A spectrophotometric assay for 6-phosphogluconolactonase involving the use of immobilized enzymes to prepare the labile 6-phosphoglucono-δ-lactone substrate

Abstract

We report the development of a new spectrophotometric assay for 6-phosphogluconolactonase. The labile substrate 6-phosphoglucono-δ-lactone is prepared from glucose 6-phosphate by enzymes co-immobilized on Sepharose beads. The assay has the advantages of high sensitivity for routine determination of enzyme activity and allows determination of both K(m) and V(max.) from a single assay. A method for estimating the contribution of spontaneous hydrolysis to total hydrolysis is described. This assay overcomes the problems encountered with all previous assays.