Preparation and physicochemical properties of functional hemoprotein P450 from mammalian tissue microsomes

Abstract

Functional hemoprotein P450 particles were isolated from rabbit liver and pig and bovine adrenocortical microsomes by tryptic digestion in 0.1 M potassium-phosphate buffer (pH 7.0) containing 2 mM EDTA and 20% (v/v) glycerol. The optical and magnetic properties of the particles were studied at three widely different temperatures. The absolute absorption spectra of functional hemoprotein P450 of liver microsomes in the oxidized and reduced forms in the visible region had absorption peaks at 360, 414, 532 and 568 mμ in the oxidized form, at 416 and 555 mμ in the reduced form and at 450 and 555 mμ in the CO complex form at room temperature. The absolute absorption spectra of hemoprotein P450 in the visible region at the temperature of liquid N 2 had absorption peaks at 350, 414, 538 and 570 mμ in the oxidized form, at 415 and 550 mμ in the reduced form and at 447 and 550 mμ in the CO complex form. The electron paramagnetic resonance (EPR) spectrum of oxidized hemoprotein P450 at the temperatures of liquid N 2 and liquid H 2 showed a low spin form, and its g galues were identical with those of hemoprotein P450 of whole microsomes, whereas the EPR spectrum of hemoprotein P420 which had been highly purified by treatment with snake venom showed a pure high spin form at these temperatures. The same results were obtained with preparations of hemoprotein P450 from pig and bovine adrenocortical microsomes. The light absorption difference spectra in the visible region of the hemoprotein P450 complex with nitrosobenzene showed peaks at 452–453 and 554–558 mμ in the oxidized form and at 422, 455, 520 and 577 mμ in the reduced form. The absorption spectra of both forms changed with the pH. the absorption spectra of complexes of hemoprotein P450 with imidazole or triazole derivatives were also investigated. The absorption spectra of the reduced hemoprotein P450-triazole complex, unlike that of the complex of reduced hemoprotein P450 with imidazole, showed splitting of the Soret bands at 425 and 445 mμ, and these were pH dependent. On the other hand the Soret band of the oxidized hemoprotein P450-triazole complex showed neither splitting nor pH dependence. The unique structure of hemoprotein P450 is not formed only on reduction, for even the oxidized form has a unique structure. The EPR spectra of oxidized hemoprotein P450 complexes with nitrogenous ligands were studied. The optical and magnetic properties of hemoprotein P450 and its complexes with nitrogenous ligands were compared withi those of hemoprotein P420 and other hemoproteins.