Immobilised lipoamide dehydrogenase. 3. Preparation and properties of an immobilised polythiolated enzyme.

Abstract

1 The dimeric flavoprotein pig heart lipoamide dehydrogenase (NADH: lipoamide oxidoreductase, EC 1.6.4.3) has been polythiolated by reaction with N-acetylhomocysteine thiolactone at pH 10.5 for 24 h. Under these conditions 5–6 mol additional thiol groups are introduced per mol FAD. 2 The free polythiolated enzyme has a 50–60% lower specific activity for lipoamide, a reduced affinity for a specific anti-lipoamide dehydrogenase antibody but an unchanged apparent Km (lipoamide). The free polythiolated enzymes containing 6 mol–SH/mol FAD and 7 mol–SH/mol FAD were 270% and 640% respectively more stable to thermal inactivation at 90°C than the free native enzyme. 3 Immobilisation of the polythiolated enzyme to thiolated 6-aminohexyl-Sepharose reduced the specific activity with lipoamide to less than 10% of that of the free native enzyme, raised the apparent Km (lipoamide) to 8.3 mM and lowered its affinity for the specific antibody. The thermal stability at 90°C was enhanced by up to 25-fold. 4 Immobilisation of the polythiolated enzyme to a short spacer group, L-cysteinyl-Sepharose, reduced the specific activity with lipoamide but enhanced the stability at 90°C and in 70% (v/v) dioxane by 800% and 770% respectively relative to the free native enzyme. These data are discussed in terms of the effects of proximity to the matrix backbone. 5 The marked improvement in stability of the polythiolated enzyme was matched by lipoamide dehydrogenase immobilised directly to CNBr-activated Sepharose at pH 7.5 and pH 9.0. However, in this case the specific activity of the immobilised enzyme was 300–350% less than that of the polythiolated enzyme. These data are discussed in terms of multiple attachment of the enzyme to the matrix and the possibility of disulphide cross-links in the polythiolated enzyme.