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1. Horse liver alcohol dehydrogenase and an NADH analogue, N6-[(6-aminohexyl)carbamoylmethyl]-NADH, have been co-immobilized to Sepharose 4B under conditions permitting binary complex formation between the enzyme and the cofactor.2. The enzyme-coenzyme-matrix preparations were assayed with a coupled oxidoreduction reaction and showed activities, prior to addition of coenzyme, that were up to 40 of that obtained in excess of free coenzyme.3. A molar ratio of 1 : 1 between the amount of bound nucleotide and bound enzyme was sufficient to obtain high activities in the absence of free coenzyme.4. The highest recycling rate obtained for the immobilized nucleotide was 3400 cycles per hour. 5. Both thermal and storage stability of alcohol dehydrogenase was increased when the enzyme was co-immobilized with the NADH analogue.6. The efficiency of the immobilized preparations (measured as product formation per minute and per assay volume) was higher (1.4 to 5 times in our assays) than the corresponding systems of free enzyme (in total enzyme units) and nucleotide in an identical assay volume.

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The coenzyme specificity of horse liver alcohol dehydrogenase assayed in mixtures of acetonitrile and buffer, 0.01 M Tris/HCI pH 7.4, was investigated. The enzyme could accept NADP' as coenzyme if it was first bio-imprinted with NADP', i.e. precipitated from an aqueous buffer with 1-propanol in the presence of NADP', dried, and then assayed in a system with less than 10% buffer. When assayed in a system with 25 % buffer, no activity with NADP' as coenzyme was observed. The activity was measured with a coenzyme regeneration assay with cinnamoyl alcohol and octylaldehyde as substrates. Other methods to prepare a binary complex of horse liver alcohol dehydrogenase and NADP', i.e. if the enzyme was immobilized on silica and NADP' added afterwards or if it was deposited on Celite together with NADP', failed to show any bio-imprinting effect. The activity of horse liver alcohol dehydrogenase bioimprinted with NADP' and assayed in acetonitrilehuffer mixtures with less than 10% buffer showed the same or higher activity than an enzyme preparation prepared by bio-imprinting with NAD'. The hypothesis is that the conformation of the active site of horse liver alcohol dehydrogenase is modified to one that is complementary to the ligand present during precipitation and drying. This is possible because of the restricted motility of the enzyme conformation in high concentrations of organic solvent. In the presence of more than 10% buffer the mobility increases in such a way that the imposed conformation with activity towards NADP' disappears.

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