Procion dyes as affinity ligands and reporter groups for dihydrofolate reductase from Walker 256 carcinoma

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Abstract
Procion dye-agarose matrices were investigated for isolation of dihydrofolate reductase (FAH2R) from Walker 256 [rat] carcinosarcoma. Cibacron blue F3GA, Procion blue MX4GD, Procion blue HERD and Procion red H3BN covalently bound to agarose adsorbed > 85% of pure FAH2R from 100 mM imidazole buffer, pH 6.3, and this enzyme was specifically and quantitatively eluted with 1 mM folate. The capacity and selectivity of the dye-agarose matrices were greater at low dye incorporation. Difference spectroscopy of the FAH2R-Cibacron blue F3GA complex indicated that 2 mol of the dye were bound in hydrophobic environments with each mole of the enzyme. NADPH and folate (at 2-fold molar excess over enzyme) or 1 M KCl displaced only 1 mol of Cibacron blue F3GA. This dye interacted stoichiometrically in a specific manner with the active site of FAH2R probably spanning the folate and NADP binding sites. The 2nd dye molecule appears to be bound in a nonspecific hydrophobic manner. Selected Procion dye-agarose matrices can be used for partial purification of FAH2R from tumor homogenate. [FAH2R is the primary intracellular target enzyme for a number of therapeutic drugs.].