Macromolecular properties and partial amino acid sequence of a Kunitz-type protease inhibitor from okra (Abelmoschus esculentus) seeds

Abstract

A Kunitz-type protease inhibitor (OPI, okra protease inhibitor) has been purified from okra (Abelmoschus esculentus) seeds by a combination of ammonium sulfate precipitation, anion-exchange chromatography and reverse-phase high-performance liquid chromatography. The protein shows an apparent mass of 21 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition. OPI exhibits inhibitory activity against trypsin. Analysis of the far-UV circular dichroism spectrum showed that the protein contains ~39% β-sheets but only ~5% α-helices. The protein is thermally quite stable, and exhibits a cooperative thermal unfolding transition at ~70°C, as determined by circular dichroism spectroscopy and differential scanning fluorimetry. De novo sequencing of OPI by nanoESI-Q-ToF mass spectrometry (MS) allowed the assignment of about 83% of its primary structure, which indicated that the protein shares 43% sequence identity with a putative 21 kDa trypsin inhibitor from Theobroma bicolor. An intramolecular disulfide linkage between Cys149 and Cys156 was also detected. The protein showed ~24 and ~25% sequence identity with α-amylase/subtilisin inhibitor from barley and soybean (Kunitz) trypsin inhibitor, respectively. Comparative structure modeling of OPI revealed a structural fold similar to other Kunitz-type TIs. The presence of Cys149–Cys156 disulfide bond as detected by MS and a second disulfide bond connecting Cys44–Cys91, conserved in all Kunitz-type TIs, is also identified in the model.