The action of iron on amino acid and protein peroxides.

Abstract

Protein oxidation by agents such as oxygen free radicals leads to changes in their stucture and function. Depending on the conditions of the oxidation, proteins can undergo chain scission and crosslinking, loss of amino acids, increase in susceptibility to proteolysis and heat denaturation and loss of biological activity [l-71. We have recently reported that, in addition to such alterations, some proteins exposed to free radicals in presence of oxygen acquire chemical reactivity characteristic of the hydroperoxide group [S]. Tests with amino acids showed that only Val, leu, ile, pro, glu and lys could be peroxidised with high efficiency, although a range of others were also susceptible to varying smaller extent. Since peroxides are potential sources of free radicals, we tested the ability of the oxidised proteins and amino acids to generate such species. Bovine serum albumin, lysozyme and the 20 common amino acids were peroxidised by irradiation with y rays. Hydrogen peroxide generated at the same time was removed with catalase and the peroxide concentrations were measured iodometrically [8]. The peroxides were decomposed by FE(I1)-EDTA in presence of the spin trap 5.5-dimethyl-1-pyrroline N-oxide (DMPO) and any ESR signals recorded on a Varian E4 spectrometer. Of the 20 amino acids tested, only those known to be susceptible to peroxidation gave ESR signals. When the oxidised amino acids or proteins were exposed to conditions which removed most of the peroxide groups (heat or NaBH4) before exposure to the chelated iron, the ESR signals were reduced in intensity or eliminated. Typical amino acid and protein ESR spectra show two overlapping triplets of equal intensities, with splitting constants of aN = 16 and a(j = 24G (Fig). The results confirm the formation of mainly alkoxyl, superoxide, carbon dioxyl and some unidentified carbon centred free radicals from the decomposing peroxides [9].