Name: Evaldo Vitor PereiraType: MSc dissertationPublication date: 24/02/2015Advisor:

Name Rolesort ascending
Alexandre Martins Costa Santos Advisor *

Examining board:

Name Rolesort ascending
Rita Gomes Wanderley Pires Internal Examiner *
Marcelo Porto Bemquerer External Examiner *
Patricia Machado Bueno Fernandes (M/D) External Examiner *
Juliana Barbosa Coitinho Goncalves Co advisor *
Alexandre Martins Costa Santos Advisor *

Summary: Bovine α-trypsin isoform was purified from commercial samples by cationic ion exchange chromatography. The purity of this sample was confirmed by MALDI-ToF and the value found was 23,312 Da. Trypsin was subjected to different concentrations of mono alcohols and DMSO and it was able to keep half of its activity in 60% v/v ethanol-buffer. Due this capacity, other subsequent tests were performed in order to a better understanding of this process. The effect of Ca2+ and Cl- ions stabilized α-trypsin till 20 mmol.L-1, in both aqueous and ethanol-buffer systems, however above this concentration, these ions tend to destabilize the protein molecule influencing the reduction in its catalytic activity. However, the structure of α-trypsin monitored by UV spectroscopy on the aromatic residues, with addition of 80% v / v ethanol, remained as in native form. A kinetic test comparing the α-trypsin activity in ethanol-buffer and buffer showed that ethanol act as a non-competitive inhibitor. However, in thermodynamic assay using the monitoring technique aromatic residue, Tyr at 285 nm, showed that only n-propanol, in the series of monoalcohols, contributed significantly to decrease the Tm as comparing to with other alcohols and buffer system. Finally, we can conclude that the organic solvent system decreases the enzymatic activity of two ways: by acting as non-competitive inhibitor and causing irreversible changes in the protein by decreasing the number of molecules available to perform catalysis.Access to document

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