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Role of disulfide linkages in structure and activity of Kunitz trypsin inhibitor from Trigonella foenum-graecum (fenugreek) seeds and its porphyrin binding studies

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Abstract

Kunitz trypsin inhibitor (TfgKTI) isolated from fenugreek (Trigonella foenum-graecum) seeds inhibited the enzyme trypsin. The inhibitor is resistant to wide range of temperatures (37–90 °C) and pHs (3–10) but reduction of the inhibitor with dithiothreitol abolished trypsin inhibitory activity. Reduction of the disulfide linkages with dithiothreitol affected the tertiary structure significantly, whereas the secondary structure was not affected considerably. The kinetic plots of the reduction as followed by protein activity and loss in tertiary structure could be comparable. Chemical denaturation of TfgKTI with guanidine hydrochloride and fluorescence quenching studies by two neutral quenchers (acrylamide and succinimide), an anionic quencher Iodide ion (I) and a cationic quencher Cesium ion (Cs+) suggested higher unfolding of protein under reduced conditions compared with native protein. Chemically denatured TfgKTI showed refolding, while it is irreversible with reduced protein. Native TfgKTI showed high binding affinity with porphyrin, but lost its binding capacity under reduced condition. Thus, disulfide linkages play a predominant role in maintaining the three-dimensional structure of the TfgKTI. The loss of inhibitory activity is correlated with loss in tertiary structure.

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Abbreviations

PIs:

Protease inhibitors

TfgKTI:

Trigonella foenum-graecum Kunitz trypsin inhibitor

DTT:

Dithiothreitol

HPLC:

High-performance liquid chromatography

BAEE:

Nα-benzoyl-l-arginine ethyl ester

CD:

Circular dichroism

Mr:

Molecular weight

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Acknowledgments

We are grateful to Prof. Lalitha Guruprasad School of Chemistry, University of Hyderabad for providing the infrastructure. We also thank School of Chemistry, University of Hyderabad for providing various equipments for the project.

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Correspondence to Oddepally Rajender.

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Pallavi, H., Rajender, O. Role of disulfide linkages in structure and activity of Kunitz trypsin inhibitor from Trigonella foenum-graecum (fenugreek) seeds and its porphyrin binding studies. J. Plant Biochem. Biotechnol. 30, 477–486 (2021). https://doi.org/10.1007/s13562-020-00634-0

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