No abstract

No 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+).

1. 2,2'-Azo-bis-amidinopropane (ABAP) thermal decomposition produces free radicals that initiate the lipid peroxidation of erythrocyte ghost membranes. 2. Addition of 6-n-propyl-2-thiouracil decreases the rate of the process, both by decreasing consumption of the natural antioxidants of the membranes and by direct interaction with the free radicals involved in the lipid peroxidation. 3. Peroxyl radicals produced in ABAP thermal decomposition inactivate lysozyme, horseradish peroxidase (HRP) and glucose oxidase, in that order. The number of enzyme molecules inactivated per radical introduced into the system increases with enzyme concentration. 4. Competitive studies employing mixtures of enzymes show that the order of reactivity of these enzymes towards the peroxyl radicals is the opposite to that obtained for the rate of enzyme inactivation. It is concluded that inactivation efficiency is determined mainly by the average number of free radicals that must react with an enzyme molecule to produce its inactivation, and that this number is directly related to the molecular weight of the enzyme.

The reactivity of a series of indole derivatives was assessed in the following systems: (i) oxidation of the indole derivatives induced by the thermolysis of 2,2'-azobis-(2-amidinopropane) (ABAP); (ii) oxidation of cumene induced by the thermolysis of 2,2'-azobis-(2-methyl propionitrile) (AIBN); (iii) lysozyme inactivation induced by the thermolysis of ABAP and (iv) brain homogenate autoxidation. In systems (ii) to (iv), addition of the indole derivatives decreases the rate of the process. The data obtained indicate that common factors (i.e., oxidation potential and presence of N-H bonds) control the reactivity of the indole derivatives in the four systems considered. However, in the brain homogenate autoxidation, hydrophobicity is an additional factor that affects the efficiency of antioxidants, as illustrated by Q1/2 values (the concentration of additive required to decrease the autoxidation rate to one half that observed in the absence of additive) of 0.1 mM and much greater than 8 mM for 3-methylindole and tryptophan, respectively.