title: Oligomerization affects the kinetics and thermodynamics of the interaction of a Bowman-Birk inhibitor with proteases.
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.
authors.affiliation: Laboratório de Espectrometria de Massa, EMBRAPA - Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil; Laboratório de Síntese e Análise de Biomoléculas, Instituto de Química, Universidade de Brasília, Brasília, DF, Brazil. Electronic address: gdbrand@unb.br.
authors.forename: G D
authors.initials: GD
authors.lastname: Brand
authors.affiliation: Laboratório de Espectrometria de Massa, EMBRAPA - Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil; Instituto Federal de Educação, Ciência e Tecnologia de Goiás, Luziânia, GO, Brazil.
authors.forename: D A T
authors.initials: DA
authors.lastname: Pires
authors.affiliation: Departamento de Polícia Federal, Unidade Técnico-Científica, Londrina, PR, Brazil.
authors.forename: J R
authors.initials: JR
authors.lastname: Furtado
authors.affiliation: School of Chemistry, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
authors.forename: A
authors.initials: A
authors.lastname: Cooper
authors.affiliation: Laboratório de Biofísica, Instituto de Biologia, Universidade de Brasília, Brasília, DF, Brazil.
authors.forename: S M
authors.initials: SM
authors.lastname: Freitas
authors.affiliation: Laboratório de Espectrometria de Massa, EMBRAPA - Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil.
authors.forename: C
authors.initials: C
authors.lastname: Bloch
mesh_terms: D000818:Animals; D002151:Calorimetry; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology; D010447:Peptide Hydrolases / Q000737:chemistry*; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D013696:Temperature; D013816:Thermodynamics; D014357:Trypsin / Q000737:chemistry; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000737:chemistry*
chemical_list: D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D010447:Peptide Hydrolases; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D014357:Trypsin
keywords: BBI; Isothermal titration calorimetry; Protease inhibitor; Protein interactions; Surface plasmon resonance