Blood cell phosphogluconolactonase: assay and properties

6-Phosphogluconolactonase (6-PGL) catalyses the second reaction of the hexosemonophosphate pathway. Although the delta-lactone of 6-phosphogluconic acid is the natural substrate for this enzyme, the more stable gamma-lactone may also be used. We prepared the gamma-lactone of 6-phosphogluconic acid from 6-phosphogluconate. When stored in dimethylsulfoxide, this material was found to be stable in liquid nitrogen for several months. A method for measuring 6-phosphogluconolactonase (6-PGL) using the gamma-lactone as substrate has been developed, after defining conditions under which spontaneous hydrolysis of the lactone is relatively slow and the enzymatic velocity is relatively rapid. The enzyme had no divalent cation requirement and was not significantly inhibited by a 50-fold excess of gluconolactone. It was distinct, therefore, from gluconolactonase. At 25 degrees C normal human red cells were found to contain approximately 50 IU of 6-phosphogluconolactonase/g Hb. The activity of the enzyme was independent of red cell age. Based on protein content, human lymphocytes, monocytes, granulocytes and platelets, contained approximately 10 times the activity of red blood cells. The activity of 6-PGL was stable for at least 6 d in red cells stored at 22 degrees C and for at least 20 d in red cells stored at 4 degrees C.