetics of RSSSR* 

*) during the incubation of HEWL in* 

*TCEP* + *R* − *SS* − *R TCEP*(*O*) + 2 *R* − *SH* (2)

The Raman spectrum of HEWL aggregates was found to change significantly in the SSS/SS vibrational signature region with the addition of TCEP (**Figure 4A**). The

ment with predominant reaction of TCEP with SSS groups. The amplitudes of both

incubation in the presence of higher concentration of TCEP (1–10 mM) indicating significant reduction of SS and SSS groups and formation of R-SH moiety in agree-

decreases after 0.5 mM TCEP addition that is in a good agree-

bands (SSS and SS, respectively) decreased as a result of HEWL

band intensity (**Figure 4B**).

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

#### **Figure 3.**

*Amyloid Diseases*

**164**

507 cm<sup>−</sup><sup>1</sup>

**Figure 2.**

*conditions (B squares).*

spectrum contains a 485 cm<sup>−</sup><sup>1</sup>

**3.4 Reduction of trisulfide bridges by TCEP**

(data not shown). As discussed in detail below, dipropyl-trisulfide (DPTS) Raman

in HEWL aggregate Raman spectrum to the SSS group. The non-resonance Raman spectroscopy of HEWL fibrillation under control conditions indicate that the

To test the hypothesis about the formation of trisulfide groups, we investigated the reaction of HEWL aggregates with TCEP reduction agent by normal Raman spectroscopy. TCEP reaction with SS groups is well known to result in oxidation of TCEP and formation of TCEP(O) and R-SH groups [58]. More recently, Cumnock et al. reported that TCEP reacted preferentially with SSS moieties in the presence of SS bridges until the majority of SSS groups were consumed according to Eqs. (1) and (2) [35]. SS bridges and thiophosphine TCEP(S) species are main products of TCEP-SSS reaction [35]. **Figure 4** shows Raman spectra of HEWL aggregates after incubation with different concentrations of TCEP (0.5, 1, 2.5, and 10 mM). The amount of aggregated HEWL molecules in these samples was kept about 3.0 mM.

peak does not change significantly during fibril formation (**Figure 3B**).

moiety that motivated us to investigate the possibility of assigning 490 cm<sup>−</sup><sup>1</sup>

*Time dependent Trp fluorescence changes of lysozyme (A) incubated in the presence of H2S for 0 minutes (dashes ---), 10 minutes (dots …), 90 minutes (long dashes ---), 48 hours (solid line \_\_\_); (B) incubated with control solution for 0 minutes (solid line \_\_\_), 40 minutes (long dashes ---), and 90 minutes (dots …); (C) Trp maximum emission wavelength of HEWL incubated with H2S (A circles) and fibrillation under control* 

Therefore, in the absence of H2S, HEWL SS bands remain intact and the g-g-g

conformation dominates, in agreement with our previous report [57].

band (**Figure 3D**) characteristic to the trisulfide

band

*Evolution of lysozyme disulfide bonds in the presence of H2S probed by normal Raman spectroscopy. Raman spectra of HEWL incubated (A) in the presence of H2S and (B) under control conditions, where 507 and 523 cm<sup>−</sup><sup>1</sup> bands correspond to g-g-g and g-g-t SS configurations, respectively. Synchronous kinetic change in the area of the 507 and 490 cm<sup>−</sup><sup>1</sup> bands is assigned to the newly formed RSSSR group. (C) The kinetics of RSSSR formation (490 cm<sup>−</sup><sup>1</sup> ) and the decrease in the amount of RSSR (507 cm<sup>−</sup><sup>1</sup> ) during the incubation of HEWL in the presence of H2S. (D) The difference spectrum between normal Raman spectra of HEWL aggregated in the presence of H2S acquired at 90- and 0-minute incubation [shown in (A), gray solid line]. The latter spectrum is represented by the expected spectral change demonstrating the disulfide-to-trisulfide transition symbolized by the inverted Raman spectrum of dipropyl disulfide (black dots and dash, -.-.-) and dipropyl trisulfide (red dots, …).*

$$T\text{CEP} + R \text{ -- SSS -- } R \stackrel{\sim}{\rightarrow} T\text{CEP} \text{(S) + } R \text{ -- SS -- } R \tag{1}$$

$$\text{TCEP} + \text{R - SS - R \Theta} \rightarrow \text{TCEP}(O) \downarrow \text{2.R - SH} \tag{2}$$

The Raman spectrum of HEWL aggregates was found to change significantly in the SSS/SS vibrational signature region with the addition of TCEP (**Figure 4A**). The SSS band at 490 cm<sup>−</sup><sup>1</sup> decreases after 0.5 mM TCEP addition that is in a good agreement with predominant reaction of TCEP with SSS groups. The amplitudes of both 490 and 507 cm<sup>−</sup><sup>1</sup> bands (SSS and SS, respectively) decreased as a result of HEWL incubation in the presence of higher concentration of TCEP (1–10 mM) indicating significant reduction of SS and SSS groups and formation of R-SH moiety in agreement with an increase in 2575 cm<sup>−</sup><sup>1</sup> band intensity (**Figure 4B**).

**Figure 4.**

*Normal Raman spectra of HEWL aggregates in the presence of reducing agent TCEP with concentration 0 mM (solid line), 0.5 mM (dotted line), 1 mM (short dashed line), 2.5 mM (dashed line), and 10 mM (dashed dotted line). Selected spectral regions with characteristic Raman bands of disulfide and trisulfide moieties (A) as well as sulfhydryl (▬ SH) group (B) are shown. Phenylalanine Raman band at 1003 cm<sup>−</sup><sup>1</sup> was used to normalize the spectra (spectral region not shown).*

#### **3.5 Hydrogen sulfide inhibition of myoglobin and hemoglobin fibril formation**

**Figures 5A** and **6A** demonstrate the formation of myoglobin and hemoglobin fibrils and its inhibition by hydrogen sulfide, respectively. The ThT fluorescence intensity associated to Mb and Hb amyloid fibrils (black) shows an initial lag process followed by a drastic increase as function of time. ThT fluorescence intensity is descriptive of prefibrillar oligomer intermediate species associated to the lag phase, while the positive slope is representative of fibrils with differing sizes and structures. Thus, the interaction of ThT with amyloid fibrils is highly specific, but neither amorphous aggregates nor soluble proteins in folded, unfolded, or partially folded states enhance ThT fluorescence [37–39]. Therefore, the data clearly show the existence of the myoglobin and hemoglobin amyloid fibril formation under the control condition of 2,2,2-trifluoroethanol (45%) and incubated at 37°C for

#### **Figure 5.**

*Myoglobin fibrils and their inhibition by hydrogen sulfide. (A) Kinetics of myoglobin amyloid formation (black) and inhibition of Mb fibril formation (blue) in the presence of H2S. (B) Far UV CD spectra of native Mb with a characteristic alpha-helical structure, Mb fibrils exhibiting beta-sheet structure, and Mb structures formed under the same condition of fibril formation, but in the presence of various H2S (blue).*

**167**

**4. Discussion**

*hemoglobin fibrils (black).*

**Figure 6.**

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

the inhibition of fibrils by H2S.

24 hours. There is a slightly difference between Mb and Hb lag phase leading to amyloid formation. However, when hydrogen sulfide is added under the control condition to Mb or Hb to generate fibrils, **Figures 5** and **6**, there is not an increase of ThT fluorescence (blue lines) for the duration of the experiment, independent of the lag phase difference between these hemeproteins. Therefore, the results demonstrate that under the experimental conditions, Mb and Hb fibrils are not observed in the presence of hydrogen sulfide. Curiously, a similar result was reported [34] for hen egg white lysozyme (HEWL), where the addition of H2S in the fibril process

Furthermore, **Figures 5B** and **6B** also show CD the spectra of native Mb and Hb with clear minima at 208 and 222 nm characteristic for α-helix conformation of protein (red line). These negative peaks at 208 and 222 nm result from n → π\* transition in the peptide bond of α-helical conformation [40, 41, 59, 60]. The presented results also indicate that both Mb and Hb form beta-sheet structures with a characteristic negative band near 218 nm and the positive band at 195 nm in CD spectra, which could be attributed to amyloid fibrils, in the presence of 45% of 2,2,2-trifluoroethanol (TFE) (black line). The data also collaborate analogous results indicating the formation of hemoglobin fibrils [28, 40, 41, 59, 60]. Nevertheless, when hydrogen sulfide is added, the CD spectra of both myoglobin and hemoglobin show 208 and 222 nm negative peaks typical for alpha-helical proteins (blue lines), suggesting

completely inhibits the formation of β-sheet of amyloid fibrils.

**4.1 Evaluation of lysozyme secondary and tertiary structure**

Lysozyme fibril formation has been extensively studied and characterized [36, 45, 61]. The most common methods used for studying the fibrillation process include AFM, ThT, and Trp fluorescence. DUVRR spectroscopy has been shown to be uniquely suitable for the structural characterization of proteins at all stages of the fibrillation process [51]. We utilized these complementary methods for

*Hemoglobin fibrils and their inhibition by hydrogen sulfide. (A) Kinetics of thioflavin T with hemoglobin amyloid formation (black) and in the presence of H2S (blue). (B) Far-UV CD studies of native hemoglobin* 

*(red); hemoglobin fibrils inhibition in the presence of various concentrations of H2S (blues) and* 

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

*Amyloid Diseases*

**Figure 4.**

*the spectra (spectral region not shown).*

**3.5 Hydrogen sulfide inhibition of myoglobin and hemoglobin fibril formation**

*as sulfhydryl (▬ SH) group (B) are shown. Phenylalanine Raman band at 1003 cm<sup>−</sup><sup>1</sup>*

*Normal Raman spectra of HEWL aggregates in the presence of reducing agent TCEP with concentration 0 mM (solid line), 0.5 mM (dotted line), 1 mM (short dashed line), 2.5 mM (dashed line), and 10 mM (dashed dotted line). Selected spectral regions with characteristic Raman bands of disulfide and trisulfide moieties (A) as well* 

 *was used to normalize* 

*Myoglobin fibrils and their inhibition by hydrogen sulfide. (A) Kinetics of myoglobin amyloid formation (black) and inhibition of Mb fibril formation (blue) in the presence of H2S. (B) Far UV CD spectra of native Mb with a characteristic alpha-helical structure, Mb fibrils exhibiting beta-sheet structure, and Mb structures* 

*formed under the same condition of fibril formation, but in the presence of various H2S (blue).*

**Figures 5A** and **6A** demonstrate the formation of myoglobin and hemoglobin fibrils and its inhibition by hydrogen sulfide, respectively. The ThT fluorescence intensity associated to Mb and Hb amyloid fibrils (black) shows an initial lag process followed by a drastic increase as function of time. ThT fluorescence intensity is descriptive of prefibrillar oligomer intermediate species associated to the lag phase, while the positive slope is representative of fibrils with differing sizes and structures. Thus, the interaction of ThT with amyloid fibrils is highly specific, but neither amorphous aggregates nor soluble proteins in folded, unfolded, or partially folded states enhance ThT fluorescence [37–39]. Therefore, the data clearly show the existence of the myoglobin and hemoglobin amyloid fibril formation under the control condition of 2,2,2-trifluoroethanol (45%) and incubated at 37°C for

**166**

**Figure 5.**

24 hours. There is a slightly difference between Mb and Hb lag phase leading to amyloid formation. However, when hydrogen sulfide is added under the control condition to Mb or Hb to generate fibrils, **Figures 5** and **6**, there is not an increase of ThT fluorescence (blue lines) for the duration of the experiment, independent of the lag phase difference between these hemeproteins. Therefore, the results demonstrate that under the experimental conditions, Mb and Hb fibrils are not observed in the presence of hydrogen sulfide. Curiously, a similar result was reported [34] for hen egg white lysozyme (HEWL), where the addition of H2S in the fibril process completely inhibits the formation of β-sheet of amyloid fibrils.

Furthermore, **Figures 5B** and **6B** also show CD the spectra of native Mb and Hb with clear minima at 208 and 222 nm characteristic for α-helix conformation of protein (red line). These negative peaks at 208 and 222 nm result from n → π\* transition in the peptide bond of α-helical conformation [40, 41, 59, 60]. The presented results also indicate that both Mb and Hb form beta-sheet structures with a characteristic negative band near 218 nm and the positive band at 195 nm in CD spectra, which could be attributed to amyloid fibrils, in the presence of 45% of 2,2,2-trifluoroethanol (TFE) (black line). The data also collaborate analogous results indicating the formation of hemoglobin fibrils [28, 40, 41, 59, 60]. Nevertheless, when hydrogen sulfide is added, the CD spectra of both myoglobin and hemoglobin show 208 and 222 nm negative peaks typical for alpha-helical proteins (blue lines), suggesting the inhibition of fibrils by H2S.

**Figure 6.**

*Hemoglobin fibrils and their inhibition by hydrogen sulfide. (A) Kinetics of thioflavin T with hemoglobin amyloid formation (black) and in the presence of H2S (blue). (B) Far-UV CD studies of native hemoglobin (red); hemoglobin fibrils inhibition in the presence of various concentrations of H2S (blues) and hemoglobin fibrils (black).*

#### **4. Discussion**

#### **4.1 Evaluation of lysozyme secondary and tertiary structure**

Lysozyme fibril formation has been extensively studied and characterized [36, 45, 61]. The most common methods used for studying the fibrillation process include AFM, ThT, and Trp fluorescence. DUVRR spectroscopy has been shown to be uniquely suitable for the structural characterization of proteins at all stages of the fibrillation process [51]. We utilized these complementary methods for

studying the effect of H2S on the morphology and structure of lysozyme aggregates. Although fibril formation was not detected by AFM and ThT fluorescence assays, the intrinsic Trp fluorescence marker suggested that significant tertiary structure changes had taken place minutes after H2S incubation began. The red shift of Trp fluorescence of greater than 10 nm is typical for unfolded lysozyme [36]. Changes were also evident for SS bridges at the same time scale, as discussed in the next section. The changes observed in the tryptophan local environment and in SS bonds indicate substantial changes in HEWL tertiary structure.

DUVRR spectroscopy was utilized to investigate changes in HEWL secondary structure during the incubation with and without H2S. It was found that H2S prevented the formation of β-sheet and resulted in a significant transition of α-helix to unordered protein. Moreover, we utilized DUVRR spectra of aggregated lysozyme to evaluate the protein secondary structure composition. Xu et al. have reported on the quantitative analysis of lysozyme DUVRR spectral changes during its denaturation [36]. According to that work, the amount of α-helix melting can be estimated from the intensity of Cα▬H bending band. This band is conveniently isolated from other Raman bands. β-Sheet and unordered structures only contribute to Cα▬H bending DUVRR band, while the α-helix does not make a noticeable input [51]. It is evident from amide I Raman bands in DUVRR spectra presented in **Figure 1D** that no fibril-type β-sheet is formed in HEWL aggregates since the Am I intensity does not increase. Therefore, the increase in the Cα▬H band intensity in the spectrum of HEWL aggregates relative to that of native protein could be assigned to newly formed unordered structures. We normalized the DUVRR spectra of HEWL aggregates and native protein with the denatured-reduced HEWL spectrum reported by Xu et al. and estimated the amount of α-helix in HEWL aggregates as 11% [36]. Assuming that the amount of β-sheet in HEWL aggregates is approximately the same as in the native protein, we estimated the secondary structural composition of HEWL aggregates as 83% unordered, 11% α-helix, and 6% β-sheet.

To summarize the results concerning the significant tertiary structural rearrangements, α-helix melting, and lack of β-sheet formation, we conclude that H2S causes more significant denaturation of lysozyme than that taking place during the initial stages of protein fibrillation, which is typically reported as *partial* protein denaturation [36]. We hypothesize that this significant lysozyme denaturation results in rapid protein aggregation, the formation of spherical species, and the prevention of the formation of β-sheets and fibrillation. In other words, H2S redirects the process to "off-pathway" aggregation, preventing fibril formation [8, 62, 63]. This observation is consistent with an earlier report by Wang and colleagues, which demonstrated that fully denatured lysozyme forms amorphous aggregates that prevent fibril formation [64]. The protein has been fully denatured by reducing SS bonds with DTTred. As a result, fully denatured lysozyme may lack the hydrophobic regions which are present in the partially unordered intermediates formed at the early stage of fibril formation. In addition, it is possible that amorphous aggregates decreased the effective concentration of HEWL available for fibril formation [64]. In agreement with Wang's report, our results suggest that lysozyme denatures strongly in the presence of H2S and forms unordered aggregates that prevent β-sheet formation and fibrillation.

#### **4.2 Formation of trisulfide bridges**

According to **Figure 3**, the contributions of both g-g-g (507 cm<sup>−</sup><sup>1</sup> band) and g-g-t (523 cm<sup>−</sup><sup>1</sup> band) conformations of SS bonds to the Raman spectrum of HEWL decreased significantly during its incubation with H2S. Simultaneously, a new peak

**169**

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

of assigning a new Raman band at 490 cm<sup>−</sup><sup>1</sup>

(**Figure 3A** and **C**). Nielsen and colleagues proposed that SSS

to the SSS moiety. Initially, we repro-

Raman band in inorganic compounds

,

stretch-

in these spectra that could

), which is generated from H2S [56],

Raman band cannot be assigned to RSSH groups.

bands

bridges can form in proteins in the presence of H2S via the thiol-disulfide exchange reaction, which is known to occur within cells [23]. We investigated the possibility

duced Raman spectra of two model compounds, dipropyl disulfide (DPDS) and dipropyl trisulfide (DPTS), shown in **Figure 3D**. In agreement with other published studies, these compounds exhibit strong Raman bands at 509 and 485 cm<sup>−</sup><sup>1</sup>

respectively, in agreement with the Raman spectra of native HEWL and HEWL spherical aggregates formed in the presence of H2S [65, 66]. Furthermore, we obtained the difference spectrum by subtracting HEWL spectra after 0 and 90 minutes of incubation in the presence of H2S and compared it to the expected spectral change representing the SS to SSS transition. The latter spectral change is depicted as a combination of dipropyl-disulfide and dipropyl-trisulfide spectra (**Figure 3D**). This spectral comparison provides further support for the hypothetical assignment

and small organic molecules containing sulfur, and we report the appearance of this band in proteins for the first time [66–69]. Wieser and Krueger have assigned the

from the SSS bend [69]. Freeman has reported the Raman spectra of organic SS and SSS compounds, found in natural products where a strong 485 cm<sup>−</sup><sup>1</sup>

ing band has been observed in cyclic and acyclic trisulfides [66]. Janz et al. have reported Raman spectra of inorganic SSS from BaS3 where 458 and 476 cm<sup>−</sup><sup>1</sup>

were assigned to the symmetric stretching of SSS [68]. It is noteworthy that these frequencies can potentially be shifted in peptides. Kimbaris et al. have reported the Raman spectra of garlic oil, which contains a variety of compounds with SS and

potentially originate from an SSS moiety, although the assignment of the band was not discussed in the article. Overall, our hypothetical assignment of the 490 cm<sup>−</sup><sup>1</sup> Raman band to the SSS moiety is in agreement with data from the literature [66, 68]. The mechanism of SSS formation in proteins is unclear despite the significant interest that this topic has gained in recent years [21, 23, 35]. There is emerging

is responsible for sulfuration through the formation of persulfide or trisulfide in proteins [71–73]. It would be interesting to investigate whether these SSS form by intra- or intermolecular processes. We are currently testing this hypothesis. It is

These groups could form as a result of disulfide bond reduction in the presence of

Approximately 50% of all extracellular proteins have disulfide bridges [61]. SS bonds preserve the three dimensional structure of proteins and their cleavage typically results in significant disruption of the native conformations of proteins [57]. It is well established that SS bonds play a significant role in amyloid fibrillation [75]. Dobson and colleagues have reported that the reduction of SS bridges significantly accelerated the rate of human lysozyme aggregation [61]. It has also been demonstrated that reduction of four SS bonds to three bonds of apo-α-LA accelerates its fibrillation and leads to the formation of a new fibril polymorph with a different morphology and structure compared to fibrils formed from the wild-type LA [57]. At the same time, SS bonds of insulin remain intact and preserve their conformation during the fibrillation process [75]. Similar to insulin, the conformation of the SS

**4.3 The mechanism of HEWL, Mb, and Hb aggregation vs. fibrillation**

Raman peak of H-SSS-H to a symmetric SS stretch with a contribution

Raman band to the SSS moiety.

SSS groups [70]. We noticed an intense band at 489 cm<sup>−</sup><sup>1</sup>

H2S by a process known as sulfuration or sulfhydration [74].

evidence indicating that sulfane sulfur (S0

noteworthy that the 490 cm<sup>−</sup><sup>1</sup>

Several studies have identified a 490 cm<sup>−</sup><sup>1</sup>

appeared at 490 cm<sup>−</sup><sup>1</sup>

of the 490 cm<sup>−</sup><sup>1</sup>

488 cm<sup>−</sup><sup>1</sup>

*Amyloid Diseases*

studying the effect of H2S on the morphology and structure of lysozyme aggregates. Although fibril formation was not detected by AFM and ThT fluorescence assays, the intrinsic Trp fluorescence marker suggested that significant tertiary structure changes had taken place minutes after H2S incubation began. The red shift of Trp fluorescence of greater than 10 nm is typical for unfolded lysozyme [36]. Changes were also evident for SS bridges at the same time scale, as discussed in the next section. The changes observed in the tryptophan local environment and in SS bonds

DUVRR spectroscopy was utilized to investigate changes in HEWL secondary structure during the incubation with and without H2S. It was found that H2S prevented the formation of β-sheet and resulted in a significant transition of α-helix to unordered protein. Moreover, we utilized DUVRR spectra of aggregated lysozyme to evaluate the protein secondary structure composition. Xu et al. have reported on the quantitative analysis of lysozyme DUVRR spectral changes during its denaturation [36]. According to that work, the amount of α-helix melting can be estimated from the intensity of Cα▬H bending band. This band is conveniently isolated from other Raman bands. β-Sheet and unordered structures only contribute to Cα▬H bending DUVRR band, while the α-helix does not make a noticeable input [51]. It is evident from amide I Raman bands in DUVRR spectra presented in **Figure 1D** that no fibril-type β-sheet is formed in HEWL aggregates since the Am I intensity does not increase. Therefore, the increase in the Cα▬H band intensity in the spectrum of HEWL aggregates relative to that of native protein could be assigned to newly formed unordered structures. We normalized the DUVRR spectra of HEWL aggregates and native protein with the denatured-reduced HEWL spectrum reported by Xu et al. and estimated the amount of α-helix in HEWL aggregates as 11% [36]. Assuming that the amount of β-sheet in HEWL aggregates is approximately the same as in the native protein, we estimated the secondary structural composition of HEWL aggregates as 83%

To summarize the results concerning the significant tertiary structural rearrangements, α-helix melting, and lack of β-sheet formation, we conclude that H2S causes more significant denaturation of lysozyme than that taking place during the initial stages of protein fibrillation, which is typically reported as *partial* protein denaturation [36]. We hypothesize that this significant lysozyme denaturation results in rapid protein aggregation, the formation of spherical species, and the prevention of the formation of β-sheets and fibrillation. In other words, H2S redirects the process to "off-pathway" aggregation, preventing fibril formation [8, 62, 63]. This observation is consistent with an earlier report by Wang and colleagues, which demonstrated that fully denatured lysozyme forms amorphous aggregates that prevent fibril formation [64]. The protein has been fully denatured by reducing SS bonds with DTTred. As a result, fully denatured lysozyme may lack the hydrophobic regions which are present in the partially unordered intermediates formed at the early stage of fibril formation. In addition, it is possible that amorphous aggregates decreased the effective concentration of HEWL available for fibril formation [64]. In agreement with Wang's report, our results suggest that lysozyme denatures strongly in the presence of H2S and forms unordered aggregates that prevent β-sheet

According to **Figure 3**, the contributions of both g-g-g (507 cm<sup>−</sup><sup>1</sup>

decreased significantly during its incubation with H2S. Simultaneously, a new peak

band) conformations of SS bonds to the Raman spectrum of HEWL

band) and

indicate substantial changes in HEWL tertiary structure.

unordered, 11% α-helix, and 6% β-sheet.

**168**

g-g-t (523 cm<sup>−</sup><sup>1</sup>

formation and fibrillation.

**4.2 Formation of trisulfide bridges**

appeared at 490 cm<sup>−</sup><sup>1</sup> (**Figure 3A** and **C**). Nielsen and colleagues proposed that SSS bridges can form in proteins in the presence of H2S via the thiol-disulfide exchange reaction, which is known to occur within cells [23]. We investigated the possibility of assigning a new Raman band at 490 cm<sup>−</sup><sup>1</sup> to the SSS moiety. Initially, we reproduced Raman spectra of two model compounds, dipropyl disulfide (DPDS) and dipropyl trisulfide (DPTS), shown in **Figure 3D**. In agreement with other published studies, these compounds exhibit strong Raman bands at 509 and 485 cm<sup>−</sup><sup>1</sup> , respectively, in agreement with the Raman spectra of native HEWL and HEWL spherical aggregates formed in the presence of H2S [65, 66]. Furthermore, we obtained the difference spectrum by subtracting HEWL spectra after 0 and 90 minutes of incubation in the presence of H2S and compared it to the expected spectral change representing the SS to SSS transition. The latter spectral change is depicted as a combination of dipropyl-disulfide and dipropyl-trisulfide spectra (**Figure 3D**). This spectral comparison provides further support for the hypothetical assignment of the 490 cm<sup>−</sup><sup>1</sup> Raman band to the SSS moiety.

Several studies have identified a 490 cm<sup>−</sup><sup>1</sup> Raman band in inorganic compounds and small organic molecules containing sulfur, and we report the appearance of this band in proteins for the first time [66–69]. Wieser and Krueger have assigned the 488 cm<sup>−</sup><sup>1</sup> Raman peak of H-SSS-H to a symmetric SS stretch with a contribution from the SSS bend [69]. Freeman has reported the Raman spectra of organic SS and SSS compounds, found in natural products where a strong 485 cm<sup>−</sup><sup>1</sup> stretching band has been observed in cyclic and acyclic trisulfides [66]. Janz et al. have reported Raman spectra of inorganic SSS from BaS3 where 458 and 476 cm<sup>−</sup><sup>1</sup> bands were assigned to the symmetric stretching of SSS [68]. It is noteworthy that these frequencies can potentially be shifted in peptides. Kimbaris et al. have reported the Raman spectra of garlic oil, which contains a variety of compounds with SS and SSS groups [70]. We noticed an intense band at 489 cm<sup>−</sup><sup>1</sup> in these spectra that could potentially originate from an SSS moiety, although the assignment of the band was not discussed in the article. Overall, our hypothetical assignment of the 490 cm<sup>−</sup><sup>1</sup> Raman band to the SSS moiety is in agreement with data from the literature [66, 68]. The mechanism of SSS formation in proteins is unclear despite the significant interest that this topic has gained in recent years [21, 23, 35]. There is emerging evidence indicating that sulfane sulfur (S0 ), which is generated from H2S [56], is responsible for sulfuration through the formation of persulfide or trisulfide in proteins [71–73]. It would be interesting to investigate whether these SSS form by intra- or intermolecular processes. We are currently testing this hypothesis. It is noteworthy that the 490 cm<sup>−</sup><sup>1</sup> Raman band cannot be assigned to RSSH groups. These groups could form as a result of disulfide bond reduction in the presence of H2S by a process known as sulfuration or sulfhydration [74].
