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

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.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemical Engineering.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2002

Author dc:creator

• Griffiths, Steven W

Advisor dc:contributor.advisor

• Charles L. Cooney.

• Chemical Engineering.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/8499

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/8499