Agarose-Bound Horse-Liver Alcohol Dehydrogenase. Dependence of Molecular Properties and Activity on Coupling Conditions

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Abstract
1 Spectroscopic methods for protein and active-site determination with the same sample of immobilised horse liver alcohol dehydrogenase have been developed. 2 The influence of pH, active-site protection of the soluble enzyme and protein concentration on coupling of alcohol dehydrogenase with cyanogen-bromide-activated Sepharose has been investigated. In phosphate buffer (pH 8.0) products with over 90% active-site retention have been synthesized. The binary complex alcohol-dehydrogenase · NADH gives a preparation with the same active-site content but a lower apparent specific activity compared to the unprotected enzyme. Increase in protein concentration yields products with the same active-site content relative to bound protein but the apparent specific activity is decreased. 3 The great similarity in spectroscopic properties of soluble and immobilised enzyme, as well as of their ternary complexes, shows that no significant conformational change has taken place during immobilisation. 4 Exchange of the non-catalytic Zn2+ against Co2+ yields a hybrid Sepharose-Co2Zn2-alcohol-dehydrogenase with over 90% active-site retention during metal exchange. The absorption spectra of the soluble and immobilised hybrid are identical.