Effect of Additives on the Inactivation of Lysozyme Mediated by Free Radicals Produced in the Thermolysis of 2, 2-Azo-Bis-(2-Amidinopropane)

Abstract

The inactivation of lysozyme caused by the radicals produced by thermolysis of 2,2'-azo-bis-2-amidinopropane can be prevented by the addition of different compounds that can react with the damaging free radicals. Compounds of high reactivity (propyl gallate, Trolox, cysteine, albumin, ascorbate, and NADH) afford almost total protection until their consumption, resulting in well-defined induction times. The number of radicals trapped by each additive molecule consumed ranges from 3 (propyl gallate) to 0.12 (cysteine). This last value is indicative of chain oxidation of the inhibitor. Uric acid is able to trap nearly 2.2 radicals per added molecule, but even at large (200 microM) concentrations, a residual inactivation of the enzyme is observed, which may be caused by urate-derived radicals. Compounds of lower reactivity (tryptophan, Tempol, hydroquinone, desferrioxamine, diethylhydroxylamine, methionine, histidine, NAD+ and tyrosine) only partially decrease the lysozyme inactivation rates. For these compounds, we calculated the concentration necessary to reduce the enzyme inactivation rate to one half of that observed in the absence of additives. These concentrations range from 9 microM (tryptophan and Tempol) to 5 mM (NAD+).