Dissociation Constants of the Binary Complex of Homogeneous Horse Liver Alcohol Dehydrogenase and Nicotiniumamide Adenine Dinucleotide.

Abstract

The coupling ofNAD+ to homogeneous liver alcohol dehydrogenase (LADH) was studied. The resulting spectrophotometric change was followed in the ultraviolet region (main peak at 281 m[mu]) by means of double difference spectrophotometry. Extreme care was taken throughout to prevent alcohol contamination. The absorption increase, induced by the coupling of [beta]-nicotiniumamide dinucleotide ([DELTA] [epsilon] at 281 m[mu] = 3.0 [plus or minus] 0.1 m[image]-1 X cm-1) with LADH was not seen with the [alpha]-isomer or with [beta]-nicotiniumamide mononucleotide or with [beta]-nicotiniumamide dinucleotide phosphate. This absorption increase provided a means of a direct determination of the dissociation constants of the B-nicotiniumamide dinucleotide complex with homogeneous liver alcohol dehydrogenase over the pH range 6 to 10. The dissociation constants thus directly determined are in good agreement with those redetermined using the previous indirect spectrofluorimetric method. Titration data indicate the presence of 2 independent and equivalent [beta]-nicotiniumamide dinucleotide-binding sites per LADH molecule throughout the pH range 6 to 10. The dissociation constants determined at various pH values could be fitted to a monovalent dissociation curve with pK = 8.75 for the proton donating group of the free enzyme, confirming the previous assumption that the ternary complexes, enzyme-imidazole-[beta]-nicotiniumamide dinucleotide and enzyme-fatty acid-[beta]-nicotiniumamide dinucleotide, are analogous to the acid and alkaline extremes of the enzyme-B-nicotiniumamide dinucleotide binary complex.