Tritium effect in peroxidation of ehtanol by liver catalase

Abstract

Simultaneous determination of the rate of appearance of 3H in water from [(1 R)-1-3H1]-ethanol and the rate of acetaldehyde formation in the presence of rat or ox liver catalase under conditions of steady-state generation of H2O2 allowed calculation of the 3H isotope effect. The mean value of 2.52 obtained for rat liver catalase at 37.degree. C and pH 6.3-7.7 was independent of both ethanol concentration and the rate of H2O2 generation over a wide range. At 25.degree. C a slightly lower mean value of 2.40 was obtained with the ox liver catalase. Neither the product, acetaldehyde, nor 4-methylpyrazole influenced the 2 rates measured in the assay. Relating the value obtained for the 3H isotope effect to a known value for the 2H isotope effect strongly supported the view that both values were close to the true isotope effect with the respective substituted compounds on the rate constant in the catalytic step involving scission of the C-H bond. The constancy of the isotope effect under various conditions made it possible to use it for interpretations in vivo. It was established that Pseudomonas fluorescens .beta.-D-galactose dehydrogenase exhibited B-specificity towards the nicotinamide ring in NAD.