Macromolecular properties and partial amino acid sequence of a Kunitz-type protease inhibitor from okra (Abelmoschus esculentus) seeds
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Date
2019-06
Journal Title
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Publisher
Indian Academy of Sciences
Abstract
A Kunitz-type protease inhibitor (OPI, okra protease inhibitor) has been purified from okra (Abelmoschus esculentus) seedsby a combination of ammonium sulfate precipitation, anion-exchange chromatography and reverse-phase high-performanceliquid chromatography. The protein shows an apparent mass of 21 kDa on sodium dodecyl sulfate-polyacrylamide gelelectrophoresis under reducing condition. OPI exhibits inhibitory activity against trypsin. Analysis of the far-UV circulardichroism spectrum showed that the protein contains *39% b-sheets but only *5% a-helices. The protein is thermallyquite stable, and exhibits a cooperative thermal unfolding transition at *70C, as determined by circular dichroismspectroscopy 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 identitywith a putative 21 kDa trypsin inhibitor from Theobroma bicolor. An intramolecular disulfide linkage between Cys149 andCys156 was also detected. The protein showed *24 and *25% sequence identity with a-amylase/subtilisin inhibitor frombarley and soybean (Kunitz) trypsin inhibitor, respectively. Comparative structure modeling of OPI revealed a structuralfold similar to other Kunitz-type TIs. The presence of Cys149–Cys156 disulfide bond as detected by MS and a seconddisulfide bond connecting Cys44–Cys91, conserved in all Kunitz-type TIs, is also identified in the model.
Description
Keywords
Abelmoschus esculentus, circular dichroism, differential scanning fluorimetry, Kunitz protease inhibitor, Malvaceae, nano-ESI mass spectrometry, RP-HPLC
Citation
Datta Debparna, Pohlentz Gottfried, Mondal Saradamoni, Divya M Bala, Guruprasad Lalitha, Mormann Michael, Swamy Musti J. Macromolecular properties and partial amino acid sequence of a Kunitz-type protease inhibitor from okra (Abelmoschus esculentus) seeds. Journal of Biosciences. 2019 Jun; 44(2): 1-11