Estudo comparativo das alterações induzidas por solventes orgânicos sobre a atividade catalítica, conformação e termodinâmica das isoformas beta- e alfa-tripsina bovinas

Abstract

Bovine trypsin has six isoforms, being the α-trypsin and β-trypsin isoforms well characterized in aqueous media. Some differences are well established as the molecular weight, the number of peptide chains and enzyme activity profile. In this work, the α- and β-trypsin isoforms were compared concerning their activities, structure, and thermodynamics properties in the aqueous-organic system. The organic polar solvents tested were methanol, ethanol, and n-propanol. Enzyme activity, conformational change, secondary structure, supramolecular organization, and thermodynamics were evaluated by different spectroscopic (UV, CD, and DLS) and calorimetric techniques. The comparative results showed that the changes induced by each solvent on the structure and activity of the same trypsin isoform occur at different concentrations. The β-trypsin isoform showed superior amidase activity in the presence of the tested organic solvents. Better results for enzyme activity are found in ethanol. No statistically significant losses were found in the content of defined secondary structures for both isoforms, but the β-trypsin isoform suffered more rearrangement. The addition of increasing concentration of organic solvent causes redistribution in the supramolecular arrangement of both isoforms, with the formation of agglomerates in 60% (v/v). In 80% (v/v) of ethanol were observed distinct behavior between the isoforms: the β-trypsin formed reversible agglomerates while the ɑ-trypsin formed insoluble aggregates. The use of different organic solvent concentrations revealed a more cooperative denaturation process for β-trypsin and a less stable native state for ɑ-trypsin. The thermodynamic studies have proved all the predictions done with the spectroscopic data and showed that the β-trypsin conformation was more resistant to the different ethanol concentrations. However, it presented more accentuated conformational differences when compared the results between aqueous and aqueous-organic media. On the other hand, the ɑ-trypsin had lower conformational stability, represented by gradual reductions in thermodynamic parameters up to 60% (v/v).