Oxidation of methionine residues in proteins of activated human neutrophils.

Abstract

A simple assay for the detection of 35S-labeled methionine sulfoxide residues in proteins is described. The assay, which is based on the ability of CNBr to react with methionine but not with methionine sulfoxide, requires the prelabeling of cellular proteins with [3S]methionine. The assay was used to study the extent of methionine oxidation in newly synthesized proteins of both activated and quiescent human neutrophils. In cells undergoing a phorbol 12-myristate 13-acetate-induced respiratory burst, about 66% of all methionine residues in newly synthesized proteins were oxidized to the sulfoxide derivative, as compared with 9% in cells not treated with the phorbol ester. In contrast, quantitation of methionine sulfoxide content in the total cellular protein by means of amino acid analysis showed that only 22% of all methionine residues were oxidized in activated cells as compared with 9% in quiescent cells. It is proposed that methionine residues in nascent polypeptide chains are more susceptible to oxidation than those in completed proteins.Methionine residues in proteins can readily be oxidized to the sulfoxide derivative in the presence of suitable oxidizing reagents, a reaction that can result in the inactivation of certain proteins (for reviews, see refs. 1 and 2). Despite indications that sulfoxidation of methionine may occur frequently under physiological conditions, the importance of this reaction remains largely unrecognized (3). The absence of a rapid facile assay for the detection of methionine sulfoxide [Met(O)] residues in proteins has hindered studies in this area. Because Met(O) is unstable to acid hydrolysis (4), long and tedious methods, involving alkaline hydrolysis or alkylation of methionine, are normally used (5). The present report describes a simple assay for the detection of Met(O) residues in proteins labeled with [3S]-methionine. The assay, which is based on the ability of CNBr to react with methionine but not with methionine sulfoxide (6), was used to examine the oxidation of methionine residues in human neutrophils. Neutrophils were chosen for these studies because of their ability to produce copious quantities of oxidizing reagents on activation. As part of their microbicidal response, neutrophils can be activated by a variety of agents such as chemotactic peptides, complement, insoluble particles, and others (7). The activated cells undergo a respiratory burst during which they produce oxidizing reagents such as superoxide anion (O°), hydrogen peroxide (H202), hydroxyl radicals, and hypochlorite ions (8-10), which are highly toxic to extracellular microorganisms and tissues (9, 11-13). These reagents can also inactivate chemotactic peptides and other polypeptides through selective oxidation of methionine residues (14-18). Recently, it has become apparent that the indiscriminate release of these oxy-radicals can damage the neutrophils themselves. Several studies have shown that the respiratory burst can lead to a decrease in neutrophil function through autooxidation (19)(20...

Proteins released from stimulated neutrophils contain very high levels of oxidized methionine

“…Generation of such high levels of oxidized Met in completed proteins by stimulated neutrophils is unique and has, so far, not been reported. Usually, Met residues are located within the hydrophobic interior of completed proteins where they seem to be more or less resistant to oxidation in contrast to Met residues in nascent polypeptide chains [6,12]. Quantitation of the Met(O) content in in- Table 1 Met ( portion of oxidized Met in the proteins released was found to be 28% (table 2).…”

“…In cells undergoing PMA-induced respiratory burst, 22% of all methionine residues in the total protein were oxidized to the sulfoxide. In contrast, about 66% of all methionine residues in proteins newly synthesized during stimulation were oxidized to the sulfoxide [6]. Newly synthesized, i.e.…”

“…Oxidation of Met in biological systems is observed during inflammation and ageing processes [5]. Fliss et al [6] analyzed the intracellular protein of neutrophils for oxidized Met. In cells undergoing PMA-induced respiratory burst, 22% of all methionine residues in the total protein were oxidized to the sulfoxide.…”

“…nascent polypeptide chains, were distinguished from completed proteins by pulse labeling with [35S]Met during incubation. It was concluded that Met residues in nascent polypeptide chains of neutrophils are more susceptible to oxidation than those in completed proteins [6].…”

Sulfur oxidation of free methionine by oxygen free radicals

“…Our suggestion that free radicals are responsible for methionine sulfoxide production from methionine is not completely new. Recent data published in [24] also suggest an oxidative role of oxygen free radicals generated by human neutrophils in the appearance of protein-bound methionine sulfoxide. Studies on the mechanism of the photo-oxidation of free amino acids showed unambiguously involvement of singlet oxygen ~O2 in this process [14].…”