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

  • Debparna Datta
  • Gottfried Pohlentz
  • Saradamoni Mondal
  • M Bala Divya
  • Lalitha Guruprasad
  • Michael Mormann
  • Musti J SwamyEmail author


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.


Abelmoschus esculentus circular dichroism differential scanning fluorimetry Kunitz protease inhibitor Malvaceae nano-ESI mass spectrometry RP-HPLC 





N-α-benzoyl-l-arginine ethyl ester


collision induced dissociation


circular dichroism


differential scanning fluorimetry




okra protease inhibitor


protease inhibitor


reverse-phase high-performance liquid chromatography


retention time


sodium dodecyl sulfate-polyacrylamide gel electrophoresis


soybean Kunitz-trypsin inhibitor


trifluoroacetic acid


trypsin inhibitor



This work was supported by a research grant from the Department of Biotechnology (India) to MJS. We thank the University Grants Commission (India) for its support through the UPE-II grant to the University of Hyderabad and the CAS program to the School of Chemistry. Support from the Department of Science and Technology under the FIST and PURSE programs is gratefully acknowledged. DD was supported by Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), India. The authors are thankful to Nano Temper technologies, Bangalore, India for the use of Prometheus NT.48 instrument and their application specialist, Ms. Saji Menon for help with the DSF experiments. This project was carried out under IRTG-MCGS (GRK 1549) financed by DFG in Germany and UGC in India.

Supplementary material

12038_2019_9859_MOESM1_ESM.docx (73 kb)
Supplementary material 1 (DOCX 72 kb)


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Copyright information

© Indian Academy of Sciences 2019

Authors and Affiliations

  1. 1.School of ChemistryUniversity of HyderabadHyderabadIndia
  2. 2.Institute for HygieneUniversity of MünsterMünsterGermany

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