Abstract: In order to investigate whether Gly33 residue in the conserved sequence of the active site of human thioredoxin hTrx-1 is essential in the process of reducing oxidative protein, the Gly33 residue of hTrx-1 was deleted or replaced (G→A) by two synthetic mutant genes. The synthetic mutant genes were then expressed in prokaryotic cells, further purified and tested for antioxidant activity. The results showed that both the deletion of Gly33 residue and the mutation to Ala, the mutated hTrx-1 could be efficiently expressed in prokaryotic cells, indicating that both mutant hTrx-1 could form a “Trx fold” structure. Antioxidant activity testing showed that in the absence of Gly33 residues, its antioxidant capacity decreased, when Gly33 mutated to Ala, its antioxidant capacity significantly decreased to near loss. Therefore, this article speculated that the change of conserved amino acid residues in the Trx-1 active center affects the geometric configuration of the active center region, resulting in difficulty in approaching the substrate protein in space. The detection results of the content of disulfide bond sulfhydryl groups involved in the formation of disulfide bonds showed that the content of disulfide bond sulfhydryl groups formed in mutant hTrx-1 was significantly lower than that in wild-type hTrx-1. It was speculated that the ability of Cys32 and Cys35 to form disulfide bonds in mutant hTrx-1 was affected.

Key words: thioredoxin 1, Trx fold, Gly33, antioxidant, disulfide bond