Oxidation of the sulfur-containing amino acids in recombinant human α1-antitrypsin

Abstract

Oxidation is one of the most prevalent forms of chemical modification, and the sulfur-containing amino acids, methionine and cysteine, are susceptible to modification by a wide array of oxidants. Aberrant oxidation reactions are of particular concern in biotechnology and medicine, where they can lead to protein inactivation or destabilization. Therefore, it is important to understand the factors that influence a protein's reactivity toward oxidants from both a bioprocessing and a biomedical perspective. This work characterizes oxidation of methionine and cysteine in recombinant human xcl-antitrypsin, a metastable protein that is a member of the serpin family of plasma protease inhibitors. Analytical techniques were developed to identify and quantify oxidation of each of al-antitrypsin's ten methionine residues, as well as its unpaired thiol, Cys232. It was found that the protein is not susceptible to oxidation during production in the E.coli cytoplasm, but does contain highly-reactive methionine and cysteine residues that are of considerable importance within the context of in vitro oxidation. At neutral pH, five methionines, Metl, Met226, Met242, Met351, and Met358, are reactive with hydrogen peroxide. With the exception of Metl, each of these residues is located in or near the protein's active site loop. Methionine oxidation studies revealed that the reactivity of these residues is dependent on environmental conditions, such as pH, that readily perturb the protein's metastable structure.

(cont.) Cys232 oxidation studies showed that oxidation of [alpha]l-antitrypsin's unpaired thiol does not lead to the formation of disulfide-linked aggregates, but rather sulfenic, sulfinic, and cysteic acids in successive steps. These species are rapidly formed in vitro as a consequence of Cys232's unusually low pKa of 6.86. Modulation of Cys232's ionization and reactivity could not be accomplished via low pH, however, due to acid-induced structural changes that enhance reactivity. In sum, analytical techniques were developed to study methionine and cysteine oxidation in [alpha]1-antitrypsin and it was found that elements of protein structure that have evolved for the physiological functioning of plasma protease inhibitors dictate oxidation susceptibility during bioprocessing and long-term storage. In the case of oxl-antitrypsin, and perhaps other proteins with similar structural biochemistries, unpaired-thiol oxidation, and not methionine oxidation, is the major contributor to oxidative degradation in vitro.