Purification of dihydrofolic reductase from chicken liver by affinity chromatography

The procedure for coupling methotrexate (4-amino-10-methylpteroylglutamic acid) to Sepharose via a six carbon chain is described. An affinity column prepared from this material quantitatively adsorbs the dihydrofolic reductase activity from a partially purified extract of chicken liver. Elution of the enzyme readily occurs with dilute K 2HPO 4 in the presence of dihydrofolate. Approximately 250-fold purification occurs by affinity chromatography yielding a preparation which appears to be homogeneous.