Solid-state photodegradation of bovine somatotropin (bovine growth hormone): Evidence for tryptophan-mediated photooxidation of disulfide bonds

Solid-state photodegradation of bovine somatotropin (bovine growth hormone): Evidence for tryptophan-mediated photooxidation of disulfide bonds
Published Online: 28 May 2003
Received: 12 December 2002; Revised: 25 February 2003; Accepted: 26 February 2003
Brian L. Miller 1, Michael J. Hageman 2, Thomas J. Thamann 2, Lorena B. Barr?n 1, Christian Sch?neich 1 *
Journal of Pharmaceutical Sciences
Volume 92, Issue 8
Wiley InterScience
1Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, Kansas 66047
2Research and Development, Pharmacia Corporation, Kalamazoo, Michigan 49007

email: Christian Sch?neich (schoneic@ukans.edu)
Keywords
photooxidation ? cystine ? tryptophan ? thiyL radicals ? bovine growth hormone
Abstract
Lyophilized recombinant bovine somatotropin (rbST; bovine growth hormone) is sensitive to photoinduced degradation. The underlying mechanisms of these processes are identified and presented. Lyophilized rbST was photolyzed with near-ultraviolet (UV) light between 305 and 410 nm, and the protein content was analyzed by various bioanalytical techniques, including tryptic mapping, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino acid analysis, and fluorescence, UV, Raman and Fourier transform infrared (FTIR) spectroscopy. The solid-state photodegradation of rbST by near-UV light exclusively targets the protein disulfide bonds. The reaction is initiated by photoionization of tryptophan (Trp) and one-electron reduction of the disulfide. However, in contrast to the behavior of other proteins in solution, rbST appears to undergo back electron transfer to restore Trp and yield a pair of cysteine (Cys) thiyL radicals, which add molecular oxygen and ultimately recombine to yield -disulfoxide, thiosulfinate, and/or thiosulfonate. Photodegradation is strictly dependent on the presence of molecular oxygen, but does not involve singlet oxygen. Between 0.4 and 10%, residual moisture levels do not affect the rate of photodegradation. Our results show a novel mechanism for Trp-mediated photodegradation of protein disulfide bonds via formation of a pair of thiyL radicals followed by addition of molecular oxygen. ? 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:1698-1709, 2003
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