Determination of structural and thermodynamic parameters of bovine α-trypsin isoform in aqueous-organic media

Abstract

The α-trypsin isoform is a globular protein that belongs to serine-protease family and has a polypeptide chain of 223 amino acid residues, six disulfide bridges and two domains with similar structures. 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 aggregate 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 ΔH) suggest a loosening 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 with catalytic activity. However from 60% v/v ethanol/buffer, population of folded partially states with less catalytic activity are predominant.