Determination of Structural and Thermodynamic Parameters of bovine α-trypsin isoform in Aqueous-Organic Media
Dayanne Pinho Rosa, Evaldo Vitor Pereira, Antonio Victor Baioco Vasconcelos, Maria Aparecida Cicilini, André Romero da Silva, Caroline Dutra Lacerda, Jamil Silvano de Oliveira, Marcelo Matos Santoro, Juliana Barbosa Coitinho, Alexandre Martins Costa Santos
International Journal of Biological Macromolecules
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.
Circular dichroism, Secondary structure, Chemical stability, Aggregation, Biochemistry