Effects of Salts, Protease Digestions, Chemical Modifications, and Heat Treatment on the Trypsin Inhibitory Activity of the Protease Inhibitor from JobVtears (Coix lacryma-jobi L. var. Ma-yuen Stapf)

Abstract

Trypsin inhibitor (JBTI) was extracted and purified from the bran of Job's-tears (Coix lacryma-jobi L. var. Ma-yuen Stapf) seeds. Its molecular weight was estimated as 16500 by SDS-PAGE. Trypsin inhibitory activity of JBTI was enhanced in the presence of divalent cations, such as Ca²⁺ or Mg²⁺, and inhibited by divalent heavy metal ions, such as Cu²⁺ or Zn²⁺ . Its activity was stable against pepsin digestion, while JBTI was denatured by subtilisin BPN' or Actynase E digestions. Its activity was stable against chemical modification of the lysyl residues, while JBTI lost its activity by the modification of the arginyl residues. The thermal unfolding of JBTI was studied in an adiabatic differential scanning calorimeter and it was found that the protein unfolds at 93°C and pH 7.0 in 50 mM phosphate buffer.