amyloids are large 
 aggregates of misfolded proteins with a highly 
 stable cross -structure , which are associated with a variety 
 of degenerative illnesses such as alzheimer s , parkinson s , and huntington s diseases . proteins with different functionalities 
 and native structures ranging from -helical and -sheet 
 rich to intrinsically unordered are able to form amyloid fibrils in vitro with a characteristic cross- core structure . 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 . steps to follow include the formation of small aggregates and a 
 -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 . for centuries , people have been 
 interested in h2s for its role as a poisonous chemical . 
 at high concentrations , more recently , it has been demonstrated that h2s has gasotransmitter functions , similar to co and no . 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 . in the past two decades , significant attention 
 has been paid to understanding the physiological role of h2s and its endogenous production . h2s is biosynthesized 
 in mammalian tissue by nonenzymatic reactions and by the enzymatic 
 degradation of cysteine by cystathione  synthetase ( cbs ) , cystathione 
  lyase ( cse ) , cysteine aminotransferase ( cat ) , and cysteine 
 lyase ( cl ) . moreover , aged garlic extract has been shown to cause a reduction 
 of in vivo a fibrils and soluble amyloid as 
 well as a decrease in tau conformational changes . 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 . there is extensive literature on the inhibitory 
 activity of various small molecules on protein fibrillation . recently , arioso 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 . 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% . 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 . it has been reported 
 that treating antibodies with h2s has resulted in ss bond 
 modifications , including the formation of trisulfide bonds ( sss ) assessed 
 by liquid chromatography and mass spectrometry ( lc - ms ) . 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 ( s ) that only binds to 
 other sulfur atoms to form polysulfides . several research groups have also reported that the sulfur atom 
 of 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 . in the current study , 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 . the effect of h2s has been investigated 
 under typical fibrillation conditions such as high temperature and 
 acidic ph using duvrr and nonresonance raman spectroscopy , fluorescence , 
 lc - ms , 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 have 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 . this discovery indicates possible new roles for h2s and trisulfide bridges in protein biochemistry in 
 vivo . louis , mo ) : 99.7% acetic acid ( 695092 ) , sodium chloride ( nacl ) ( s771 - 3 ) , 
 hewl ( l6876 ) , sodium sulfidenonahydrate salt ( 208043 ) , dipropyl disulfide 
 ( 149225 ) , and trisulfide ( 6028 - 61 - 1 ) . 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 . 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 the range of 4001800 cm . a laser power of approximately 11.5 mw was used to avoid sample photodegradation . a reaction with tris(2-carboxyethyl)phosphine 
 ( tcep ) reducing agent hen egg white lysozyme ( hewl ) in native and 
 aggregated form was incubated at ph 2.0 and room temperature for 90 
 min in the presence of tcep . lc - ms experiments were performed 
 on thermo ltq orbitrap xl mass spectrometer ( thermo , bremen , germany ) . samples were injected using an agilent 1200 nano - hplc system ( agilent , 
 palo alto , ca ) and agilent 1200 autosampler . agilent zorbax c18 column 
 ( 150  4.6 mm ) was used for separation of mixtures . the injection 
 volume was up to 8 l , and a flow rate of the mobile phase was 
 250 l / min . mobile phase consisted of 0.2% of formic acid ( solvent 
 a ) and 0.2% formic acid in acetonitrile ( solvent b ) with the gradient 
 from 5% to 40% of solvent b in 40 min followed by washing the column 
 by 90% of solvent b and equilibration . the mass spectrometer operated 
 in esi mode in the mass range m / z 100500 with detection of positively and negatively charged 
 ions . the resolution was 30  000 , and the accuracy of 
 mass measurements was better than 2 ppm . powder samples of hewl 
 aggregates incubated at different concentrations of tcep were prepared 
 for nonresonance raman spectroscopic analysis by drying the corresponding 
 solutions under a nitrogen flow at room temperature . 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 . ) each 
 spectrum recorded an average of 20 accumulations with 30 s acquisition 
 time . grams / ai 7.0 software ( thermo galactic , salem , nh ) was used 
 for data processing . fluorescence spectra 
 were measured in a jobinyvon fluoromax-3 spectrofluorometer ( jobinyvon , 
 edison , nj ) . intrinsic tryptophan fluorescence of 25 mg / ml hewl was 
 measured in a 10 m path length cell without dilutions . the 
 uv absorption was < 0.05 at an excitation wavelength of 295.5 nm . three spectral accumulations were taken , and the spectra were averaged 
 for each sample . hewl fibrils formed after 90 min 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 min , centrifugation for 4 min at 13  000 rpm , 
 the removal of supernatant liquid , and resuspension in an acetic acid 
 solution . 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 . 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 
 1025 nm tip curvature radius . human 
 neuroblastoma sh - sy5y cells were 
 cultured in a 1:1 ( v / v ) mixture of dmem ( sigma - aldrich , st . louis , 
 mo ) and ham s f-12 nutrient mixture ( sigma - aldrich , st . louis , 
 mo ) supplemented with 10% fbs ( sigma - aldrich , st . louis , mo ) and antibiotic - antimitotic 
 solution ( sigma - aldrich , st . the cells were incubated 
 at 37 c in an incubator with 95% humidified air and 5% co2 . cells were plated onto flat - bottom 
 96-well plates ( 1  10 cells per well ) and incubated 
 overnight at 37 c . after cell attachment , the initial medium 
 was replaced with serum - free medium using different concentrations 
 of mature hewl fibrils or hewl aggregates ( formed with h2s ) and incubated for 24 h. cytotoxicity was assessed using the mtt 
 reduction inhibition assay . mtt was added 
 to the culture medium to yield a final concentration of 0.5 mg / ml . 
 cells were incubated with the mtt medium for 4 h in a co2 incubator , followed by the addition of 200 l of an isopropanol 
 0.04 n hcl solution to dissolve formazan crystals . absorbance was 
 measured at 540 nm using a synergy h1 plate reader ( biotek , vermont ) . control experiments were performed by exposing cells to solutions 
 of an equivalent volume of the same initial buffer for the same length 
 of time . cell viability was measured relative to control cells that 
 were not exposed to the hewl aggregates / hewl fibrils solutions . 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 rodlike 
 fibrils was evident after 90 min 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 ) . lysozyme forms -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 min 
 and ( b ) hewl aggregates formed after incubation of the solution in 
 the presence of h2s for 48 h ; scale bars are 1 m . ( 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 ) for comparison . the 
 amide i vibrational mode ( am i ) is dominated by c = o stretching , 
 with minor contributions from c amide ii ( am ii ) and amide iii 
 ( am iii ) bands involve significant c the 
 ch bending vibrational mode involves ch 
 symmetric bending and c  c stretching . tht fluorescence intensity increased dramatically after 70 
 min 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 h. 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 . 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 . a noticeable increase in the intensity and sharpness of the am i band ( approximately 1672 cm ) indicated 
 the appearance of -sheets due to the formation of fibrils . the duvrr spectrum of fibrillated lysozyme under 
 control conditions confirmed the formation of -sheets . the 
 spectrum of hewl after 30 min of incubation under control conditions 
 ( figure 1d , red ) is similar to that reported 
 previously for hewl fibrils . however , 
 the duvrr spectrum of lysozyme incubated in the presence 
 of h2s confirmed the lack of -sheet formation . in 
 this case , the am i band ( approximately 1670 cm ) 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 . this was further supported by the increase in ch band intensity at 1390 cm that was 
 indicative of -helix melting . a 
 significant change in raman bands for am iii ( approximately 1250 cm ) and am ii ( approximately 1555 cm ) was consistent with the transition of -helix to unordered 
 protein . therefore , afm , tht fluorescence and duvrr spectroscopy indicated 
 the formation of unordered spherical aggregates of hewl by contrast 
 with -sheet - rich fibrils in the presence of h2s . tryptophan ( trp ) fluorescence is an efficient 
 intrinsic marker of local environments , which is often used for monitoring 
 tertiary structural changes in proteins . native lysozyme at neutral ph shows a maximum trp emission at 340 
 nm . 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 , c ) . to confirm that the intrinsic trp fluorescence is dominated 
 by the signal from hewl fibrils , the solutions ( after incubation for 
 40 and 90 min ) were sonicated , centrifuged and resuspended in 20% 
 acetic acid to remove possible monomeric and oligomeric forms of the 
 protein ( figure 2b ) . time - dependent trp fluorescence 
 changes of lysozyme ( a ) incubated 
 in the presence of h2s for 0 min ( -  -  - ) , 
 10 min (  ) , 90 min (    ) , and 
 48 h (  ) and ( b ) incubated with control solution for 0 min 
 (  ) , 40 min    ) , and 90 min (  ) . ( c ) trp maximum emission wavelength of hewl incubated with h2s ( a ,  ) and fibrillation under control conditions ( b ,  ) . a significant shift of the trp 
 emission maximum , from 345 to 357 
 nm , was observed after 90 min of lysozyme incubation in the presence 
 of h2s ( figure 2a , c ) , with no further 
 changes for at least 48 h. 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 . 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 . nonresonance raman 
 spectroscopy of proteins offers a unique opportunity for characterizing 
 the conformation of disulfide bridges . the ss symmetric stretching vibrational mode is 
 typically represented as a strong raman band in the range of 505550 
 cm . the raman spectrum of hewl was found to change 
 significantly in the ss signature region with incubation time ( figure 3a ) . a strong 507 cm 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 peak can be attributed to the gauche  gauche trans 
 ( g - g - t ) configuration of the fourth ss bond of lysozyme . the amplitudes of these peaks decreased , and a new peak appeared 
 at 490 cm as a result of hewl incubation in the 
 presence of 12 mm h2s , indicating significant rearrangements 
 of ss bonds ( figure 3a ) . 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 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 bands 
 is assigned to the newly formed rsssr group . ( c ) kinetics of rsssr 
 formation ( 490 cm ) and the decrease in the amount 
 of rssr ( 507 cm ) during the incubation of hewl 
 in the presence of h2s . ( d ) difference spectrum between 
 normal raman spectra of hewl aggregated in the presence of h2s acquired at 90 and 0 min 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 ( -    - ) 
 and dipropyltrisulfide ( red  ) . 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 . the 1003 cm 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 bands with incubation time up to 90 min . no further changes were 
 observed during 48 h of additional incubation in the presence of h2s ( data not shown ) . as discussed in detail below , the dipropyl 
 trisulfide ( dpts ) raman spectrum contains a 485 cm band ( figure 3d ) characteristic of the trisulfide 
 moiety that motivated us to investigate the possibility of assigning 
 the 490 cm band in the hewl aggregate raman spectrum 
 to the sss group . the nonresonance raman spectroscopy of hewl fibrillation 
 under control conditions indicates that the 507 cm peak does not change significantly during fibril formation ( figure 3b ) . therefore , in the absence of h2s , 
 the hewl ss bands remain intact and the g - g - g conformation dominates , 
 in agreement with our previous report . 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 and mass spectrometry . tcep reaction with ss groups 
 is well - known to result in oxidation of tcep and formation of tcep(o ) 
 and r  sh groups . 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 . 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.12 normal 
 raman spectra of hewl aggregates in the presence of reducing 
 agent tcep with concentration 0 mm (  ) , 0.5 mm (  ) , 
 1 mm ( -  -  - ) , 2.5 mm (    ) , and 
 10 mm ( -    - ) . selected spectral regions with characteristic 
 raman bands of disulfide and trisulfide moieties ( a ) as well as sulfhydryl 
 ( sh ) group ( b ) are shown . the phenylalanine raman band at 
 1003 cm was used to normalize the spectra ( spectral 
 region not shown ) . 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 decreases after 0.5 mm tcep addition which is in a good agreement 
 with predominant reaction of tcep with sss groups . the amplitudes 
 of both 490 and 507 cm bands ( sss and ss , respectively ) 
 decreased as a result of hewl incubation in the presence of higher 
 concentration of tcep ( 110 mm ) , indicating significant reduction 
 of ss and sss groups and formation of r  sh moiety in agreement 
 with an increase in 2575 cm band intensity ( figure 4b ) . lc - ms was utilized to monitor the formation 
 of tcep(s ) and tcep(o ) 
 as products of sss and ss reduction , respectively ( eqs 1 and 2 ) . the reduction of native hewl 
 with an excess of tcep ( 10 mm ) resulted in products with retention 
 times of 6.68 , 9.01 , and traces at 17.51 min ( figure 5a ) . accurate mass measurements confirmed that the products 
 correspond to tcep , tcep(o ) , and tcep(s ) , respectively . hewl aggregates 
 treated with 0.5 mm tcep showed tcep(o ) and tcep(s ) , with retention 
 times of 9.0 and 17.53 min ( figure 5b ) . all 
 initial tcep was completely consumed due to the reaction with sss 
 and ss groups . combined lc - ms ( esi in negative ion mode ) chromatograms 
 of ions 
 at m / z 249 , m / z 265 , and m / z 281 corresponding 
 to deprotonated tcep , tcep(o ) , and tcep(s ) , respectively , for native 
 lysozyme incubated with 10 mm tcep ( a ) and lysozyme aggregates incubated 
 with 0.5 mm tcep ( b ) . the solution of native hewl incubated with an excess of tcep 
 contained 
 significant amounts of tcep and tcep(o ) and traces of tcep(s ) . the 
 main expected products of tcep reaction with rssr are tcep(o ) and 
 2rsh . we believe that the traces of tcep(s ) 
 could form due to a prolonged exposure of native hewl to the excess 
 of tcep prior to lc - ms analysis . this suggestion is based on the reported 
 reaction 3 of triphenylphosphine ( ph3p ) with dialkyl disulfides ( rssr ) , which resulted in formation of 
 ph3p(s ) . we hypothesize that 
 the trace amount of tcep(s ) is formed in the reaction of tcep with 
 native hewl due to a similar mechanism . however , the amount of tcep(s ) 
 is low because the second step in reaction 3 is slow and reaction 2 dominates.3overall , the analysis of reaction 
 products 
 resulting from the incubation of native and aggregated lysozyme in 
 the presence of tcep using lc - ms is consistent with the presence of 
 sss in aggregated hewl . the variation in the amplitude of the 490 
 cm raman band with the concentration of tcep is 
 in agreement with its assignment to the sss group as well . to compare the cytotoxicity 
 of hewl fibrils and spherical aggregates 
 formed in the presence of h2s , samples of mature fibrils 
 and spherical aggregates were posteriorly transferred to pbs buffer 
 at ph 7.4 in order to avoid drastic changes in the ph of the cells . 
 to achieve this , mature fibrils were washed three times in pbs buffer , 
 and spherical aggregates were dialyzed against pbs buffer . the latter 
 procedure did not change fibrils and spherical aggregates according 
 to their normal raman spectra ( data not shown ) . specifically , the 
 490 cm band remained intact after buffer exchange 
 to pbs . the incubation of confluent sh - sy5y cells with mature fibrils 
 ( 0.536 m ) for 24 h resulted in a significant reduction 
 ( 40% ) of the cellular viability . mean cell viability and one standard deviation of two independent 
 experiments performed in triplicate are presented . 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 . 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 . 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 -sheet 
 and resulted in a significant transition of -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 . according 
 to that work , the amount of -helix melting can be estimated 
 from the intensity of ch bending band . -sheet 
 and unordered structures only contribute to ch 
 bending duvrr band , while the -helix does not make a noticeable 
 input . it is evident from amide i raman 
 bands in duvrr spectra presented in figure 1d that no fibril - type -sheet is formed in hewl aggregates 
 since the am i intensity does not increase . therefore , the increase 
 in the ch 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 -helix in hewl aggregates as 11% . assuming that 
 the amount of -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% -helix , 
 and 6% -sheet . to summarize the results concerning the 
 significant tertiary structural 
 rearrangements , -helix melting , and lack of -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 . we hypothesize 
 that this significant lysozyme denaturation results in rapid protein 
 aggregation , the formation of spherical species , and the prevention 
 of the formation of -sheets and fibrillation . in other words , 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 . 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 . in agreement with wang s report , our 
 results suggest that lysozyme denatures strongly in the presence of 
 h2s and forms unordered aggregates that prevent -sheet 
 formation and fibrillation . according to figure 3 , the contributions of 
 both g - g - g ( 507 cm band ) and g - g - t ( 523 cm band ) conformations 
 of ss bonds to the raman spectrum of hewl decreased significantly 
 during its incubation with h2s . simultaneously , a new peak 
 appeared at 490 cm ( figure 3a , c ) . nielsen and colleagues proposed that sss bridges can form in 
 proteins in the presence of h2s via the thiol we investigated the possibility of assigning 
 a new raman band at 490 cm 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 , respectively , in agreement with the raman spectra 
 of native hewl and hewl spherical aggregates formed in the presence 
 of h2s . furthermore , we obtained the difference spectrum 
 by subtracting hewl spectra after 0 and 90 min 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 raman band to the sss moiety . several studies have identified a 490 cm raman 
 band in inorganic compounds and small organic molecules containing 
 sss , and we report the appearance of this band in proteins for the first 
 time . wieser and krueger have assigned the 488 cm raman peak of h  sss  h to a symmetric ss stretch with 
 a contribution from the sss bend . freeman 
 has reported the raman spectra of organic ss and sss compounds , found 
 in natural products where a strong 485 cm stretching 
 band has been observed in cyclic and acyclic trisulfides . janz et al . have reported raman spectra of inorganic 
 sss from bas3 where 458 and 476 cm bands 
 were assigned to the symmetric stretching of sss . kimbaris et al . have reported the gc / ms and raman spectra of garlic oil , which contains 
 a variety of compounds with ss and sss groups . we noticed an intense 
 band at 489 cm 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 raman band to the sss moiety is 
 in agreement with data from the literature . the mechanism of sss formation in proteins is unclear despite the 
 significant interest that this topic has gained in recent years . there is emerging evidence indicating 
 that sulfane sulfur ( s ) , which is generated from h2s , is responsible for sulfuration 
 through the formation of persulfide or trisulfide in proteins . it would be interesting to investigate whether these sss form by 
 intra- or intermolecular processes . it is noteworthy that the 490 cm raman band 
 can not 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 . however , r  sh and r  ssh groups have a characteristic 
 raman band at 2575 cm which is not evident in 
 the experimental spectra ( figures s1 and s2 ) . in addition , these groups do not have a vibrational mode at 490 
 cm . there is evidence 
 in the literature that indicates the possibility of ss bond reduction 
 in the presence of h2s and the formation of rssh groups 
 in basic environments . the results allowed us to eliminate rssh as a possible candidate for 
 newly formed species with a characteristic 490 cm raman band . ss bonds preserve the three - dimensional structure 
 of proteins , and their cleavage typically results in significant disruption 
 of the native conformations of proteins . it is well established that ss bonds play a significant role in 
 amyloid fibrillation . dobson and colleagues 
 have reported that the reduction of ss bridges significantly accelerated 
 the rate of human lysozyme aggregation . it has also been demonstrated that reduction of four ss bonds to 
 three ss bonds of apo--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 . at the same time , ss bonds of insulin 
 remain intact and preserve their conformation during the fibrillation 
 process . similar to insulin , the conformation 
 of the ss bonds in hewl remains intact during the fibrillation of 
 hewl in control solution , as we have described here . it has 
 been suggested that a partial denaturation of lysozyme precedes fibril 
 formation because the native tertiary 
 structure would not allow rearrangement to the cross--sheet 
 structure due to steric constraints . it 
 has also been reported that partial denaturation , the first step of 
 lysozyme fibrillation , is an irreversible process . at the same time , a fully denatured lysozyme forms amorphous 
 aggregates that prevent fibril formation . it is believed that the fully denatured protein lacks the hydrophobic 
 side chains present in partially unordered intermediates . in addition , 
 amorphous aggregates potentially decrease the effective concentration 
 of hewl available for fibril formation . in agreement with these observations , our results suggest that lysozyme 
 denatures strongly in the presence of h2s and forms unordered 
 aggregates that prevent -sheet formation and fibrillation . amyloid fibrils are associated with many neurodegenerative 
 diseases 
 including alzheimer s , parkinson s , and systemic amyloidosis . it has recently been found that there is a reduced amount of hydrogen 
 sulfide ( h2s ) in the brain tissue of patients with alzheimer s 
 disease , leading us to investigate the 
 effects of h2s on the formation of amyloid fibrils . our 
 objective was to utilize several complementary techniques , including 
 raman spectroscopy , afm , and intrinsic tryptophan and tht fluorescence 
 for studying the kinetics of hewl fibrillation , a well - characterized 
 model amyloidogenic protein , in the presence of h2s . lysozyme 
 forms typical -sheet - rich fibrils after incubation in 20% acetic 
 acid solution at 60 c for approximately 70 min . the addition 
 of 12 mm h2s in a molar ratio of 5:1 ( h2s : hewl ) 
 completely prevented the formation of the -sheet conformation 
 as measured by deep uv resonance raman ( duvrr ) spectroscopy and tht 
 fluorescence . by contrast , the melting of the -helix resulted 
 in unordered protein in the form of spherical aggregates , which were 
 distinctly different from amyloid fibrils . according to the intrinsic 
 tryptophan fluorescence , hewl exhibited a more significant perturbation 
 of its tertiary structure in the presence of h2s than that 
 which occurred during the partial protein denaturation at the early 
 stage of fibrillation . lysozyme has four ss bonds , which stay 
 intact during the fibrillation 
 process . however , based on our data from nonresonance raman spectroscopy , 
 ss bonds exhibit significant rearrangements in the presence of h2s . the peak area of the 507 cm raman band 
 corresponding to the ss segment in the g - g - g conformation was reduced significantly , and a new band appeared 
 at 490 cm . we assigned this new band to the sss 
 group based on previously reported data , the raman spectra we acquired 
 for model compounds , and lc - ms analysis of tcep reduction products . cytotoxicity tests revealed that the spherical aggregates formed by 
 lysozyme in the presence of h2s are nontoxic to cells by 
 contrast with fibrils . overall , we report for the first time 
 that h2s prevents 
 fibrillation of hewl in vitro and results in the 
 formation of small spherical aggregates of unordered protein . we also 
 hypothesize that the mechanism of this h2s effect involves 
 the formation of sss bridges . our findings that 
 h2s inhibits fibril formation and 
 that putative sss forms open a new and intriguing topic of biochemical 
 and biomedical research . the fact that the spherical aggregates formed 
 are nontoxic to cells by contrast with fibrils is worth further investigation in vivo . given that there are new drugs under development 
 for a slow release of h2s with a variety of therapeutic 
 targets , including s - sildenafil for urological conditions , s - diclofenac 
 for inflammation , s - latanoprost for neurodegenerative illnesses , s - levodopa 
 for parkinson s disease , and s - aspirin for cardiovascular conditions , our results suggest possible new roles of sss in vivo .