Thermodynamics of acid-proteins-chitosan soluble complexes formation: an isothermal titration calorimetry study

BOERIS, V.,; BURGOS I.,; FIDELIO G.D.,; PICO, G.

BUZIOS, RJ

Congreso; 67th Calorimetry Conference ICCT 2012; 2012

Calorimetry Conference and Inernatinal conference on chemical thermodynamics (ICCT)

Proteins can interact with polyelectrolytes depending on several factors related to the protein (isoelectric pH, superficial aminoacid distribution, size), the polyelectrolyte (chemical nature of the charged groups, flexibility, electrical density) and also to the medium conditions (pH, ionic strength, presence of cosolutes). The interaction may result in the formation of soluble complexes or non-soluble macroaggregates. The formation of these complexes has been used for different purposes as protein separation, immobilization and stabilization. The ability of different synthetic and natural polyelectrolytes to interact with proteins has been studied using techniques such as: spectroscopy, turbidimetry and light scattering. Nevertheless, the desire to understand what drives protein ? polyelectrolyte association has increasingly led to the use of isothermal titration calorimetry (ITC) to characterize the process.Chitosan (Chi), a natural polycation, is commonly used for the separation and purification of enzymes by means of precipitation. Chi forms complexes with acid proteins by coulombic attraction, since the positive charge of the amine polymer groups interacts with the negatively charged groups in the protein. These complexes are soluble at low pH value (below the pH of the soluble?non-soluble complex equilibrium) while at higher pH, they are non-soluble. This property has made Chi useful for its application in separation and purification of enzymes by precipitation.Bovine liver catalase (CAT), bovine serum albumin (BSA) and bovine abomasums pepsin (PEP) are known to interact with Chi to form soluble or non-soluble complexes. We have used ITC to thermodynamically characterize the first step of these acid proteins - Chi complexes formation.Chi 5 mg/L was titrated with CAT, BSA and PEP 2.5 mg/mL in 50 mM acetic medium pH 4.00. In all cases, a classical saturation behavior was observed and it applies to the binding of the proteins to Chi with N identical and independent binding sites. The stoichiometry and thermodynamic parameters associated to the proteins-Chi soluble complexes formation were calculated:The values of the affinity constant suggest a moderately strong interaction, in agreement with a coulombic mechanism. We have detected a high negative enthalpy change of interaction between the proteins and Chi. On the other hand, the entropic change was always positive, contributing to decrease the value of the free energy associated to the soluble complex formation.The results of this work allowed us to better understand the thermodynamic and molecular mechanism of interaction between these proteins and Chi. These findings may be useful in different protocols as a protein isolation strategy, immobilization or in purification of a target protein using Chi.