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

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

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-β sheet structure due to steric constraints [8, 76]. It has also been reported that partial denaturation, the first step of lysozyme fibrillation, is an irreversible process [46]. At the same time, a fully denatured lysozyme forms amorphous aggregates that prevent fibril formation [64]. 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 [64]. In agreement with these observations, our results suggest that lysozyme denatures strongly in the presence of H2S and forms unordered aggregates that prevent β-sheet formation and fibrillation.

Regarding this, it has been shown that Hb under physiological conditions and in presence of 45% 2,2,2-trifluoethanol (TFE) produces amyloid-like fibril species [28]. The mechanism surrounding these fibril events remains almost unknown. Curiously, myoglobin and hemoglobin do not have any S-S moiety in their chemical structures, and **Figures 5** and **6** show that the fibrillation inhibition effect of H2S depends on its concentration. Specifically, the Mb and Hb α-helix assemblies are almost preserved at higher H2S concentrations. Therefore, in these hemeproteins, it is not clear the inhibition mechanism by H2S, since CD indicates that hydrogen sulfide prevents β-sheet formation and fibrillation without altering significantly the α-helical structure of Mb or Hb. Also similar to HEWL, the addition of H2S to Mb or Hb fibrils does not revert the β-sheet amyloid fibrils to the native α-structure. These results are consistent with the ThT findings that β-sheets are present in Mb and Hb amyloid-like fibrils in the presence of 45% TFE and that increasing concentration of H2S inhibits β-sheet formation. The findings demonstrate the same H2S effect on to the fibrillation of Mb monomer and Hb tetramer, although some quantitative kinetic differences may be evident and need further study.
