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

Abstract

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 Cu2+ and Fe3+, 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.

Biochemistry and pathology of radical-mediated protein oxidation

“…These observations have been confirmed and extended [24,99,100,110,117]. HOd and O # − d act synergistically [99], possibly as a result of additional decay pathways for the α-carbon peroxyl radicals by reaction with O # − d\HOOd The reasons for selective cleavage are not entirely clear ; they may include differences in accessibility of sites to the attacking species, the stability of the protein radicals formed, and the occurrence of radical transfer reactions.…”

“…Thus BSA exposed to HOd (from radiolysis) in the presence of O # yields only small amounts of molecules of changed size, as detected by nondenaturing HPLC gel filtration, even though dose-dependent generation of smaller fragments can be seen by SDS\PAGE [99,100]. In the absence of O # , HOd causes substantial crosslinking.…”

“…Like most partially denatured proteins, modestly oxidized proteins are usually more sensitive to proteolytic attack by most proteinases [39,99,117], whereas heavily oxidized proteins generally show decreased susceptibility [101,117]. The latter may depend on local chemical and structural changes rendering parts of the molecules poorly digestible [170], in agreement with the fact that certain aldehyde derivatives of proteins can be less susceptible to proteolysis than the parent protein ; for example 4-HNE-(but not malondialdehyde-) modified apoB in LDL [171].…”

Peroxynitrite Increases the Degradation of Aconitase and Other Cellular Proteins by Proteasome

“…A close relationship between protein oxidation and susceptibility to proteolysis has been well demonstrated in a variety of biological systems (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). A series of publications have demonstrated the enhanced degradation of proteins oxidized by exposure to the superoxide anion radical (O 2 .…”

“…A series of publications have demonstrated the enhanced degradation of proteins oxidized by exposure to the superoxide anion radical (O 2 . ), to hydrogen peroxide (H 2 O 2 ), or to the hydroxyl radical ( ⅐ OH), in isolated systems as well as in various cell types (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). These studies were performed in erythrocytes and reticulocytes of various species (3,6), in Escherichia coli (7,9), in rat muscles in vitro (1,4), in primary cultures of rat hepatocytes (13,14), and recently in dividing cell lines (15,16).…”