Determinação de parâmetros estruturais e termodinâmicos da isoforma a-tripsina bovina em solventes aquo-orgânicos

Abstract

Organic solvents are common in industrial processes that use enzymes but, at the same time, it is known that they change the properties thereof, thus the effects of aqueous-organic solvent (ethanol) in different concentration on the α-trypsin structure have been investigated by spectroscopic techniques and thermodynamic data analysis. The results from spectroscopic measurements, including far-UV Circular Dichroism, UV-Vis absorption spectroscopy, intrinsic tryptophan fluorescence and dynamic light scattering (DLS) suggest the formation of partially folded states, instead of aggregates states, at high ethanol concentration (> 60% v/v ethanol), with little loss of secondary structure, but with significant tertiary structure changes. The thermodynamic data (Tm and ∆H) suggest a loosen of intramolecular weak interactions, which reflects in a flexibility increase such that the catalytic capacity can be increased or decreased according to the ethanol concentration into the system. Overall results we suggest that in range of 0-60% v/v ethanol/buffer, α-trypsin undergoes reversible multimerization phenomena maintaining its catalytic activity. However from 60% v/v ethanol/buffer, population of folded partially states with less catalytic activity are predominant.