Immobilised Lipoamide Dehydrogenase

1 Pig heart lipoamide dehydrogenase (NADH : lipoamide oxidoreductase, EC 1.6.4.3) has been immobilised to Sepharose 4B by thiol‐disulphide interchange to yield a preparation containing 2.6 nmol enzyme/g moist weight gel. 2 The specific activity of approximately 1900 μmol NADH min ‐1 (μmol FAD) ‐1 at pH 6.5 with dl ‐lipoamide as substrate represented 24% of the specific activity of ths soluble native enzyme under the same conditions. The immobilised enzyme exhibited an elevated diaphorase activity. 3 The pH optima, apparent Michaelis constant for lipoamide and thermal stability of the native and Sepharose‐bound enzyme were similar. In contrast, the native enzyme modified by reaction with 5,5′‐dithio‐bis‐(2‐nitrobenzoic acid) was considerably less stable at 90 °C and had a lower energy of activation (127.7 kJ mol −1 ) than either the native enzyme (341.5 kJ mol −1 ) or the immobilised enzyme (392.9 kJ mol −1 ). 4 Both the native and immobilised enzyme were equally susceptible to the effects of urea and guanidine hydrochloride whilst the immobilised enzyme was approximately twice as stable to dioxane concentrations up to 80% (v/v). 5 Where properties of the immobilised enzyme were different to the native enzyme they were believed to be due to modification of the reactive thiol used for immobilisation rather than immobilisation per se . Thus, the ratio of NADH : lipoamide dehydrogenase activity to diaphorase activity decreased from 54.2 in the native enzyme to 8.9 in the immobilised enzyme and 9.8 in the soluble enzyme modified by reaction with 5,5′‐dithio‐bis(2‐nitrobenzoic acid). Furthermore, the immobilised enzyme was relatively insensitive to inactivation by cupric ions in marked contrast to the native enzyme. 6 The remarkable similarity in the properties of the native and immobilised enzyme attests to the lack of significant conformational constraints imposed by the matrix.