Purification of human leucocyte pyruvate kinase

The M2 form of pyruvate kinase (M2-PK) was purified from human leucocytes by a new method involving a succession of two different Dyematrex agarose chromatographies. The main step consisted of an orange dye affinity column with elution by fructose-1,6-diphosphate. This purification procedure allowed us to obtain M2-PK with a specific activity of 433 I.U./mg of protein, i.e. a 188-fold purification with an overall yield of 33%. The homogeneity of this preparation was verified by sodium dodecyl sulphate polyacrylamide gel electrophoresis and double immunodiffusion in Ouchterlony plates. Anti-M2-PK antibodies obtained from rabbit neutralized the enzyme activity. Their specificity with regard to other types of PK showed that anti-M2-PK also reacted with M1-PK but not with R-PK.