title: Analysis of the interactions between Eudragit® L100 and porcine pancreatic trypsin by calorimetric techniques.
abstract: Flexible-chain polymers with charge (polyelectrolytes) can interact with globular proteins with a net charge opposite to the charge of the polymers forming insoluble complexes polymer-protein. In this work, the interaction between the basic protein trypsin and the anionic polyelectrolyte Eudragit(®) L100 was studied by using isothermal calorimetric titrations and differential scanning calorimetry. Turbidimetric assays allowed determining that protein-polymer complex was insoluble at pH below 5 and the trypsin and Eudragit(®) L100 concentrations required forming the insoluble complex. DSC measurements showed that the T(m) and denaturalization heat of trypsin increased in the polymer presence and the complex unfolded according to a two-state model. ΔH° and ΔS° binding parameters obtained by ITC were positives agree with hydrophobic interaction between trypsin and polymer. However, ionic strength of 1.0M modified the insoluble complex formation. We propose a mechanism of interaction between Eudragit(®) L100 and trypsin molecules that involves both hydrophobic and electrostatic interactions. Kinetic studies of complex formation showed that the interaction requires less than 1 min achieving the maximum quantity of complex. Finally, a high percentage of active trypsin was precipitated (approximately 76% of the total mass of protein). These findings could be useful in different protocols such as a protein isolation strategy, immobilization or purification of a target protein.
authors: Laboratory of Physical Chemistry Applied to Bioseparation, Faculty of Biochemical and Pharmaceutical Sciences, National University of Rosario (UNR), Rosario, Argentina.
authors: Mauricio
authors: M
authors: Braia
authors: Gisela
authors: G
authors: Tubio
authors: Bibiana
authors: B
authors: Nerli
authors: Watson
authors: W
authors: Loh
authors: Diana
authors: D
authors: Romanini
mesh_terms: D000818:Animals; D000838:Anions; D002151:Calorimetry / Q000379:methods; D002152:Calorimetry, Differential Scanning / Q000379:methods; D004573:Electrolytes; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D009391:Nephelometry and Turbidimetry / Q000379:methods; D010179:Pancreas / Q000201:enzymology*; D011108:Polymers / Q000737:chemistry; D011109:Polymethacrylic Acids / Q000737:chemistry* / Q000378:metabolism; D013552:Swine; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry*
chemical_list: D000838:Anions; D004573:Electrolytes; D007477:Ions; D011108:Polymers; D011109:Polymethacrylic Acids; C038300:methylmethacrylate-methacrylic acid copolymer; D014357:Trypsin