Spectrophotometric assay of bisphosphoglycerate mutase: a reexamination of Rapoport-Luebering's method.

The saturation by substrates and cofactors, the effects of pH and the influence of salts and auxiliary enzymes have been studied. The linear NAD+ reduction observed before addition of haemolysate to the assay system was proportional to pH, being higher with phosphate than with Tris-HCl buffer. In the presence of bisphosphoglycerate mutase, an optimal pH (7.8-8.1) was obtained and the inhibition by sulfate ions could be confirmed. It can then be suggested that the absence of an equilibrium, the pH used by several authors and sulfate inhibition could be sources of error in the spectrophotometric assay of bisphosphoglycerate mutase activity. Once optimal conditions have been established, activities found in both human and rat erythrocytes are similar to those given by other accurate methods.