Efficient Peroxide-Mediated Oxidative Refolding of a Protein at Physiological pH and Implications for Oxidative Folding in the Endoplasmic Reticulum
- 1 May 2009
- journal article
- research article
- Published by Mary Ann Liebert Inc in Antioxidants and Redox Signaling
- Vol. 11 (5), 963-970
- https://doi.org/10.1089/ars.2008.2326
Abstract
The majority of secreted and outer membrane eukaryotic proteins contain disulfide bonds, formed by complex interdependent pathways in the endoplasmic reticulum. The current model for the major route of disulfide formation is the regulated flow of oxidizing equivalents from molecular oxygen to the membrane-associated enzyme Ero1 to protein disulfide isomerase, and hence to substrate proteins. One molecule of hydrogen peroxide is produced by Ero1 per disulfide bond made. This peroxide is usually considered to be a dangerous by-product. Here we show that peroxide, added to a refolding buffer or generated enzymatically in situ, results in the efficient refolding of a model protein to the native state. At pH 7.0, the kinetics of obtaining the native folded state are more efficient using peroxide than by the use of a glutathione redox buffer. Disulfide bond formation by peroxide is kinetically favored over oxidation of cysteine to cysteine sulfinic acid and over the oxidation of other amino acids in the proteins such as methionine. Hence, unless peroxides are added in excess, oxidative damage to the folding protein is minimal. Our results offer insights into potential mechanisms for disulfide bond formation in vivo. Antioxid. Redox Signal. 11, 963–970.Keywords
This publication has 43 references indexed in Scilit:
- Low reduction potential of Ero1α regulatory disulphides ensures tight control of substrate oxidationThe EMBO Journal, 2008
- A novel disulphide switch mechanism in Ero1α balances ER oxidation in human cellsThe EMBO Journal, 2008
- Protein Sulfenation as a Redox SensorMolecular & Cellular Proteomics, 2007
- Intracellular catalysis of disulfide bond formation by the human sulfhydryl oxidase, QSOX1Biochemical Journal, 2007
- Managing and exploiting stress in the antibody factoryFEBS Letters, 2007
- Detection and Mapping of Widespread Intermolecular Protein Disulfide Formation during Cardiac Oxidative Stress Using Proteomics with Diagonal ElectrophoresisPublished by Elsevier BV ,2004
- Two Conserved Cysteine Triads in Human Ero1α Cooperate for Efficient Disulfide Bond Formation in the Endoplasmic ReticulumPublished by Elsevier BV ,2004
- What the papers say: Protein folding pathways determined using disulphide bondsBioEssays, 1992
- Selective Oxidation of Methionine Residues in Kunitz-Type Protease InhibitorsBiological Chemistry Hoppe-Seyler, 1989
- Principles that Govern the Folding of Protein ChainsScience, 1973