INVESTIGADORES
NERLI Bibiana Beatriz
congresos y reuniones científicas
Título:
Calorimetric analysis of the interaction between trypsin and EUDRAGIT® L100
Autor/es:
BRAIA, MAURICIO; TUBíO, GISELA; NERLI, BIBIANA; LOH, WATSON; ROMANINI, DIANA
Lugar:
Buzios
Reunión:
Conferencia; Calorimetric analysis of the interaction between trypsin and EUDRAGIT ® L100. Conference on Chemical Thermodynamics (ICCT 2012); 2012
Institución organizadora:
Organizing Committee for The 22nd International Conference on Chemical Thermodynamics
Resumen:
Abstract: Trypsin (TRP) is a serin-protease with a molecular weight of 23 kDa and a pI close to 11. It is widely used in the food and pharmaceutical industries and in molecular biology. Eudragit® L100 (EL100) is a synthetic flexible-chain polymer with a molecular weight of 135 kDa. It has ionizable acrylic groups that are negatively charged at high values of pH. It is widely use in the pharmaceutical industry to coat drugs for oral delivery formulations. At pH 5.00, TRP has a positive density of charges and EL100 has negative charges. Both molecules interact at pH 5.00 to form insoluble protein-polymer complexes. Since these complexes can be used for industrial and scientific purposes, it is very important to know the mechanism of interaction between TRP and EL100, and the structural stability of the enzyme. ITC experiments were performed at pH 5.00 in order to study the formation of the insoluble complex. DSC experiments allowed determining the thermal stability of the enzyme at pH 5.00 in absence and presence of EL100.Trypsin (TRP) is a serin-protease with a molecular weight of 23 kDa and a pI close to 11. It is widely used in the food and pharmaceutical industries and in molecular biology. Eudragit® L100 (EL100) is a synthetic flexible-chain polymer with a molecular weight of 135 kDa. It has ionizable acrylic groups that are negatively charged at high values of pH. It is widely use in the pharmaceutical industry to coat drugs for oral delivery formulations. At pH 5.00, TRP has a positive density of charges and EL100 has negative charges. Both molecules interact at pH 5.00 to form insoluble protein-polymer complexes. Since these complexes can be used for industrial and scientific purposes, it is very important to know the mechanism of interaction between TRP and EL100, and the structural stability of the enzyme. ITC experiments were performed at pH 5.00 in order to study the formation of the insoluble complex. DSC experiments allowed determining the thermal stability of the enzyme at pH 5.00 in absence and presence of EL100.® L100 (EL100) is a synthetic flexible-chain polymer with a molecular weight of 135 kDa. It has ionizable acrylic groups that are negatively charged at high values of pH. It is widely use in the pharmaceutical industry to coat drugs for oral delivery formulations. At pH 5.00, TRP has a positive density of charges and EL100 has negative charges. Both molecules interact at pH 5.00 to form insoluble protein-polymer complexes. Since these complexes can be used for industrial and scientific purposes, it is very important to know the mechanism of interaction between TRP and EL100, and the structural stability of the enzyme. ITC experiments were performed at pH 5.00 in order to study the formation of the insoluble complex. DSC experiments allowed determining the thermal stability of the enzyme at pH 5.00 in absence and presence of EL100. ITC experiments indicate that 15 moles of TRP interact per mol of EL100 with high affinity. ÄH° and ÄS° indicates that the interaction is endothermic and that the disorderÄH° and ÄS° indicates that the interaction is endothermic and that the disorder of the system increases. This is probably due to the release of structured-water molecules to the medium. DSC experiments show that EL100 stabilize the structure of trypsin against thermal denaturation, since Tm and ÄH°cal shift to higher values when the enzyme is in the presence of the polyelectrolyte. Moreover, the unfolding mechanism of the trypsin in the absence and the presence of EL100 is different. In the absence of EL100, trypsin shows a non-two-state model, with three transitions, indicating intramolecular cooperation between the domains of the enzyme. In the presence of EL100, trypsin shows a two-state model indicating that there is equilibrium between the native and denature states, with no intermediates. All this information (along with turbidimetric, kinetic and enzymatic activity data) can be applied to design a method for isolating, concentrating the trypsin by decantation of the insoluble trypsin-EL100 complex, or immobilized the enzyme in a solid matrix.m and ÄH°cal shift to higher values when the enzyme is in the presence of the polyelectrolyte. Moreover, the unfolding mechanism of the trypsin in the absence and the presence of EL100 is different. In the absence of EL100, trypsin shows a non-two-state model, with three transitions, indicating intramolecular cooperation between the domains of the enzyme. In the presence of EL100, trypsin shows a two-state model indicating that there is equilibrium between the native and denature states, with no intermediates. All this information (along with turbidimetric, kinetic and enzymatic activity data) can be applied to design a method for isolating, concentrating the trypsin by decantation of the insoluble trypsin-EL100 complex, or immobilized the enzyme in a solid matrix.