The effect of Hofmeister anions and protein concentration on the activity and stability of some immobilized NAD-dependent dehydrogenases.
The effect of several factors on the activity and stability of alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and 20beta-hydroxysteroid dehydrogenase, both free and immobilized on CNBr-activated Sepharose 4B, was investigated. Enzymes were im- mobilized under different conditions including various degrees of matrix activation, variable amounts of protein, in the presence, or in the absence of, additives (coenzymes, dithioth- reitol, salts). Activity recovery was in general satisfactorily high with 20beta-hydroxysteroid dehydrogenase, low with glyceraldehyde-3-phosphatedehydrogenase, and markedly linked to the concentration of immobilized protein with alcohol dehydrogenase. In the latter case the advantageous stabilizing effect of high enzyme concentrations was notably diminished by the parallel decrease of the effectiveness factor. The effect of high concentrations of anions of the Hofmeister series was examined. It was found that 1M phosphate and 0.5M sulfate dramatically stabilize both free and immobilized enzymes against inactivation by temperature and urea. K(m), values of apolar substrates were considerably lowered by the two anions while K(m) values of polar substrates were not affected. In some cases V(max) values also were influenced by high concentrations of these anions. The present results appear of interest particularly in view of enzyme utilization for analytical as well as for preparative purposes.
Istituto di Chimica degli Ormoni, CNR, Via Mario Bianco 9, 20131 Milano, Italy.
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Carrea
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Bovara
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Pasta
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Cremonesi