Toxic Interaction Mechanism Between Signaling Molecule H₂S And Allinase

Hydrogen sulfide?H₂S?was suggested to act as a gaseous signaling molecule in a variety of physiological processes.Its molecular mechanism of action was proposed to involve protein S-sulfhydration,that is,conversion of cysteinyl thiolates?Cys-S-?to persulfides?Cys-S-S-?.In this paper,alliinase,L-lactic dehydrogenase?L-LDH?and D-lactic dehydrogenase?D-LDH?were used as the protein target,and three kinds of H₂S-donor reagents?Na₂S,NaHS,K₂S_n?werechosen.TheinteractionsoftheseH₂S-donorreagentswith alliinase/L-LDH/D-LDH were disclosed by molecular fluorescent assays to real-time monitoring them activity.As Na₂S/NaHS/K₂S_n?Polysulfides,PS?concentrations increased from 0.125 mmol/L to 0.5 mmol/L,a significant increase of fluorescence intensity at about480 nm was observed,suggesting that Na₂S,NaHS and K₂S_n could inhibit activity of them in vitro in a concentration-dependent manner.With H₂O₂?1 mmol/L?oxidized alliinase/L-LDH/D-LDH or NEM?1 mmol/L?treated alliinase/L-LDH/D-LDH,DTT?5mmol/L?was capable of partially reactivating the enzyme.DTT analysis confirmed H₂O₂-induced disulfide bond formation in alliinase,thus indicating reversible oxidation of the active site cysteine.Then the experiments were compared inhibition of the thiol-reactive alkylating agent N-Ethylmaleimide?NEM?,Na₂S/NaHS/PS with that of other biologically relevant oxidants,Hydrogen Peroxide?H₂O₂?/Oxidized Glutathione?GSSG??1 mmol/L?.On an equimolar basis,NEM exhibited the strongest inhibited effect of all compounds tested.The results of the SDS-PAGE showed that H₂S might not interact with alliinase/L-LDH to form disulfide/trisulfide bonding.Moreover,the results identified polysulfides?S_n?formed in Na₂S/NaHS/K₂S_n solutions as the oxidizing species using UV-Visible Spectrophotometer,and presented evidence that ulfur-containing PS?polysulfide?was a stronger protein S-sulfurating agent than the other two sulfides.The results showed that H₂S might lead to modification of the secondary structure of alliinase/L-LDH/D-LDH.Native alliinase/L-LDH/D-LDH has a high?-helical content and very little?-sheet structure,and K₂S_n showed the most significant changes in secondary structures composition,with the lowest?-helical content,the highest?-sheet structure.In conclusion,the effects that have been attributed to H₂S in previous reports might in fact have been mediated by polysulfides,to achieve reactions with cysteine thiols of proteins.Finally,Using MODAL-TOF-MS/MS to verify whether H₂S was targeting alliinase and L-LDH cysteine sulfhydryl derivatives to form a disulfide bond.MALDI-TOF-MS/MS study showed some of the cysteine sites existed S-sulfuration in alliinase/L-LDH.In conclusion,H₂S exerts its biological effects as a gasotransmitter through its reactions with cysteine thiols in proteins by S-sulfuration.