THE PRIMARY STRUCTURE AND PROPERTIES OF THIOLTRANSFERASE (GLUTAREDOXIN) FROM HUMAN RED-BLOOD-CELLS

Thioltransferase (glutaredoxin) was purified from human red blood cell s essentially as described previously (Mieyal JJ et al., 1991a, Bioche mistry 30:6088-6097). The primary sequence of the HPLC-pure enzyme was determined by tandem mass spectrometry and found to represent a 105-a mino acid protein of molecular weight 11,688 Da. The physicochemical a nd catalytic properties of this enzyme are common to the group of prot eins called glutaredoxins among the family of thiol:disulfide oxidored uctases 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 nu mber of significant differences. Most notably an additional cysteine r esidue (Cys-7) occurs near the N-terminus 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 consisten t with isoelectric focusing analysis, which revealed 2 distinct bands for either the oxidized or reduced forms of the protein. Because the e nzyme was prepared from blood combined from a number of individual don ors, it is not clear whether this Asp/Asn ambiguity represents inter-i ndividual variation, gene duplication, or a deamidation artifact of pu rification.