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

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 alpha-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.