Double-ternary complex affinity chromatography: preparation of alcohol dehydrogenases.
A general affinity chromatographic method for alcohol dehydrogenase purification has been developed by employing immobilized 4-substituted pyrazole derivatives that isolate the enzyme through formation of a specific ternary complex. Sepharose 4B is activated with 300 mg of cyanogen bromide/ml of packed gel and coupled to 4-[3-(N-6-aminocaproyl)aminopropyl]pyrazole. From crude liver extracts in 50 mM phosphate-0.37 mM nicotinamide adenine dinucleotide, pH 7.5, alcohol dehydrogenase is optimally bound at a capacity of 4-5 mg of enzyme/ml of gel. Addition of ethanol, propanol, or butanol, 500 mM, results in the formation of a second ternary complex, which allows the elution of bound enzyme in high yield and purity. This double-ternary complex affinity chromatography has been applied successfully to human, horse, rat, and rabbit liver extracts to isolate the respective homogeneous alcohol dehydrogenases.
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification*; D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology; D009243:NAD; D011485:Protein Binding; D013329:Structure-Activity Relationship
D008024:Ligands; D009243:NAD; D000429:Alcohol Oxidoreductases