Characterization of chicken liver dihydrofolate reductase after purification by affinity chromatography and isoelectric focusing.

Chicken liver dihydrofolate reductase purified to apparent homogeneity by affinity chromatography contains tightly bound dihydrofolate. The most effective method for removal of the bound substrate is by electrofocusing. This procedure also removes previously unsuspected contaminants. In addition, the isoelectric profile revealed as many as four distinct peaks of enzyme activity. The major peak (pI = 8.4) represents 60–75% of the total activity, is devoid of bound substrate, and exhibits an $\text{A}{}_{280}\text{A}{}_{260}$ ratio approaching 1.9 and a specific activity of 14 units/mg. The peak of activity at the isoelectric point of 7.4 contains bound dihydrofolate. The major isoelectric band is shown to be homogeneous by the usual criteria. Notable features of the amino acid composition include a single cysteine, three tryptophans, and an excess of acidic residues. The N-terminal residue is valine. The molecular weight as determined by sedimentation equilibrium is 22,474. The s_20,w⁰ is 2.07. A frictional coefficient of 1.2 indicates that the enzyme approximates a sphere. Circular dichroism measurements suggest a low α-helical content and a high degree of β-structure. The molar extinction coefficient was determined to be 28,970.