Molecular Pharmacology, Vol 16, 1046-1058, Copyright © 1979 by the American Society for Pharmacology and Experimental Therapeutics

Characterization of the Interaction of the Aromatic Hydrocarbons Benzene and Toluene with Human Hemoglobin

¹ Department of Pharmacology, Northwestern University Medical and Dental Schools, Chicago, Illinois 60611

The longitudinal relaxation rates, T₁^-1, and transverse relaxation rates, T₂^-1, were determined for the protons of benzene and the methyl and phenyl protons of toluene in aqueous media in the presence of varying concentrations of human ferrihemoglobin. The T₁^-1 relaxation rate of the protons of 18.8 mM benzene and the methyl and phenyl protons of 8.2 mM toluene increased from 0.065 sec^-1, 0.083 sec^-1 and 0.14 sec^-1 in aqueous medium in the absence of hemeprotein to 2.5 sec^-1, 5.6 sec^-1, and 5.3 sec^-1, respectively, at 140 µM human ferrihemoglobin. Similar increases were also observed in the transverse relaxation rates with values of T₂^-1 for the protons of benzene and the methyl and phenyl protons of toluene increasing from 1.1 sec^-1, 2.7 sec^-1 and 2.3 sec^-1 in the absence of hemeprotein to 3.2 sec^-1, 6.7 sec^-1, and 6.3 sec^-1 in the presence of 140 µM ferrihemoglobin. Formation of cyanoferrihemoglobin in situ, which changed the paramagnetic spin state of the heme iron atom from S = [unknown] to S = , decreased the T₁^-1 values for the protons of benzene and the methyl and phenyl protons of toluene. For example, the methyl and phenyl proton T₁ relaxation rates of toluene decreased from 5.6 sec^-1 and 5.3 sec^-1 in the presence of 140 µM ferrihemoglobin to 2.0 sec^-1 and 2.1 sec^-1, respectively, in the presence of 140 µM cyanoferrihemoglobin. In contrast, formation of fluoroferrihemoglobin in situ, which enhanced the paramagnetic effect, increased the T₁ relaxation rates for the protons of benzene and the methyl and phenyl protons of toluene in comparison with an equal concentration of ferrihemoglobin. For example, T₁^-1 for the methyl and phenyl protons of toluene increased dramatically from 3.8 sec^-1 and 3.6 sec^-1 at 91 µM ferrihemoglobin to 7.7 sec^-1 and 13.0 sec^-1 in the presence of 91 µM fluoroferrihemoglobin. The in situ formation of fluoroferrihemoglobin produced dramatic changes in the relative relaxation rates of the phenyl and methyl protons of toluene with methyl > phenyl. In all cases carbonmonoxyferrohemoglobin, the diamagnetic form of the hemeprotein, was used to determine the paramagnetic contributions (T_11p^-1, T_2p^-1) to the observed T₁ and T₂ relaxation rate increases. T₁^-1 for the protons of benzene and the methyl and phenyl protons of toluene decreased from 2.4 sec^-1, 5.6 sec^-1 and 5.3 sec^-1 in the presence of 140 µM human ferrihemoglobin to 0.64 sec^-1, 1.4 sec^-1, and 1.4 sec^-1, respectively, in the presence of 140 µM carbonmonoxyferrohemoglobin. The relaxation rates of the protons of benzene and the methyl and phenyl protons of toluene in the presence of cyanoferrihemoglobin were significantly greater than those observed in the presence of an identical concentration of carbonmonoxyferrohemoglobin, suggesting that these aromatic ligands are not displaced by cyanide but continue to interact with cyanoferrihemoglobin in proximity to the paramagnetic center. T₂^-1 changes for the protons of benzene and the methyl and phenyl protons of toluene in the presence of cyano- or fluoroferrihemoglobin or carbonmonoxyferrohemoglobin agreed qualitatively with those observed in the longitudinal relaxation rate, T₁^-1. Variable temperature experiments confirmed that exchange between free ligand and the hemeprotein-complexed ligand occurred in the fast exchange region of the variable temperature profile. Although both paramagnetic and diamagnetic components contribute to the observed enhancement of relaxation rate the paramagnetic contribution is approximately an order of magnitude greater. Thus these results support an interaction between benzene or toluene and human hemoglobin and show that this interaction occurs at or in proximity to the heme iron atom.

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ACKNOWLEDGMENT The authors wish to express their gratitude to Drs. P. H. Stern and K. P. Vatsis for critical review of this manuscript.