Solid-State Photodegradation of Bovine Somatotropin (Bovine Growth Hormone): Evidence for Tryptophan-Mediated Photooxidation of Disulfide Bonds

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 410nm, 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 α-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