CHYMOTRYPSIN INTERACTION WITH POLYANIONS: THERMODINAMICS, SOLOBILITY AND SIZE OF THE COMPLEXES

PICÓ GUILLERMO; WOITOVICH VALETTI NADIA; CAPELLA LAURA; LOMBARDI, JULIA; BOERIS VALERIA

Laussana

Simposio; 9th International Symposium on polyelectrolytes (ISP). 2012; 2012

Ecole Polytecnique du Laussana

The complex formation between chymotrypsin (ChTRP), a basic protein, and the following polyanions: carrageenan (Carr), polyvinylsulphonate (PVS), polyacrilate (PAA), Eudragit S100 (EuS) and L100 (EuL) was studied by turbidimetry, dynamic light scattering (DLS) and isothermal titration calorimetry (ITC). Thermodynamical thermal stability of the enzyme was also assayed. The net positive electrical charge of the protein is partially neutralized by the negative electrical charges of the different polyelectrolytes (PEL). The differences in the structure of the PEL, such as rigidity, molecular weight (MW), lineal charge density and acidity are related to the pH range of solubility, the stoichiometry of interaction and the size of the soluble complexes and non-soluble macroaggregates. The pH of maximum ChTRP precipitation was: 4.5 (Carr), 2.5 (PVS), 4.5 (PAA), 5.4 (EuS) and 4.6 (EuL). ChTRP precipitates with Carr and PVS in acid media below pH 6.0, while it only precipitates with the weak polyacids (PAA, EuS and EuL) above pH 3.0, when the polymer is ionized. The stoichiometry of the non-soluble macroaggregates depends on the MW of the polyanions and also on the electrical charge density: PVS is the most effective PEL to precipitate ChTRP (14.3 mg PVS/ g ChTRP) and EuS the least effective (up to 600 mg EuS/ g ChTRP), among the assayed PEL. The increase on the ionic strength of the media inhibits the ChTRP-PEL precipitation more for Carr and PVS and less in the case of the weak polyacids. The hydrodynamic diameter –determined by DLS- of the soluble complexes formed by these PEL and ChTRP is always lower than the diameter of these same PEL in solution. The size of the complexes was also dependent on the pH. The more alkaline the pH (from 6.8 to 8.5), the more extended is the complex conformation. This can be explained by taking into account the net positive electrical charge of ChTRP in that range. As the pH is increased, the enzyme becomes less efficient in neutralizing the PEL negative electrical charges, being the repulsion among the negative electrical charges in the PEL backbone enough to open the structure. ITC was used to determine the binding constant, stoichiometry, enthalpy and entropy of the formation of ChTRP-PEL soluble complexes at neutral pH. The binding constant was higher than 105 M-1 in all cases. The stoichiometry varied from 2200 mol ChTRP/mol PVS to 33 mol ChTRP/ mol EuS, in agreement with the ability of ChTRP precipitation of these PEL. The binding of ChTRP to PVS was exothermic, while the binding of ChTRP to both EuL and EuS was slightly endothermic, being the entropic factor the responsible of the soluble complexes formation. No modifications of the secondary and tertiary structure and biological activity of ChTRP were observed when it was forming soluble complexes with all the assayed PEL. The thermal thermodynamical stability of ChTRP was increased when it was bound to the PEL.