Glutaredoxin from calf thymus. Purification to homogeneity.

The protein glutaredoxin, required for GSH-dependent ribonucleotide reduction, has been purified to homogeneity from calf thymus. The preparative method consisted of ammonium sulfate precipitation and three chromatography steps on DEAE-cellulose, Sephadex G-50, and CM-Sepharose. Calf thymus glutaredoxin was assayed on the basis of its inherent GSH-disulfide transhydrogenase activity. Glutaredoxin, purified 3000-fold was demonstrated to be homogeneous by polyacrylamide gel electrophoresis and high performance liquid chromatography. It behaved as a neutral or slightly basic molecule having a Mr of around 11,000. The apparent Km value of glutaredoxin with calf thymus ribonucleotide reductase at 4 mM GSH was 6.0 X 10(-7) M. With ribonucleotide reductase from Escherichia coli, calf thymus glutaredoxin had a Km value of 1.9 X 10(-6) M and a molecular activity that was only 10% of that achieved with the calf thymus enzyme.