An Inhibitor of Liver Alcohol Dehydrogenase in Preparations of Reduced Diphosphopyridine Nucleotide

Abstract

Different samples of reduced diphosphopyridine nucleotide (β-DPNH, Sigma Chemical Co.) have been found to give significantly different maximum rates of reduction of aldehyde catalysed by liver alcohol dehydrogenase. The crystalline enzyme was prepared from horse liver¹ and measurements of initial rates were made with a recording fluorimeter^1,2 at 23.5°. In Table 1 relative values of initial rates with 1.3 × 10⁻³ M acetaldehyde and 2 × 10⁻⁵ M DPNH in pH 6 phosphate buffer (ionic strength 0.1) are collated with assays of the samples of DPNH. The percentage by weight of anhydrous disodium salt was calculated from direct extinction measurements at 340 mµ (column 2), and also from the decrease of absorption of light at this wave-length (ΔE340) accompanying complete oxidation of the coenzyme by excess acetaldehyde and liver alcohol dehydrogenase (column 3), using in each case the value 6.22 (ref. 3) for the millimolar extinction coefficient. The second value is taken to be the true DPNH content. The difference between the two values is due to the presence of a substance which closely resembles DPNH in both light absorption and fluorescence emission, but is not coenzymically active with liver (or yeast) alcohol dehydrogenase. The results indicate that the samples which give the higher initial rates have the greater coenzyme content and a smaller excess of total adenine over coenzyme (column 4). Ribose and total phosphate analyses were also made ; calculating the total adenine content from the extinction at 260 mµ (pH 7) and a millimolar extinction of 15 (ref. 4), the molar ratios of ribose and phosphate to adenine were 2.1 ± 0.1 for all the samples.