A spectrophotometric assay for 6-phosphogluconolactonase involving the use of immobilized enzymes to prepare the labile 6-phosphoglucono-delta-lactone substrate.
We report the development of a new spectrophotometric assay for 6-phosphogluconolactonase. The labile substrate 6-phosphoglucono-delta-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 Km and Vmax. 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.
Moir
R D
RD
Department of Biochemistry, University of Western Australia, Perth, Australia.
Stokes
G B
GB
D002265:Carboxylic Ester Hydrolases / Q000032:analysis*; D004800:Enzymes, Immobilized; D005942:Gluconates; D005954:Glucosephosphate Dehydrogenase; D005980:Glutathione Reductase; D006868:Hydrolysis; D007700:Kinetics; D010734:Phosphogluconate Dehydrogenase; D013053:Spectrophotometry / Q000379:methods*
C048453:6-phosphoglucono-gamma-lactone; D004800:Enzymes, Immobilized; D005942:Gluconates; D010734:Phosphogluconate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D005980:Glutathione Reductase; D002265:Carboxylic Ester Hydrolases; C022363:6-phosphogluconolactonase