title: Characterization of the Interaction Between Pancreatic Trypsin and an Enteric Copolymer as a Tool for Several Biotechnological Applications.
abstract: Protein-polyelectrolyte complexes are very interesting systems since they can be applied in many long-established and emerging areas of biotechnology. From nanotechnology to industrial processing, these complexes are used for many purposes: to build multilayer particles for biosensors; to entrap and deliver proteins for pharmaceutical applications; to isolate and immobilize proteins. The enteric copolymer poly(methacrylic acid-co-methyl methacrylate) 1:2 (MMA) has been designed for drug delivery although its chemical properties allow to use it for other applications. Understanding the interaction between trypsin and this polymer is very important in order to optimize the mechanism of formation of this complex for different biotechnological applications.The formation of the trypsin-MMA complex was studied by spectroscopy and isothermal titration calorimetry. Structural analysis of trypsin was carried out by catalytic activity assays, circular dichroism and differential scanning calorimetry. Isothermal titration calorimetry experiments showed that the insoluble complex contains 12 trypsin molecules per MMA molecule at pH 5 and they interact with high affinity to form insoluble complexes. Both electrostatic and hydrophobic forces are involved in the formation of the complex. The structure of trypsin is not affected by the presence of MMA, although it interacts with some domains of trypsin affecting its thermal denaturation as seen in the differential scanning calorimetry experiments. Its catalytic activity is not altered. Dynamic light scattering demonstrated the presence of a soluble trypsin-copolymer complex at pH 5 and 8. Turbidimetric assays show that the insoluble complex can be dissolved by low ionic strength and/or pH in order to obtain free native trypsin.
authors.affiliation: Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ)-CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Mauricio Javier
authors.initials: MJ
authors.lastname: Braia
authors.affiliation: Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ)-CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Dana Belén
authors.initials: DB
authors.lastname: Loureiro
authors.affiliation: Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ)-CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Gisela
authors.initials: G
authors.lastname: Tubio
authors.affiliation: Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ)-CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina. Electronic address: dromanini@conicet.gov.ar.
authors.forename: Diana
authors.initials: D
authors.lastname: Romanini
mesh_terms: D001709:Biotechnology; D002151:Calorimetry; D002942:Circular Dichroism; D006801:Humans; D010179:Pancreas / Q000201:enzymology*; D011108:Polymers / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry*
chemical_list: D011108:Polymers; D014357:Trypsin
keywords: Bioseparation; Calorimetric techniques; Immobilization; Polymer; Trypsin