Reactions of para-Substituted Nitrosobenzenes with Human Hemoglobin

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Abstract
Bio-monitoring the covalent binding of nitrosoarenes to the SH groups of human hemoglobin has been proposed at a reliable approach to get an integral parameter for exposure control and possibly risk assessment of persons exposed to aromatic amines and nitro compounds. Availability of nitrosoarenes to bind to the cysteine residues is greatly influenced by the competition of hemoglobin iron with nitrosoarenes. In contrast to earlier reports, we found that nitrosobenzene has a 14 fold higher affinity for "stripped" human hemoglobin than oxygen. The binding mode is similar to gaseous ligands and exhibits the same free energy of cooperation and sensitivity to heterotrophic effectors like inositol hexaphoshate. To elucidate the electronic influence of para substituents, 4-chloronitrosobenzene, 4-nitrosotoluene and 4-nitrosophenetole were tested. A linear free energy relationship was found for all equilibrium parameters within a reaction constant .rho. = 3, when using Hammett .**GRAPHIC**. contants. Similarly, the apparent second order rate constants for binding of para-substituted nitrosobenzenes to the cysteine residues (Cys.beta.93) in hemoglobin followed the Hammett relationship with lgk - lgk0 = 1.7 .times. .**GRAPHIC**. (r2 = 0.99). In case of 4-chloronitrosobenzene covalent binding proceeding biphasically and a "semimercaptal"-like intermediate was observed. The affinities for hemoglobin iron and for the SH groups were highest with 4-chloronitrosobenze and lowest with 4-nitrosophenetole. All nitrosobenzenes were capable to produce ferrihemoglobin. In the absence of oxygen, 4-chloronitrosobenzene hemoglobin decayed with formation of ferrihemoglobin. Presumably the nitroxide radical anion is formed as an intermediate which comproportionates into the azoxy derivative. It is assumed that the efficiency of the microscopic compartmentation of nitrosoarenes by binding to hemoglobin iron has important impacts on the toxicokinetics of these compounds.