Glutathione Reductase from Human Erythrocytes.

1. From 90 1 erythrocytes 200 mg glutathione reductase was purified in four steps with an overall yield of 40%. The specific activity rose from 3.71 U glutathione reductase per g hemoglobin in the erythrocytes to 235 U/mg protein in the isolated enzyme. The purification procedure which was completed in 5 days, included (a) denaturation of hemoglobin by a mixture of butan-1-oI and chloroform [Scott. E. M. (1976) Prep. Biochem. 6, 147-152]. (b) precipitation of glutathione reductase with acetone, (c) extraction of the precipitate and fractionation of the extract with ammonium sulfate, (d) affinity chromatography on adenosine-2',5'-bisphosphate -Sepharose 4B. 2. The 280/460 nm absorbance ratio was found to be 5.9 for the purified enzyme; all other studies on physical and chemical properties of the enzyme confirmed the results of Worthington and Rosemeyer [Eur. J. Biochem. 67, 231-238 (1976) and earlier papers]. 3. The new purification procedure provided crystals suitable for X-ray diffraction analysis at high resolution. Furthermore sufficient amounts for sequencing the enzyme were isolated. As a first step in the determination of the protein's primary structure, CNBr-produced fragments of the carboxymethyiated enzyme were fractionated. 4. A chain segment containing the catalytic disulfide of the native protein was isolated. Its sequence was determined to be Leu-Gly-Gly-Thr-Cys-Val-Asn-Val-Gly-Cys-Val-Pro-Lys. An identical sequence is present in yeast glutathione reductase, and lipoamide dehydrogenases contain an active site peptide with very similar sequence [Williams, C. H. Jr (1976) The Enzymes 13, 89 - 173]. Thus the long-recognized mechanistic similarities between glutathione reductases and lipoamide dehydrogenases are reinforced by sequence similarities around the two redox-active cysteine residues. The sequenced regions are, however, too short to allow speculations on phylogenetic relationships within this category of proteins. [ABSTRACT FROM AUTHOR]