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

Datta D, Pohlentz G, Mondal S, Divya MB, Guruprasad L, Mormann M, Swamy MJ

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 similar to 39\% beta-sheets but only similar to 5\% alpha-helices. The protein is thermally quite stable, and exhibits a cooperative thermal unfolding transition at similar to 70 degrees 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 Cys(149) and Cys(156) was also detected. The protein showed similar to 24 and similar to 25\% sequence identity with alpha-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 Cys(149)-Cys(156) disulfide bond as detected by MS and a second disulfide bond connecting Cys(44)-Cys(91), conserved in all Kunitz-type TIs, is also identified in the model.