Oligomerization affects the kinetics and thermodynamics of the interaction of a Bowman-Birk inhibitor with proteases

Brand, G.D., Pires, D.A.T., Furtado, J.R., Cooper, A., Freitas, S.M. and Bloch Jr., C. (2017) Oligomerization affects the kinetics and thermodynamics of the interaction of a Bowman-Birk inhibitor with proteases. Archives of Biochemistry and Biophysics, 618, pp. 9-14. (doi:10.1016/j.abb.2017.01.009) (PMID:28132757)

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Abstract

The black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) forms concentration dependent homomultimers, as previously demonstrated by Light scattering and Atomic Force Microscopy. Considering that these self-aggregates might influence their binding to cognate enzymes, we investigated the interaction of BTCI at picomolar concentrations using surface immobilized Chymotrypsin (α-CT) and Trypsin (T) by Surface Plasmon Resonance. Our results indicate that BTCI has subnanomolar affinity to both immobilized enzymes, which is approximately two orders of magnitude higher than previously reported. Moreover, we probed the influence of temperature on protein binding equilibria in order to investigate their interaction energetics. While the BTCI/T interaction concurs with the canonical entropy-driven mechanism described for BBI interactions with serine proteinases, the BTCI/α-CT interaction does not. Our measurements indicate that bimolecular BTCI/α-CT complexes form with a negative enthalpy change and a moderate entropic increase. Direct calorimetric evaluation is in accord with the van't Hoff approximation obtained by SPR. We demonstrate that as protein concentrations increase to the micromolar range, secondary endothermic events become prevalent and affect both the kinetics and thermodynamics of protein associations. Our study reinforces that BBI interactions with serine proteinases should be studied in dilute solutions to abridge often neglected secondary interactions.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Brand, Mr Guilherme and Cooper, Professor Alan
Authors: Brand, G.D., Pires, D.A.T., Furtado, J.R., Cooper, A., Freitas, S.M., and Bloch Jr., C.
College/School:College of Science and Engineering > School of Chemistry
Journal Name:Archives of Biochemistry and Biophysics
Publisher:Elsevier
ISSN:0003-9861
ISSN (Online):1096-0384
Published Online:26 January 2017
Copyright Holders:Copyright © 2017 Elsevier Inc.
First Published:First published in Archives of Biochemistry and Biophysics 618:9-14
Publisher Policy:Reproduced in accordance with the copyright policy of the publisher

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