Preparation of an affinity chromatographic system for the separation of ADP binding proteins.
[4-(3-Bromoacetylpyridinio)-butyl]adenosine pyrophosphate as a structural analog of NAD+ reacts covalently with the sulfhydryl groups of thiopropyl agarose. 10-20 mumol can be bound to 1 ml gel. Stabilization of the insoluble coenzyme is attained by treatment with sodium boro hydride (NaBH4). This complex when applied to column chromatography, allows the separation of various dehydrogenases as a result of their different complex stability coefficients. Alcohol dehydrogenase from liver, lactate dehydrogenase, and adenylate kinase, which all bind to the ADP-analog residues of the gel matrix, can thus be separated by different salt gradients. Alcohol dehydrogenase from yeast, however, does not form a complex and can easily be eluted from the column with phosphate buffer. Glyceraldehyde-3 phosphate and aldehyde dehydrogenases can be eluted by the addition of NAD+ or NADH to the buffer. The uncharged 1,4-dihydropyridine ring of the reduced coenzyme produces a more stable complex with the dehydrogenases than the oxidized form.
D000244:Adenosine Diphosphate / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D009243:NAD; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification*; D010084:Oxidation-Reduction
D002352:Carrier Proteins; D009243:NAD; D000244:Adenosine Diphosphate; D009247:NADH, NADPH Oxidoreductases