Characterization of Disulfide Bond Oxidation in Peptides and Proteins by Myeloperoxidase-Derived Oxidants

Abstract

Activated neutrophils play an important role in the immune response to invading pathogens and bacteria. These cells generate large fluxes of reactive oxidants via an oxidative burst to kill pathogens. Inappropriate or misdirected stimulation of this mechanism, such as occurs during inflammation, can damage the host tissue. This inflammation-induced damage has been linked to the development and/or progression of multiple diseases such as atherosclerosis, arthritis, asthma and some cancers. One of the major sources of these reactive oxidants at sites of inflammation is the heme enzyme myeloperoxidase (MPO) that is released by activated leukocytes. Hydrogen peroxide (H2O2) is generated from multiple sources, including the NADPH oxidase system of activated leukocytes. MPO uses H2O2 in the presence of halide (Cl ͞ ,Br ͞ )and pseudo-halide (SCN ͞ ) to generate highly reactive oxidants hypohalous acids (HOX) including hypochlorous acid (HOCl, the major component of household bleach), hypothiocyanous acid (HOSCN) and at lower quantity hypobromous acid (HOBr). HOCl is a strong oxidant with antibacterial properties but excessive or misplaced production of HOCl have been reported to be involved in several diseases. HOCl has many biological targets, but proteins are major targets due to their abundance and their high reactivity. Previous studies have shown that the most reactive protein residues with hypohalous acids are the sulfur- and selenium-containing amino acids cysteine (Cys), methionine (Met) and selenocysteine (Sec). Lysine (Lys) side-chains, disulfide bonds and the aromatic side-chains of tryptophan (Trp) and histidine (His) also prone to damage. The order of second-order rate constant for the various side-chains present in proteins and peptides was determined to be: Cys > Met >> Cystine ~ His ~ α-amino > Trp >Lys >> Tyr ~ Arg > Gln ~ Asn. These data show that disulfides (e.g. cystine) also react, but the values of k2 and the effect of structure and environment on these values have not been examined in detail. Disulfides are among the most important bonds in proteins and peptides, which are conventionally known as elements to stabilize proteins structure but many studies have categorized disulfides in two subproteomes. One group provides structural stabilizing and a second redox-active group which provides catalytic activity. Insight into disulfide oxidation e.g. the kinetics, specificity and mechanism of this oxidation by hypohalous acids is of pivotal importance, for example in understanding protein stability. The studies reported in Chapter 3 show that the second-order rate constant for the reaction of a variety of model disulfides with HOCl determined using stopped-flow instrumentation and competition kinetic methodology are highly variable. The k2 values determined for the reaction of HOCl with acyclic (linear) disulfides vary from 6×103 to 5×105 M– s–1. Further studies confirmed this high reactivity but showed that for cyclic disulfides the variation is ~ 10,000-fold. The second-order rate constants for reaction of HOCl with 5-membered ring species (e.g., lipoic acid, lipoamide) were determined as ~ 1×108 M–1 s–1. These data suggest that a conformation of a disulfide bond has a dramatic effect on its oxidation kinetics. The higher rate constant of the 5-membered ring species compared with 6-membered rings and acyclic species is particularly notable. α-Lipoic acid and α-lipoamide may, therefore, be significant targets for these oxidants. These suggest that if proteins contain strained disulfide bonds then these may be more susceptible to oxidation than thermodynamically stable disulfides. The high rate constant of these compounds makes them potential protective agents against damage to proteins with which they are associated. The kinetic values of two protein model compound with several disulfide bonds (insulin and α-lactalbumin) are significantly higher than for any of the model compounds with the exception of the 5-membered ring structures. The wide spectrum of products was reported to be generated from Cys reaction with hypohalous acids including disulfides (RSSR'); sulfenic (RSOH), sulfinic (RSO2H) or sulfonic (RSO3H) acids, sulfinamides (RS(O)NR'), sulfonamides (RS(O)2NR') and RS-NO species. In contrast to the abundance of data on Cys and Met, little is known about the oxidation products of cystine residues and particularly those in proteins. The studies in Chapter 4 assessed and identified disulfide bond oxidation products and kinetics of a fluorescently tagged model peptide (Naph-SS) on reaction with HOCl. Oxidation of Naph-SS with HOCl demonstrated the formation of disulfide-S-oxides, sulfenic, sulfinic and sulfonic acids as well as peptide cleavage between Trp and Arg. The reaction of the oxidized products with thiols (GSH, N-Ac-Cys) did not result in a repair but generated mixed disulfide products with Naph-SS and the cleaved peptide which may exacerbate the damage. This study was subsequently extended to two other compounds; a synthetic dimer of the tripeptide (GSH) with a disulfide bond between the Cys residues and the α-amino groups blocked with N-acetyl groups ((N-Ac)2GSSG) and the biologically relevant protein insulin. The primary oxidation products detected on reaction with HOCl and HOBr were the RS(O)SR and, the disulfide-S-dioxide, which have been characterized by mass spectrometry. Higher oxidation state products including sulfinic and sulfonic acids were characterized by LC-MS/MS. The studies reported in Chapter 6 investigated whether there is a possibility to visualize disulfide bond oxidation products by Raman spectroscopy. In a wide range of inflammatory diseases, MPO-derived oxidants are implicated. 3-chlorotyrosine is commonly used as a marker of HOCl formation which is a relatively minor product, but very specific. Analysis of HOCl-treated model peptides containing sulfur and amine groups by Raman spectroscopy showed dose-dependent changes in the wavelength and intensity of characteristic Raman bands assigned to disulfides (500–550 cm-1), and the formation of a new Raman absorption at ~ 2255 cm-1 which has been tentatively assigned to bond stretching of nitrile groups. The latter peak was detected in a dose-dependent manner on the HOCl treatment of Lys-containing protein and peptide models, N-acetyl-histidine and oxidized glutathione (GSSG). Thus HOCl-mediated oxidation of proteins and peptides modifies protein and peptide side-chains in a manner that can be readily detected by Raman spectroscopy in isolated systems. The absorption band attributed to C≡N stretching at ~ 2255 cm-1 is well removed from other features in the Raman spectra, making it a potential marker of HOCl-mediated protein oxidation, chloramine formation and decomposition. Overall the studies presented in this thesis are consistent with selective damage to the specific disulfides, such as in proteins, with this resulting in structural perturbation. Oxidation of protein disulfides by HOCl may alter the stability of protein structures and enhance unfolding by incorporation of oxygen atoms and, disulfide or backbone cleavage. This may be of particular relevance for proteins such as integrins and extracellular proteins, where cystine residues are abundant, and Cys and Met levels low.