Molecular Pharmacology, Vol 17, 225-232, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics

Mechanistic Aspects of the Hematin-Mediated Increases in Brain Monooxygenase Activities

¹ Department of Pharmacology, School of Medicine, University of Washington, Seattle, Washington 98195

Addition of micromolar concentrations of hematin to reaction vessels containing 9000g supernatant fractions of rabbit brain homogenates together with cofactors necessary for monooxygenation resulted in up to 70-fold increases in rates of benzo[a]pyrene hydroxylation relative to rates observed for incubations performed in the absence of hematin. Time course determinations revealed the hematin-independent activity to be linear with time for at least 2 hr while the hematin-dependent activity was nonlinear, following an upward hyperbolic course of product formation. Preincubation of samples in the presence of substrate and cofactors before the additions of hematin resulted in increased initial rates for the hematin-dependent reaction. However, preincubations of supernatant fractions with hematin alone did not result in increases in initial reaction rates. At equivalent heme concentrations, hemoglobin was approximately one-ninth as effective as hematin in eliciting the stimulatory response, whereas protoporphyrin IX, biliverdin, myoglobin, catalase, and FeCl₂ were essentially ineffective in producing enhanced rates of reaction. Hematin did not produce increases in metabolic rates when incubated with hepatic homogenates. Both hematin-dependent and hematin-independent activities were found to be inhibited by carbon monoxide, aniline, cytochrome c, 17-estradio1, 7,8-benzoflavone, butylated hydroxyanisole, and 2-mercaptoethanol. Increasing enzymatic activities by pretreating animals with phenobarbital, 3-methylcholanthrene, or Aroclor 1254 caused a decrease in the stimulatory capabilities of hematin in vitro. Hematin also produced increases in rates of oxidative metabolism of 7,12-dimethylbenz[a]anthracene and 17-estradiol, but not of N-2-fluorenylacetamide. Metabolic profiles obtained with high-pressure liquid chromatography and benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene as substrate illustrated that additions of hematin increased the quantity of individual metabolites produced without causing qualitative changes in the metabolic profiles.