Characterization of disulfide (cystine) oxidation by HOCl in a model peptide: Evidence for oxygen addition, disulfide bond cleavage and adduct formation with thiols

Highlights

• Disulfide bonds play a key role in stabilizing proteins by cross-linking secondary structure.

• Kinetic, mechanistic and product data are presented for HOCl-induced disulfide oxidation.

• Low HOCl levels give disulfide cleavage and oxy acids via reactive intermediates.

• The intermediates react with thiols to give adducts at the former disulfide bond.

• Disulfide bond oxidation provides a novel pathway to peptide glutathionylation.

Abstract

Disulfide bonds play a key role in stabilizing proteins by cross-linking secondary structures. Whilst many disulfides are effectively unreactive, it is increasingly clear that some disulfides are redox active, participate in enzymatic reactions and/or regulate protein function by allosteric mechanisms. Previously (Karimi et al., Sci. Rep. 2016, 6, 38752) we have shown that some disulfides react rapidly with biological oxidants due to favourable interactions with available lone-pairs of electrons. Here we present data from kinetic, mechanistic and product studies for HOCl-mediated oxidation of a protected nine-amino acid model peptide containing a N- to C-terminal disulfide bond. This peptide reacts with HOCl with k₂ 1.8 × 10⁶ M⁻¹ s⁻¹, similar to other highly-reactive disulfide-containing compounds. With low oxidant excesses, oxidation yields multiple oxidation products from the disulfide, with reaction predominating at the N-terminal Cys to give sulfenic, sulfinic and sulfonic acids, and disulfide bond cleavage. Limited oxidation occurs, with higher oxidant excesses, at Trp and His residues to give mono- and di- (for Trp) oxygenated products. Site-specific backbone cleavage also occurs between Arg and Trp, probably via initial side-chain modification. Treatment of the previously-oxidised peptide with thiols (GSH, N-Ac-Cys), results in adduction of the thiol to the oxidised peptide, with this occurring at the original disulfide bond. This gives an open-chain peptide, and a new mixed disulfide containing GSH or N-Ac-Cys as determined by mass spectrometry. Disulfide bond oxidation may therefore markedly alter the structure, activity and function of disulfide-containing proteins, and provides a potential mechanism for protein glutathionylation.

Introduction

Disulfide bonds (cystine residues) are formed in proteins of mammalian cells as they mature in the endoplasmic reticulum, Golgi complex, post-Golgi complex vesicles and mitochondrial intermembrane space [1]. Although disulfides appear to play a key role in many proteins as stabilizing elements that maintain protein structure by cross-linking secondary structures [2,3], a number of studies have provided evidence for a second disulfide proteome. This second group are redox active and catalyze enzymatic reactions or regulate protein function by allosteric reactivity. It is, therefore, clear that disulfide bonds play important additional roles in addition to being (intra- or inter-molecular) structural “staples” that connect separate parts of a protein's structure [4]. Strain and conformational constraints imposed on a disulfide bridge by the surrounding protein are believed to influence the redox potential of disulfide/thiol systems and hence their reactivity [2,3].

The redox balance of cells and tissues is dependent on whether, and to what extent, cells are exposed to endogenous or exogenous stimuli that generate oxidant species. In many cells, electron leakage from the electron transport chains of mitochondria (and also the endoplasmic reticulum, Golgi and plasma membranes) is a major sources of superoxide radicals (O2−.), and hydrogen peroxide (H2O2) formed via O2−. dismutation [5]. At sites of inflammation, activated leukocytes (e.g. neutrophils, monocytes and macrophages) generate multiple powerful oxidants including peroxynitrous acid (ONOOH) and hypohalous acids (e.g. hypochlorous acid, HOCl). The former is formed from diffusion-controlled reaction of O2−. with NO., generated by NADPH oxidases and nitric oxide synthases respectively [6,7]. HOCl, and related hypohalous acids (HOBr, HOI, HOSCN) are formed via the action of the heme enzyme myeloperoxidase (MPO) in the presence of H2O2 and halide and pseudohalide ions [8]. MPO is released in to phagolysosomes, and also extracellularly by stimulated neutrophils, monocytes, and some tissue macrophages. Elevated levels of MPO, and the oxidants it generates, have been associated with host tissue damage at sites of inflammation, and in multiple human pathologies (reviewed [8,9]).

HOCl is a powerful two-electron oxidant and reacts rapidly with many biological targets [8,9]. Proteins are favoured targets due to their high abundance (both inside and external to cells), and the high rate constants for reaction of HOCl with particular amino acid side-chains [10]. Sulfur-containing amino acids (cysteine (Cys), methionine (Met) and cystine) are favoured targets, with the apparent second order rate constants k₂, for reaction of HOCl with the free amino acids being in the range 10⁸–10⁵ M⁻¹ s⁻¹, and decreasing in the order Cys > Met > cystine [[10], [11], [12]]. These data indicate that within cells, where there are high levels of Cys residues (both on proteins, and in the tripeptide glutathione, GSH), these will be major targets. External to cells, where the environment is typically more oxidizing, cystine residues predominate over Cys. This is exemplified by the much higher levels of disulfide (cystine) bonds in serum proteins (e.g. human serum albumin: 17 disulfides and one Cys), many extracellular matrix proteins [e.g. the epidermal growth factor (EGF) domains of laminin and fibronectin], integrins involved in cell binding, mitogenic and developmental proteins (e.g. Notch and Delta), blood clotting factors (Factors VII, IX, X) and receptors (e.g. the low-density lipoprotein receptor, epidermal growth factor receptor, insulin receptor) [13,14].

We have shown that k₂ for reaction of disulfide bonds with HOCl, and also other hypohalous acids, vary by up to 10,000-fold (~10⁴ to ~10⁸ M⁻¹ s⁻¹) with the environment and disulfide structure [12]. In some proteins, high rate constants have also been reported, consistent with the presence of particularly reactive disulfides ([12] L. Carroll et al., unpublished data). Two factors are responsible for this enhanced reactivity: stabilization of the electron-deficient reaction centre by suitably-placed remote electron pairs (e.g. carboxylates, alcohols and neutral amines) and favourable overlap with the lone pairs of the neighbouring sulfur atom in the disulfide bond [12].

Previous studies on model disulfides indicate that mono-oxygenated thiosulfinates RSS(=O)R’ (also known as disulfide-S-monoxides) are the initial products formed by H₂O₂, peracids and HOCl [[15], [16], [17], [18], [19], [20]]. Thiosulfinates typically have relatively modest half-lives (minutes – hours) in solution, and can undergo further reactions resulting in cleavage of the disulfide bond [17,19,21,22]. The mechanism by which this occurs remains to be established. The formation of thiosulfinates on proteins is less well characterized [17,21], though similar reactions of such species might be predicted to have major impacts on protein structure and function. Such reactions may be a major factor in loss of activity of protein-based pharmaceuticals [23], and contribute to the accumulation of dysfunctional, mis-folded or aggregated proteins during human pathologies (e.g. Ref. [24,25]).

The aim of the studies reported here was to investigate disulfide oxidation by HOCl, and the mechanism and consequences of this process. Studies were carried out using a nine amino acid peptide (N-Ac-Cys-Nle-Arg-His-(D)2-Nal-Arg-Trp-Gly-Cys-NH₂; Fig. 1; referred to as Naph-SS from hereon) containing a fluorescent a 2-naphthylated Ala residue; (D)2-Nal) and a disulfide-bond between Cys1 and Cys9. This peptide was chosen for these studies as it is readily available at high purity, reacts rapidly with HOCl (see below), has a relatively long elution time on reverse phase chromatogrsphy columns (allowing ready detection of more polar, earlier eluting, products), contains no Cys or Met residues and a blocked N-terminal amine, that would otherwise by major alternative targets for HOCl [10], and can be readily detected as a result of the strong UV and fluorescent properties of the (unreactive) (D)2-Nal residue.