Abstract
Thioltransferase (glutaredoxin) was purified from human red blood cells essentially as described previously (Mieyal JJ et al., 1991a, Biochemistry 30:6088β6097). The primary sequence of the HPLCβpure enzyme was determined by tandem mass spectrometry and found to represent a 105βamino acid protein of molecular weight 11,688 Da. The physicochemical and catalytic properties of this enzyme are common to the group of proteins called glutaredoxins among the family of thiol: disulfide oxidoreductases that also includes thioredoxin and protein disulfide isomerase. Although this human red blood Cell glutaredoxin (hRBC Grx) is highly homologous to the 3 other mammalian Grx proteins whose sequences are known (calf thymus, rabbit bone marrow, and pig liver), there are a number of significant differences. Most notably an additional cysteine residue (Cysβ7) occurs near the Nβterminus of the human enzyme in place of a serine residue in the other proteins. In addition, residue 51 of hRBC Grx displayed a mixture of Asp and Asn. This result is consistent with isoelectric focusing analysis, which revealed 2 distinct bands for either the oxidized or reduced forms of the protein. Because the enzyme was prepared from blood combined from a number of individual donors, it is not clear whether this Asp/Asn ambiguity represents interβindividual variation, gene duplication, or a deamidation artifact of purification.