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

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 o...

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Bibliographic Details
Published in:Archives of biochemistry and biophysics 2017-03, Vol.618, p.9-14
Main Authors: Brand, G.D., Pires, D.A.T., Furtado, J.R., Cooper, A., Freitas, S.M., Bloch, C.
Format: Article
Language:English
Subjects:
Animals
BBI
Calorimetry
Cattle
Chymotrypsin - chemistry
Isothermal titration calorimetry
Kinetics
Pancreas - enzymology
Peptide Hydrolases - chemistry
Protease inhibitor
Protein Binding
Protein interactions
Surface Plasmon Resonance
Temperature
Thermodynamics
Trypsin - chemistry
Trypsin Inhibitor, Bowman-Birk Soybean - chemistry
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Summary: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.
ISSN:0003-9861
1096-0384
DOI:10.1016/j.abb.2017.01.009