Interaction of nitrobenzoates with haemoglobin in red blood cells and a haemolysate

Haemoglobin, either in the intact red blood cells or in their haemolysate, readily reacts with mono- and di-nitrobenzoates. For all the nitroaromatics considered, the rate of the process is faster in the haemolysate than in the whole red blood cell. At low (< 8 mM) concentrations, almost quantitative production of methaemoglobin is observed and the process follows second order kinetics. At higher concentrations, the kinetics become complex and other haemoglobin derivatives are produced. The bimolecular rate constants obtained at low substrate concentrations show little relationship to the nitroaromatic reduction potential. The data indicate that mono-nitrobenzoate derivatives are very active in oxidizing haemoglobin in in vitro erythrocyte suspensions, the activities being similar to that of 3,5-dinitrobenzoate. The measured reactivity follows the order m-nitrobenzoate > 3,5-dinitrobenzoate > p-nitrobenzoate > o-nitrobenzoate and the reactivity of all the compounds is considerably larger than that of nitrobenzene. The present results constitute the first kinetic data bearing on the reactivity of nitroaromatics with haemoglobin, both free and incorporated in the intact red cell. Furthermore, they indicate that the interaction of the nitroaromatics with haemoglobin, leading to total oxidation and transformation, in spite of the total disruption of the membrane, does not produce significant lipid-peroxidation, as measured by chemiluminescence emission, production of TBA reactive material and oxygen consumption.