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+).

Inhibition of protein hydroperoxide formation by protein thiols

“…While these data confirmed other studies showing peroxyl radical scavenging by cysteine, 13 the data did not prove that protein-bound thiols would have the same properties. Though BSA contains a free thiol residue, DTNB thiol analysis showed this residue was largely oxidised in the samples obtained from the supplier.…”

“…The sulphonyl radicals could react with further cysteine thiol to generate sulphonic acid and more thiyl radical. 12,13 It is intriguing that we have observed the same type of reaction occurring with the protein-bound thiols. Even though the thiol is fixed to a large protein chain, the resulting thiyl radicals can still interact to form a chain reaction.…”

“…Radical scavenging by cysteine has previously been reported to cause a thiol-mediated chain reaction. 12,13 Studies on cysteine protection of lysozyme activity from AAPH-mediated inactivation calculated a chain length for cysteine oxidation of eight, 13 while an earlier study using just AAPH and cysteine calculated a chain length of between 4-5. 12 The hypothesised reaction involves peroxyl scavenging by cysteine to form the thiyl radical (RS ) which will react with oxygen to give the sulphonyl radical (RSO 2 ).…”

“…12,13 EPR studies have suggested the presence of a free thiol in BSA can reduce Fe 2+ -EDTA/H 2 O 2 -generated hydroxyl radical-mediated protein damage by acting as a radical sink. 14 In this study, we have investigated the ability of protein thiols to inhibit the formation of protein hydroperoxide during peroxyl and hydroxyl mediated oxidation.…”

Reaction of 5-Aminosalicylic Acid with Peroxyl Radicals: Protection and Recovery by Ascorbic Acid and Amino Acids

“…For these two AA, a relatively similar reactivity was previously seen when tested in the protection of lysozyme against its inactivation promoted by peroxyl radicals (17) and in their reaction against ABTS radical cation (18). Interestingly, the present study shows that in addition to protecting 5-ASA against peroxyl radicals, Cys also has the ability to partially regenerate 5-ASA from its oxidation products (quinone-imine).…”