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. 1986 Mar 1;234(2):399–403. doi: 10.1042/bj2340399

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

S P Wolff, R T Dean
PMCID: PMC1146578  PMID: 3718475

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Cockle S. M., Dean R. T. Distinct proteolytic mechanisms in serum-sufficient and serum-restricted fibroblasts. Transformed 3T3 cells fail to regulate proteolysis in relation to culture density only during serum-sufficiency. Biochem J. 1984 Jul 1;221(1):53–60. doi: 10.1042/bj2210053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Creeth J. M., Cooper B., Donald A. S., Clamp J. R. Studies of the limited degradation of mucus glycoproteins. The effect of dilute hydrogen peroxide. Biochem J. 1983 May 1;211(2):323–332. doi: 10.1042/bj2110323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dean R. T., Pollak J. K. Endogenous free radical generation may influence proteolysis in mitochondria. Biochem Biophys Res Commun. 1985 Feb 15;126(3):1082–1089. doi: 10.1016/0006-291x(85)90296-7. [DOI] [PubMed] [Google Scholar]
  4. Dean R. T., Roberts C. R., Forni L. G. Oxygen-centred free radicals can efficiently degrade the polypeptide of proteoglycans in whole cartilage. Biosci Rep. 1984 Dec;4(12):1017–1026. doi: 10.1007/BF01116694. [DOI] [PubMed] [Google Scholar]
  5. Dean R. T., Roberts C. R., Jessup W. Fragmentation of extracellular and intracellular polypeptides by free radicals. Prog Clin Biol Res. 1985;180:341–350. [PubMed] [Google Scholar]
  6. Fligiel S. E., Lee E. C., McCoy J. P., Johnson K. J., Varani J. Protein degradation following treatment with hydrogen peroxide. Am J Pathol. 1984 Jun;115(3):418–425. [PMC free article] [PubMed] [Google Scholar]
  7. Gutteridge J. M., Wilkins S. Copper salt-dependent hydroxyl radical formation. Damage to proteins acting as antioxidants. Biochim Biophys Acta. 1983 Aug 23;759(1-2):38–41. doi: 10.1016/0304-4165(83)90186-1. [DOI] [PubMed] [Google Scholar]
  8. Habeeb A. F. Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. Anal Biochem. 1966 Mar;14(3):328–336. doi: 10.1016/0003-2697(66)90275-2. [DOI] [PubMed] [Google Scholar]
  9. Halliwell B., Gutteridge J. M. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J. 1984 Apr 1;219(1):1–14. doi: 10.1042/bj2190001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jentoft N., Dearborn D. G. Labeling of proteins by reductive methylation using sodium cyanoborohydride. J Biol Chem. 1979 Jun 10;254(11):4359–4365. [PubMed] [Google Scholar]
  11. McCord J. M. Free radicals and inflammation: protection of synovial fluid by superoxide dismutase. Science. 1974 Aug 9;185(4150):529–531. doi: 10.1126/science.185.4150.529. [DOI] [PubMed] [Google Scholar]
  12. Monboisse J. C., Braquet P., Randoux A., Borel J. P. Non-enzymatic degradation of acid-soluble calf skin collagen by superoxide ion: protective effect of flavonoids. Biochem Pharmacol. 1983 Jan 1;32(1):53–58. doi: 10.1016/0006-2952(83)90651-2. [DOI] [PubMed] [Google Scholar]
  13. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  14. Rivett A. J., Roseman J. E., Oliver C. N., Levine R. L., Stadtman E. R. Covalent modification of proteins by mixed-function oxidation: recognition by intracellular proteases. Prog Clin Biol Res. 1985;180:317–328. [PubMed] [Google Scholar]
  15. Schuessler H., Schilling K. Oxygen effect in the radiolysis of proteins. Part 2. Bovine serum albumin. Int J Radiat Biol Relat Stud Phys Chem Med. 1984 Mar;45(3):267–281. doi: 10.1080/09553008414550381. [DOI] [PubMed] [Google Scholar]
  16. Shaklai N., Garlick R. L., Bunn H. F. Nonenzymatic glycosylation of human serum albumin alters its conformation and function. J Biol Chem. 1984 Mar 25;259(6):3812–3817. [PubMed] [Google Scholar]

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