A Simple Procedure for Covalent Immobilization of NADH in a Soluble and Enzymically Active Form

Using a new and simplified technique for covalent immobilization of adenine nucleotides [Fuller, C. W. and Bright, H. J., J. Biol. Chem. 252 , 6631 (1977)], we have prepared immobilized NADH using two water‐soluble polymers and tested these preparations for activity with several dehydrogenases. The first polymer used, having a weight‐average molecular weight of about 350000, is a copolymer of methacrylyl choline and the epoxide‐containing monomer 3‐[4‐(2,3‐epoxypropoxy)butoxy]‐2‐hydroxypropyl acrylate. Immobilization of NADH onto this copolymer was accomplished in three steps, namely, alkylation of NAD at N‐1, reduction of the nicotinamide moiety with dithionite and Dimroth rearrangement of the alkyl linkage from the N‐1 to the C‐6 amino position. The second copolymer tested was a copolymer of the same epoxide‐containing monomer and N ‐methacrylyl‐2‐glucosamine. Using this copolymer, immobilization of NADH through the adenine C‐6 amino position was accomplished in a single step. Measurements of the steady‐state kinetics of five dehydrogenases at pH 7 and pH 9 showed that, on the average, V and K m values obtained with the immobilized NADH were, respectively, about one third and twice those found for the free coenzyme. In general, when compared with the free coenzyme, the immobilized NADH had greater relative enzymic reactivity at pH 9 than at pH 7. The simplicity of this method, the general enzymic reactivity, and the ability to be recycled enzymically suggest that this immobilized NADH may be useful in creating enzyme reactors for synthetic, analytical and other purposes.