Abstract

The relationship between the free radical-induced loss of lysozyme activity and the occurrence of oxidative modifications, evidenced as gain in DNPHreactive carbonyl groups (CO) and loss of tryptophan-associated fluorescence (TAF), was addressed in vitro.2,2'-azobis (2-amidino propane)(20 raM, AAPtt) and a mixture of Fe+2/Ascorbate (Fe+2/A, 0.1/25 mM) were used as peroxyl and oxygen-de rived free radical sources. Lysozyme incubated (2h, 37C) in the presence of AAPH underwent a 75% decrease in its activity, a concomitant 50% loss of TAF, and a 6-fold rise in CO levels. Such a change in CO levels, however, could account at the most for the inactivation of 6% of the enzyme molecules. Further suggesting a dissociation between CO formation and inactivation, the enzyme's activity and the TAF remained totally unaltered upon exposure of the protein to Fe+2/A in spite of an almost 5-fold rise in CO. The effects induced by either AAPH or Fe+2/A were all inhibitable by antioxi dant addition. Nevertheless, depending on the antioxidant used, we observed that the enzyme inactivation and TAF loss could be prevented or not, at short times (20 rain), with or without any effect of these compounds on CO accumulation. In (onclusion, our data support the concept that CO formation in proteins may or may not occur concomitantly with enzyme inactivation, and that both processes are not necessarily linked mechanistically. Fondecyt 1950258.