Reaction-Rate Assay of Serum Sorbitol Dehydrogenase Activity at 37 °C

Abstract

The sorbitol dehydrogenase (EC 1.1.1.14) activity of human serum and liver was investigated at 37 °C. Various buffers were examined but tris(hydroxymethyl)aminomethane HCl in a final concentration of 90 mmol/liter (pH 6.6, at 37 °C) was the one with which activity was the greatest. The enzyme is inhibited by its substrate, D-fructose, and activity was greatest with a substrate concentration (in the final assay mixture) of 500 mmol/ liter and an NADH concentration of 247 µmol/liter. The specimen volume was 100 µl. The enzyme from liver and serum was shown to have similar K_m values and activation energies, and we conclude that the liver enzyme was unchanged on passing into the extrahepatic space. Normal values for this assay are 0-2.6 U/liter (2.5 and 97.5 percentiles)