Reactions of 4-dimethylaminophenol with hemoglobin, and autoxidation of 4-dimethylaminophenol

Abstract

The reactions between 4-dimethylaminophenol and hemoglobin were studied with 4-dimethylaminophenol 14C-labelled either in the methyl groups or in C 1 of the ring. In the absence of oxygen 4-dimethylaminophenol was stable in red cell suspensions or hemoglobin solutions. In the presence of oxygen oxyhemoglobin rapidly oxidized 4-dimethylaminophenol. The following reaction products were found in incubates of 4-dimethylaminophenol with red cells or hemoglobin: ferrihemoglobin, formaldehyde, dimethylamine, and hemoglobin with derivatives of 4-dimethylaminophenol covalently bound to its protein moiety. 4-Dimethylaminophenol catalytically transferred electrons from ferrohemoglobin to oxygen. It was oxidized by oxyhemoglobin, and oxidized 4-dimethylaminophenol was reduced to 4-dimethylaminophenol by ferrohemoglobin with formation of ferrihemoglobin. Hydrolysis of oxidized 4-dimethylaminophenol, N, N-dimethylquinonimine, and its covalent binding to globin limited the catalytic ferrihemoglobin formation by 4-dimethylaminophenol to an average between 50 and 100 electron transfers per molecule of 4-dimethylaminophenol, when 4-dimethylaminophenol concentration was low and hemoglobin concentration was high. Since 4-dimethylaminophenol reduced ferrihemoglobin to ferrohemoglobin, though more slowly than the catalytic cycle produced it, the increase in ferrihemoglobin content does not indicate the amount of ferrihemoglobin produced. In red cell suspensions at 37° 4-dimethylaminophenol, 0.58 m M, disappeared in 10 min, but dimethylamine continued to be formed, obviously from protein-bound derivative(s) of 4-dimethylaminophenol. The rate of autoxidation of 4-dimethylaminophenol was found to be much lower that the rate of oxidation of 4-dimethylaminophenol by oxyhemoglobin. After autoxidation of 4-dimethylaminophenol several products were isolated and identified which were not detected in incubates of 4-dimethylaminophenol with oxyhemoglobin, namely hydroquinone, 4-methylaminophenol, 4-aminophenol, 2-dimethylamino-1, 4-benzoquinone, a purple and a yellow dye. Nuclear magnetic resonance (NMR), mass spectroscopy, and synthesis from 1,4-benzoquinone and 4-methylaminophenol proved the purple dye to be 2-( N- methyl- N-( p-hydroxyphenyl)-amino-1,4-benzoquinone. The structure of the yellow dye, which is produced also by oxidation of the purple dye with hydrogen peroxide, was not proved unequivocally. IR, NMR spectra and the product of hydrogenation with Pd-charcoal and acetylation showed the compound to be an epoxide of 2-( N-methyl- N-( p-hydroxyphenyl)-amino)-benzoquinone.