Enzymatic micromethod for measuring galactose-1-phosphate uridylyltransferase activity in human erythrocytes.

A kinetic microspectrophotometric assay for galactose1-phosphate uridylyitransferase (EC 2.7.7. 12) activity in erythrocytes is described. BlOOd is collected in ammonium heparinized microhematocrit tubes, centrifuged and the erythrocytes are lysed with water. Galactose-1-phosphate uridylyltransferase activity is determined by mixing 25 ul of hemolysate with a reagent consisting of galactose1-phosphate, uridine diphosphogiucose, NADP, ethylenediaminetetraacetate (disodium salt), phosphoglucomutase, and glucose-6--phosphate dehydrogenase. The reaction medium is maintained at 37 #{176}C. The increase in absorption of the NADPH formed (340 nm) is recorded for 9 mm. Under these conditions two moles of NADPH are produced per mole of glucose-6-phosphate oxidized. Activity is referred to hemoglobin, measured as cyanmethemoglobin. The chelator is added to activate the enzyme. Stability studies show that the transferase is stable for several days in frozen erythrocytes. Good comparisons were obtained when this assay was compared to the uridine diphosphogiucose consumption method. Because the method requires only a small amount of blood and is rapid, it can be used routinely to quantitate erythrocyte galactose-1 -phosphate uridylyltransf erase activity in newborn infants.