Oxidative Modification of Proteins: Oxidation of Tryptophan and Production of Dityrosine in Purified Proteins Using Fenton's System

E. E. Dubinina^1*, S. V. Gavrovskaya¹, E. V. Kuzmich¹, N. V. Leonova¹, M. G. Morozova¹, S. V. Kovrugina¹, and T. A. Smirnova²

¹Bekhterev Psychoneurological Research Institute, ul. Bekhtereva 3, St. Petersburg, 193019 Russia; fax: (812) 567-7127; E-mail: spbinstb@infopro.spb.su

²Mechnikov St. Petersburg State Medical Academy, Piskarevskii pr. 47, St. Petersburg, 195067 Russia

^* To whom correspondence should be addressed.

Received February 19, 2001; Revision received May 30, 2001

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Specific features of metal-catalyzed oxidation (MCO) of purified proteins (human serum albumin and human erythrocyte superoxide dismutase) were analyzed by the oxidation level of tryptophan and tyrosine. The production of dityrosine cross-links and the oxidation of tryptophan residues were recorded by fluorescence. The degree of oxidative modification of the amino acid residues of the proteins depended on the concentration of the Fenton's medium components and on the incubation time. These changes were different in different proteins. By electrophoresis and gel-permeation chromatography, changes in the superoxide dismutase structure are shown to be caused by oxidative modification of the enzyme and to be accompanied by a decrease in its activity. Findings with OH^* scavengers (mannitol and ethanol) suggest that oxidative modification of the proteins in Fenton's medium should be associated not only with hydroxyl radical but also with ferryl and perferryl ions and with the radical CO₃^-*.

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KEY WORDS: oxidative modification of proteins, dityrosine cross-links, superoxide dismutase, human serum albumin, hydroxyl radical, hydrogen peroxide, radical scavengers, CO₃^-*

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