6] Affinity chromatography of NAD on immobilized dehydrogenase columns

Abstract

This chapter discusses the results of affinity chromatography of nicotinamide adenine dinucleotide (NAD) on immobilized dehydrogenase columns. For immobilization of alcohol dehydrogenase to glutaraldehydetreated AE-cellulose, 150 ml of 0.5 M NaOH are added to 10 g (dry weight) of support material. All NaOH is then washed off with water. The material is resuspended in 150 ml of 0.1 M phosphate buffer, pH 7.0. To this 10 ml of 50% glutaraldehyde in water is added. The mixture is then covered and kept at room temperature for 2 h, with constant stirring, after which it is centrifuged at 10,000 g . It is then washed six times, with the coupling buffer, to dilute out the remaining glutaraldehyde. The treated support material is resuspended in 150 ml of the same buffer. To 15 ml (1 g AE-cellulose) of this suspension, a desired amount of alcohol dehydrogenase is added for coupling and the mixture is kept for 16 h at 4° under constant stirring. This chapter discusses the procedure for the attachment of lactate and alcohol dehydrogenase to S-triazine activated diethylaminoethyl (DEAE) cellulose. An overview of the assay methods, including alcohol dehydrogenase assay, lactate dehydrogenase assay, NAD assay, assay of immobilized lactate dehydrogenase, etc., is also discussed in this chapter.