Abstract
Phenylhydrazine interacted with oxidized and reduced cytochrome P-450 of rat liver microsomes to produce binding difference spectra typical of many nitrogenous compounds. The phenylhydrazine-induced difference spectrum observed with oxidized microsomal cytochrome P-450 was converted, in a time-dependent process, to yield a new spectral intermediate with an absorbance maximum around 480 nm. The time required to form this new phenylhydrazine-induced spectral intermediate was decreased from hours to minutes when either NADPH or NADH was added to the reaction mixture. Phenyldiazene generated by addition of the decarboxylation product of methyl phenyldiazenecarboxylate or by addition of potassium ferricyanide and phenylhydrazine (2:1 molar equivalents) instantly formed the new spectral intermediate. This suggests that phenyldiazene is formed during the NADPH-dependent reaction. The appearance of the new spectral intermediate occurred concomitant with the loss of CO-reactive cytochrome P-450 (less than 90%) and loss of absorbance at 418 nm. The interpretation of the optical spectral changes was supported by a loss of the low spin signals characteristic of oxidized cytochrome P-450 as determined by electron paramagnetic resonance spectroscopy. The loss of CO-reactive cytochrome P-450 apparently resulted from the formation of a binary complex of phenyldiazene and the heme of oxidized cytochrome P-450 giving rise to the 480 nm spectral intermediate. In addition, the diazene-bound heme of cytochrome P-450 apparently was modified irreversibly in the presence of oxygen. The effects observed with phenylhydrazine could be produced to a lesser degree by other hydrazine derivatives. The possible role of phenylhydrazine as a new type of suicide substrate is discussed.
Highlights
Phenylhydrazine interacted with oxidized and reduced cytochrome P-450 of rat liver microsomes to produce binding difference spectra typicalofmany nitrogenous compounds.The phenylhydrazine-induced differencespectrumobserved with oxidizedmicrosomal cytochrome P-450 was converted, in a time-denm. pendent process, t o yield a new spectral intermediate with an absorbance maximum aroun4d80 The time required to form this new phenylhydrazine-induced spectral intermediatewas decreased from hours to minutes when either NADPH o r NADH w a s added t o the reaction mixture
1-Acetyl-2-phenylhydrazinewas purchased from Matheson, Coleman, and Bell, Cincinnati, OH; hydrazine dihydrochloride was obtained from American Scientific Products, McGaw Park, IL. l-Methyl-1phenylhydrazine was obtained from Eastman Kodak, methyl phenyldiazenecarboxylate and p-chloromercuribenzoate were purchased from Calbiochem, and disodiumtetraacetatewas obtained from J
Binding Spectraof Phenylhydrazine to Oxidized and Reduced Cytochrome P-450-Fig. 1 shows the spectral changes which occurred when phenylhydrazine was added to a suspen
Summary
Phenylhydrazine interacted with oxidized and reduced cytochrome P-450 of rat liver microsomes to produce binding difference spectra typicalofmany nitrogenous compounds.The phenylhydrazine-induced differencespectrumobserved with oxidizedmicrosomal cytochrome P-450 was converted, in a time-denm. pendent process, t o yield a new spectral intermediate with an absorbance maximum aroun4d80 The time required to form this new phenylhydrazine-induced spectral intermediatewas decreased from hours to minutes when either NADPH o r NADH w a s added t o the reaction mixture. Phenyldiazene generated by addition of the decarboxylation product of methyl phenyldiazenecarboxylate or by addition of potassium ferricyanide and phenylhydrazine (2:l molar equivalents) instantly formed the new spectral intermediate. This suggests that phenyldiazene is formed duringthe NADPH-dependent reaction. The reaction of phenylhydrazine and other hemolytic agents with hemoglobin generates hydrogen peroxide and concomitantly destroys hemoglobin through the formation of oxidized derivatives and free radicals of the hydrazine [6, 7]. The oxidized derivatives of hydrazines, in particular the unstable and oxygen-sensitive diazenes,have been reported to bind to heme compounds [7,8, 11]. Earlystudies by Kat0 et al [12] and Clark et al [13]
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