Quantitative Determination by Esr of the Arylaminyl Free Radical During the Reaction of N,N,N',N'-Tetramethyl-P-Phenylenediamine with Oxyhemoglobin

Abstract

Aromatic amines with electron-donating substituents are directly activated by pure oxyhemoglobin with formation of ferrihemoglobin. Of these xenobiotics the N-alkylated p-phenylenediamines are particularly active. With N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) cooxidation with oxyhemoglobin was observed by ESR spectroscopy with formation of the arylaminyl free radical (TMPD ^+*). Since the radical is rapidly reduced by ferrohemoglobin, a catalytic cycle of ferrihemoglobin formation is sustained with initially very low steady-state concentrations of the radical, e.g. below 0.1%. Ferrihemoglobin is also able to oxidize TMPD to the radical, hence the steady-state concentration of TMPD^+* rises with increasing ferrihemoglobin. Radicals of the Wurster's type tend to disproportionate at high rates generating reactive quinonediiminium cations which oxidize and arylate cellular thiols like GSH and protein SH groups. Because the disproportionation rate depends on the square of the radical concentration, quenching of the radicals by ferrohemoglobin to protect cellular thiols will be effective as long as the capacity of the methemoglobin reductase system is not overwhelmed. The results indicate that erythrocytes may play a critical role in activation and detoxication of p-phenylenediamines.