IIBYT   23944
INSTITUTO DE INVESTIGACIONES BIOLOGICAS Y TECNOLOGICAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
The use of Isothermal Titration Calorimetry to measure enzyme kinetics in molecular crowded systems
Autor/es:
NOLAN, MV; JM SANCHEZ; M.A. PERILLO
Lugar:
Tucumán
Reunión:
Encuentro; XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012
Institución organizadora:
Sociedad Argentina de biofísica
Resumen:
The cellular cytoplasm is composed of different molecular species that generate a crowded environment. Macromolecules occupy an important volume fraction that cannot be occupied by other molecules. As a consequence of this, macromolecules undergo steric and diffusional restrictions that affect their properties. The effects of crowding on biochemical reaction rates are complex because although crowding reduces diffusion, it increases thermodynamic activities.beta-galactosidase (beta-Gal) is an enzyme that catalyzes lactose hydrolysis. It has been extensively studied because of its nutritional, biotechnological and therapeutic impact. Not only in the cellular milieu but also in situations of technological interest the activity of b-Gal has to be evaluated in crowded systems.Determination of enzyme kinetics is traditionally measured with a variety of methods including spectroscopy. Using spectral methods when measuring enzyme kinetics in crowded solutions are limited due to scattering and/or absorption of the light. The use of calorimetry to measure the rate of enzymatic reactions appears as a good option in these cases. Isothermal titration calorimetry (ITC) measures the heat-flow associated with a given chemical reaction. The rate of the enzymatic reaction is proportional to this heat flow which in turn results the primary experimental observable. Since practically all reactions produce or consume heat, enzyme activity can be monitored in real time without the need of probes, substrate analogues, etc.The aims of this study were: i) to investigate the effect of macromolecular crowding on the kinetics of hydrolysis of o-nitrophenyl galactopyranoside (ONPG) catalyzed by b-Gal, ii) to compare ONPG hydrolysis data obtained by UV-visible spectroscopy and ITC, to validate the ITC technique to study enzyme kinetics. Molecular crowding was simulated with solutions of polyethylene glycol at different concentrations. The results showed that Vmax was not affected, while the affinity of the enzyme (KM) suffered a significant decrease at growing molecular crowding levels. It could be due to the effect of crowing on the thermodynamic activity of water associated to changes in the solvent structure1. The results obtained by the two techniques (spectroscopy and ITC) were not significantly different, allowing us to validate the use of ITC as a technique to study the kinetics of reactions mediated by enzymes both in diluted and in crowded solutions1 Clop et al, J. Phys. Chem. B(2012) 116, 11953−11958Acknowledgements: CONICET, SeCyT-UNC, Mincyt-Prov.Córdoba y Foncyt.