An improved assay for hexokinase activity in human tissue homogenates.
An improved method has been developed for the assay of hexokinase (EC 2.7.1.1) levels in human tissue homogenates. The enzyme is quantitated by the spectrophotometric measurement, at 340 nm, of NADPH formed according to the reaction scheme: [formula: see text] In tissue homogenates a number of enzymes are present which can interfere with the assay by reacting with substrates or products of the assay reactions. In the described procedure hexokinase is assayed directly in homogenates under conditions in which the effect of possible contaminating enzymes (glucose dehydrogenase, EC 1.1.1.47; glucose 6-phosphatase, EC 3.1.3.9; glucose phosphate isomerase, EC 5.3.1.9; 6-phosphogluconate dehydrogenase EC 1.1.1.44; and NADP-reducing enzymes) are eliminated. Precision studies on the assay gave within-day reproducibility of 4.3% (CV) on a tissue having a mean activity of 1.68 U/g of tissue, and day-to-day variability of 15% (CV) for a tissue averaging 1.83 U/g of tissue.
Department of Pathology, LSU Medical Center, New Orleans 70112, USA.
W D
WD
Scheer
H P
HP
Lehmann
M F
MF
Beeler
D042843:Glucose 1-Dehydrogenase; D005948:Glucose Dehydrogenases / Q000032:analysis; D006593:Hexokinase / Q000032:analysis* / Q000097:blood; D006801:Humans; D007202:Indicators and Reagents; D009249:NADP; D010734:Phosphogluconate Dehydrogenase / Q000032:analysis / Q000097:blood; D015203:Reproducibility of Results; D013053:Spectrophotometry
D007202:Indicators and Reagents; D009249:NADP; D005948:Glucose Dehydrogenases; D010734:Phosphogluconate Dehydrogenase; D042843:Glucose 1-Dehydrogenase; D006593:Hexokinase