Affinity chromatography of enzyme cofactors: the separation of NAD on immobilised dehydrogenase colums.
1. Alcohol dehydrogenase (EC 1.1.1.1.) has been immobilised to aminoethyl-cellulose by glutaraldehyde, to DEAE-cellulose by an s-triazine derivative and to agarose using CNBr. Lactate dehydrogenase has been immobilised to the latter two supports. 2. Their use for affinity chromatography of NAD was compared and alcohol dehydrogenase immobilised to CNBr-activated agarose chosen for detailed study due to the efficient coupling of applied enzyme and the specific nature of binding. 3. The efficiency of coupling of alcohol dehydrogenase dropped from 94.5 to 72.2% when the applied load was increased from 18 to 54 mg/g activated agarose. Activity relative to free enzyme fell from 21 to 11%. The binding of NAD was maximal between pH 5.5 and 6. With the lowest loading of enzyme, NAD binding fell from 450 to 320 mug/g support when the linear flow rate was increased from 0.84 to 3.95 cm/min. 4. NAD was completely separated from a mixture with ATP, ADP and AMP. Separation from NMN and hydrolysed RNA and DNA was evidently possible. Immobilised alcohol dehydrogenase used for 34 binding experiments over a period of weeks maintained 60% of its original enzyme activity. 5. The method was applied to yeast NAD following mechanical disruption of yeast, clarification and either ultrafiltration or hollow-fibre dialysis to permit separate purification of macromolecules and nucleotides.
D000429:Alcohol Oxidoreductases; D002846:Chromatography, Affinity / Q000379:methods; D003636:DEAE-Cellulose; D003956:Dialysis; D005069:Evaluation Studies as Topic; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase; D009243:NAD / Q000302:isolation & purification*; D009537:Nicotinamide Mononucleotide / Q000032:analysis; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000032:analysis; D012685:Sepharose; D014462:Ultrafiltration
D009243:NAD; D009537:Nicotinamide Mononucleotide; D012685:Sepharose; D003636:DEAE-Cellulose; D000429:Alcohol Oxidoreductases; D007770:L-Lactate Dehydrogenase