Purification of dihydrofolate reductase from human leukocytes and from mouse liver and electrofocusing on thin-layer polyacrylamide gel.

A method for the purification of dihydrofolate reductase starting upon such small quantities as 0.05--0.3 U is described. By ammonium sulfate precipitation, gel filtration and affinity chromatography on folate-aminohexyl-Sepharose extracts of leukocytes and mouse liver could be purified 1,500 and 830-fold, respectively. Electrofocusing on thin-layer polyacrylamide gels revealed two isoenzymes of mouse liver dihydrofolate reductase with pI values of 8.65 and 8.15. The purified leukocyte enzyme showed one band at pI 7.5. Addition by dihydrofolic acid or methotrexate prior to the isoelectric focusing did not change the behavior of the enzymes. Only addition of NADPH caused a slight decrease of the liver pI 8.15 and of the leukocyte band and the appearance of a faint band at pI 5.4 and 5.0, respectively.