The isolation of dihydrofolate reductases by affinity chromatography on folate-Sepharose

Abstract

Folate-Sepharose affinity column material has been prepared and used for the isolation of dihydrofolate reductases from three sources: (a) amethopterin-resistant L1210 cells in culture; (b) amethopterin-resistant Lactobacillus casei; and (c) human leukemic leukocytes. The column material was synthesized in two steps: (i) condensation of folate (1 part) and diaminohexane (10 parts) promoted by 1-ethyl-3(3′-dimethylaminopropyl) carbodiimide (5 parts) in dimethylsulfoxide; and (ii) combination of the product, 1-aminohexyl-6-amidofolate, with cyanogen bromide-activated Sepharose. Application of a crude cellular homogenate from each source to the affinity matrix led to preferential enzyme retention. After an initial wash with solutions of increased ionic strength, recovery of the two mammalian enzymes was achieved in a greater than 65% yield by elevation of the pH and addition of folate to the elution buffer. The L1210 enzyme was homogeneous by polyacrylamide electrophoresis ( R f = 0.20). The L. casei enzyme (specific activity ∼9 unit/mg) was recovered by merely increasing the eluting buffer strength. However, preliminary passage of the cell extract through DEAE-cellulose followed by affinity chromatography gave improved purification (specific activity ∼16 unit/mg). The reductases from both the L1210 and L. casei sources were recovered as a single form of the enzyme after affinity chromatography, that with no bound NADPH.