Studies on the disulfide region of α1-protease inhibitor

Abstract

The single disulfide bond of purified human α₁-protease inhibitor was reduced with dithiothreitol in the absence of denaturant and the resultant sulfhydryl groups were alkylated with iodoacetamide-1-C¹⁴. The product was found to be fully functional as an inhibitor of trypsin and elastase in esterolytic and proteolytic assays. The modified protein was also found to be nearly identical to native α₁-protease inhibitor when analyzed by immunological, electrophoretic, and spectral methods. The performic acid oxidized inhibitor, on the other hand, was devoid of any enzyme inhibitory activity. Analysis of the derivatized protein by amino acid analysis and by radioactive counting revealed only a single cysteine-containing peptide. The alkylated inhibitor was digested with cyanogen bromide and then trypsin, and subjected to two-dimensional peptide mapping. A single cysteine-containing peptide was recovered and shown to have the sequence Phe-Asn-Ile-Gln-His-Cys-Lys. A variety of experiments involving gel filtration or dialysis of reduced or oxidized α₁-protease inhibitor indicate that this Cys-peptide is covalently bound to either free cysteine or to glutathione via a disulfide bridge.