Fragmentation of proteins by free radicals and its effect on their susceptibility to enzymic hydrolysis

Defined radical species generated radiolytically were allowed to attack proteins in solution. The hydroxyl radical (OH^.) in the presence of O₂ degraded bovine serum albumin (BSA) to specific fragments detectable by SDS/polyacrylamide-gel electrophoresis; fragmentation was not obvious when the products were analysed by h.p.l.c. In the absence of O₂ the OH^. cross-linked the protein with bonds stable to SDS and reducing conditions. The superoxide (O₂^-.) and hydroperoxyl (HO₂^.) radicals were virtually inactive in these respects, as were several other peroxyl radicals. Fragmentation and cross-linking could also be observed when a mixture of biosynthetically/labelled cellular proteins was used as substrate. Carbonyl and amino groups were generated during the reaction of OH^. with BSA in the presence of O₂. Changes in fluorescence during OH^. attack in the absence of O₂ revealed both loss of tryptophan and changes in conformation during OH^. attack in the presence of O₂. Increased susceptibility to enzymic proteolysis was observed when BSA was attacked by most radical systems, with the sole exception of O₂^-.. The transition-metal cations Ca²⁺ and Fe²⁺, in the presence of H₂O₂, could also fragment BSA. The reactions were inhibited by EDTA, or by desferal and diethylenetriaminepenta-acetic acid ('DETAPAC') respectively. The increased susceptibility to enzymic hydrolysis of radical-damaged proteins may have biological significance.