Analysis Of The Interactions Between Eudragit ® L100 And Porcine Pancreatic Trypsin By Calorimetric Techniques

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 1min 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. © 2011 Elsevier B.V.501180186Boeris, V., Spelzini, D., Peleteiro Salgado, J., Picó, G., Romanini, D., Farruggia, B., (2008) BBA Gen. Subj., 1780, pp. 1032-1037Hilbrig, F., Fretag, R., (2003) J. Chromatogr. B, 790, pp. 79-90Freitag, R., Schumacher, I., Hilbrig, F., (2007) Biotechnol. J., 2, pp. 685-690Rodrigues, A., Cabral, J., Taipa, M.A., (2002) Enzyme Microb. Technol., 31, pp. 133-141Zhang, C., Lillie, R., Cotter, J., Vaughan, D., (2005) J. Chromatogr. A, 1069, pp. 107-112Park, J.M., Muhoberac, B.B., Dubin, P.L., Xia, J., (1992) Macromolecules, 25, pp. 290-295Mattison, K., Brittain, I.J., Dubin, P.L., (1995) Biotechnol. Prog., 11, pp. 632-637Ladbury, J., Chowdhry, B.Z., (1998) Biocalorimetry: Applications of Calorimetry in the Biological, , J. Wiley & Sons, Inc., HardcoverLesk, A., Fordham, W., (1996) J. Mol. Biol., 258, pp. 501-537Transue, T., Krahn, J.M., Gabel, S.A., DeRose, E.F., London, R.E., (2004) Biochemistry, 43, pp. 2829-2834Beynon, R., Bond, J.S., (2001) Proteolytic Enzymes Practical Approach, , Oxford University PressFlora, J., Baker, B., Wybenga, D., (2008) Chemosphere, 70, pp. 1077-1084Mora-Huertas, C.E., Fessi, H., Elaissari, A., (2010) Int. J. Pharm., 385, pp. 113-142LCooper, C., Dubin, P.L., Kayitmazer, A.B., Turksen, S., (2005) Curr. Opin. Colloids Interface Sci., 10, pp. 52-78Gildberg, A., Overbo, K., (1990) Comp. Biochem. Physiol. B, 97, pp. 775-782Takahashi, D., Kubota, Y., Kokai, K., Izumi, T., Hirata, M., Kokufuta, E., (2000) Langmuir, 16, pp. 3133-3140Romanini, D., Braia, M., Giatte Angarten, R., Loh, W., Picó, G., (2007) J. Chromatogr. B, 857, pp. 25-31Patrickios, C.S., Sharma, L.R., Armes, S.P., Billingham, N.C., (1999) Langmuir, 15, pp. 1613-1620Kim, W., Yamasaki, Y., Kataoka, K., (2006) J. Phys. Chem. B, 110, pp. 10919-10925Jha, N.S., Kishore, N., (2009) Thermochim. Acta, 482, pp. 21-29Porfiri, M.C., Braia, M., Farruggia, B., Picó, G., Romanini, D., (2009) Process Biochem., 44, pp. 1046-1049(2001) Protein-Ligand Interactions: Hydrodynamics and Calorimetry, pp. 287-319. , Oxford University Press Inc., New York, A. Cooper, M.A. Nutley, A. Wadood, S.E. Harding, B.Z. Chowdhry (Eds.)Privalov, P.L., (1979) Adv. Protein Chem., 33, pp. 167-241Sturtevant, J.M., (1987) Annu. Rev. Phys. Chem., 38, pp. 463-488Braia, M., Porfiri, M.C., Farruggia, B., Picó, G., Romanini, D., (2008) J. Chromatogr. B, 873, pp. 139-143Santos, A.M.C., Santana, M.A., Gomide, F.T.F., Miranda, A.A.C., Oliveira, J.S., Vilas Boas, F.A.S., Vasconcelos, A.B., Santoro, M.M., (2008) Int. J. Biol. Macromol., 42, pp. 278-284Kumara, A., Srivastavaa, A., Yu Galaevb, I., Mattiasson, B., (2007) Prog. Polym. Sci., 32, pp. 1205-1237Arvind, L., Aruna, N., Roshnnie, J., Devika, T., (2000) Process Biochem., 35, pp. 777-785de Vries, R., (2004) J. Chem. Phys., 120, pp. 3475-348

Interaction of lysozyme with negatively charged flexible chain polymers

The complex formation between the basic protein lysozyme and anionic polyelectrolytes: poly acrylic acid and poly vinyl sulfonic acid was studied by turbidimetric and isothermal calorimetric titrations. The thermodynamic stability of the protein in the presence of these polymers was also studied by differential scanning calorimetry. The lysozyme-polymer complex was insoluble at pH lower than 6, with a stoichiometric ratio (polymer per protein mol) of 0.025-0.060 for lysozyme-poly vinyl sulfonic acid and around 0.003-0.001 for the lysozyme-poly acrylic acid. NaCl 0.1 M inhibited the complex precipitation in agreement with the proposed coulombic mechanism of complex formation. Enthalpic and entropic changes associated to the complex formation showed highly negative values in accordance with a coulombic interaction mechanism. The protein tertiary structure and its thermodynamic stability were not affected by the presence of polyclectrolyte. (c) 2007 Elsevier B.V. All rights reserved.8571253

Analysis of the interactions between Eudragit (R) L100 and porcine pancreatic trypsin by calorimetric techniques

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)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 (R) 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 (R) 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. Delta H degrees and Delta S degrees binding parameters obtained by ITC were positives agree with hydrophobic interaction between trypsin and polymer. However, ionic strength of 1.0 M modified the insoluble complex formation. We propose a mechanism of interaction between Eudragit (R) 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. (C) 2011 Elsevier B.V. All rights reserved.501180186FonCyT [PICT 2008-0186]Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FonCyT [PICT 2008-0186]CAPES [BR/09/10

The Averno 2 fissure eruption: a recent small-size explosive event at the Campi Flegrei Caldera (Italy)

The Averno 2 eruption (3,700 ± 50 a B.P.) was an explosive low-magnitude event characterized by magmatic and phreatomagmatic explosions, generating mainly fall and surge beds, respectively. It occurred in the Western sector of the Campi Flegrei caldera (Campanian Region, South Italy) at the intersection of two active fault systems, oriented NE and NW. The morphologically complex crater area, largely filled by the Averno lake, resulted from vent activation and migration along the NE-trending fault system. The eruption generated a complex sequence of pyroclastic deposits, including pumice fall deposits in the lower portion, and prevailing surge beds in the intermediate-upper portion. The pyroclastic sequence has been studied through stratigraphical, morphostructural and petrological investigations, and sub- divided into three members named A through C. Member A was emplaced during the first phase of the eruption mainly by magmatic explosions which generated columns reaching a maximum height of 10 km. During this phase the eruption reached its climax with a mass discharge rate of 3.2 106 kg/s. Intense fracturing and fault activation favored entry of a significant amount of water into the system, which produced explosions driven by variably efficient water-magma inter- action. These explosions generated wet to dry surge deposits that emplaced Member B and C, respectively. Isopachs and isopleths maps, as well as areal distribution of ballistic fragments and facies variation of surge deposits allow definition of four vents that opened along a NE oriented, 2 km long fissure. The total volume of magma extruded during the eruption has been estimated at about 0.07 km3 (DRE). The erupted products range in composition from initial, weakly peralkaline alkali-trachyte, to last-emplaced alkali-trachyte. Isotopic data and modeling suggest that mixing occurred during the Averno 2 eruption between a more evolved, less radiogenic stored magma, and a les

The Averno 2 fissure eruption: a recent small-size explosive event at the Campi Flegrei caldera (Italy)

The Averno 2 eruption (3,700±50 a B.P.) was an explosive low-magnitude event characterized by magmatic and phreatomagmatic explosions, generating mainly fall and surge beds, respectively. It occurred in the Western sector of the Campi Flegrei caldera (Campanian Region, South Italy) at the intersection of two active fault systems, oriented NE and NW. The morphologically complex crater area, largely filled by the Averno lake, resulted from vent activation and migration along the NE-trending fault system. The eruption generated a complex sequence of pyroclastic deposits, including pumice fall deposits in the lower portion, and prevailing surge beds in the intermediate-upper portion. The pyroclastic sequence has been studied through stratigraphical, morphostructural and petrological investigations, and subdivided into three members named A through C. Member A was emplaced during the first phase of the eruption mainly by magmatic explosions which generated columns reaching a maximum height of 10 km. During this phase the eruption reached its climax with a mass discharge rate of 3.2 106 kg/s. Intense fracturing and fault activation favored entry of a significant amount of water into the system, which produced explosions driven by variably efficient water-magma interaction. These explosions generated wet to dry surge deposits that emplaced Member B and C, respectively. Isopachs and isopleths maps, as well as areal distribution of ballistic fragments and facies variation of surge deposits allow definition of four vents that opened along a NE oriented, 2 km long fissure. The total volume of magma extruded during the eruption has been estimated at about 0.07 km3 (DRE). The erupted products range in composition from initial, weakly peralkaline alkali-trachyte, to last-emplaced alkali-trachyte. Isotopic data and modeling suggest that mixing occurred during the Averno 2 eruption between a more evolved, less radiogenic stored magma, and a less evolved, more radiogenic magma that entered the shallow reservoir to trigger the eruption. The early phases of the eruption, during which the vent migrated from SW to the center of the present lake, were fed by the more evolved, uppermost magma, while the following phases extruded the less evolved, lowermost magma. Integration of the geological and petrological results suggests that the Averno 2 complex eruption was fed from a dyke-shaped shallow reservoir intruded into the NE-SW fault system bordering to the west the La Starza resurgent block, within the caldera floor