Amino acid free radicals in oxidised metmyoglobin

Abstract

1. 1.|A technique is described for distinguishing by electron spin resonance (ESR) spectrometry between aromatic and aliphatic amino acids. It is employed to determine the nature of the free radicals which are formed when metmyoglobin or methaemoglobin is oxidized by H 2O 2. The results obtained strongly suggest that the first free radical produced is that of a phenylalanine or histidine residue close to the porphyrin on the distal side, and that the free radical site is rapidly transferred to a tyrosine residue. 2. 2.|Ultraviolet absorption spectrophotometry shows that in the presence of excess H 2O 2 some of the aromatic units are converted to DOPA and DOPAquinonelike residues, but the two tryptophan units are not quick to oxidize. 3. 3.|Horse methaemoglobin behaves qualitatively like horse metmyoglobin during oxidation but the free radicals are destroyed more rapidly. Whale metmyoglobin differs significantly from horse metmyoglobin; the differences are probably explainable in terms of the protruding tyrosine residue in the former. 4. 4.|The preservation of myoglobin against oxidation damage in vivo is attributed to the existence of a means of transferring free radical sites from the vicinity of the haem to a tyrosine residue which is accessible to external reducing agents. 5. 5.|Under the specific conditions used for generation of free radical sites in individual amino acids and in peptides the greatest ease of formation and stability are observed in the radicals from tyrosine, tryptophan and the products of their hydroxylation. ESR signals due to hydroxy-aromatic radicals display a strong tendency to saturate which is increased by further hydroxylation. As first formed the phenylalanine radical does not give a saturated signal, but in the presence of excess H 2O 2 it undergoes further oxidation and is therefore recognized as aromatic by the saturated signal of the final product.