Red cell pyruvate kinase deficiency: an optimised assay.
As a result of using a variety of assay techniques, rather different values for the rate of the pyruvate kinase (PK) reaction were obtained. Kinetic measurements performed with red cell PK using the stopped-flow as well as the "classical" spectrophotometric method demonstrated that only the initiation of the reaction with ADP yields reproducible results in the standard assay. Moreover, the influence of substrate concentrations, activator concentrations, ionic strength, buffer system, temperature and duration of preincubation were thoroughly investigated and were shown to play an important role. As a consequence of our kinetic measurements, a modified optimised assay composition together with new normal values for erythrocyte PK activity in haemolysate are presented. For better characterisation and comparability of PK variants, we suggest the use of the optimised assay composition described and that the reaction be initiated with ADP.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000746:Anemia, Hemolytic, Congenital Nonspherocytic / Q000175:diagnosis*; D004912:Erythrocytes / Q000201:enzymology*; D005635:Fructosediphosphates / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000097:blood / Q000172:deficiency*; D012964:Sodium / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet
D005635:Fructosediphosphates; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D012964:Sodium; D011770:Pyruvate Kinase; D008274:Magnesium; D011188:Potassium