Transketolase has been purified for the first time from human leukocytes, according to a new procedure which consists of three conventional steps. The enzyme was finally detached from CM-cellulose by specific elution with a ~-xylulose-5-phosphate/~-ribose-5-phosphate mixture and the isolated product exhibited a specific activity of about 10 units/mg protein at 37 "C. Transketolase preparations are contamination-free, except for a slight residual activity of phosphohexose isomerase. Kinetic constants for D-xylulose 5-phosphate and o-ribose 5-phosphate were found to be 0.19 mM and 0.63 mM, respectively. Pure transketolase migrates on SDSjPAGE as a single band, with a molecular mass of about 66 kDa.The isoelectrophoretic heterogeneity of transketolase was assessed either by activity staining or immunovisualization with anti-transketolase antisera, previously induced in rabbits. These techniques yielded two practically overlapping patterns consisting of 6 -8 distinct bands within a PI range of 6.5 -8.5. Both pure and crude transketolase preparations showed a similar heterogeneous profile, thus confirming the stability of the enzyme throughout purification.The occurrence of multiple enzyme forms in fresh human white cells has also been established by the analysis of transketolase in isolated populations of either lymphocytes or polymorphonuclear leukocytes, from individual healthy subjects.Transketolase is a key enzyme of the nonoxidative pentose cycle and catalyzes the reversible transfer of a keto group between several donor and acceptor substrates [l, 21. Both magnesium ions and thiamine pyrophosphate are required as cofactors for enzyme activity [I -41. There is some interest in human transketolase, whose activity in blood cells is used as a marker of thiamine deficiency and poor nutritional status [5, 61. In addition, it has been inferred that transketolase is altered in alcoholics [7] and in patients with WernickeKorsakoff syndrome [8], where the occurrence of peculiar isoenzyme patterns was also reported [9].However, owing to the little information available on pure enzyme preparations, the properties of human transketolase have not yet been clearly established. For instance, the occurrence of multiple forms and variants [lo] Abbreviations. Ery4P, D-erythrose 4-phosphate; XluSP, D-XYIUlose 5-phosphate; RibSP, D-ribose S-phosphate; Glc6P, D-glucose 6-phosphate; Fru6P, D-fructose 6-phosphate; Sed7P, D-sedoheptulose 7-phosphate; DEAE-cellulose, diethylaminoethyl cellulose; CMcellulose, carboxymethyl cellulose; PMN, polymorphonuclear leukocytes; NaCl/Pi buffer, phosphate-buffered saline.Enzymes. Transketolase (EC 2.2.1.1); phosphoribose isomerase (EC 5.3.1.6); D-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1); transaldolase (EC 2.2.1.2); ~-glucose-6-phosphate dehydrogenase (EC 1.1.1.49); hexokinase (EC 2.7.1 .I); glycerol-3-phosphate dehydrogenase (EC 1 1.1.8); phosphohexose isomerase (EC 5.3.1.9).years, all of which concern erythrocytes from outdated blood. We now suggest a new procedure for a high-yield purific...