Determination of structural and thermodynamic parameters of bovine α-trypsin isoform in aqueous-organic media.
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 (Tmand 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.
Pos-Graduate Program at Biochemistry and Pharmacology, Federal University of Espírito Santo, Vitória, ES, 29043090, Brazil.
Dayanne Pinho
DP
Rosa
Pos-Graduate Program at Biochemistry and Pharmacology, Federal University of Espírito Santo, Vitória, ES, 29043090, Brazil.
Evaldo Vitor
EV
Pereira
Pos-Graduate Program at Biochemistry and Pharmacology, Federal University of Espírito Santo, Vitória, ES, 29043090, Brazil.
Antonio Victor Baioco
AVB
Vasconcelos
Physiological Science Department, Federal University of Espírito Santo, Vitória, ES, 29040090, Brazil.
Maria Aparecida
MA
Cicilini
Federal Institute of Education, Science and Technology of Espírito Santo, Aracruz, ES, 29192733, Brazil.
André Romero
AR
da Silva
Pos-Graduate at Biological Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, 21044020, Brazil.
Caroline Dutra
CD
Lacerda
Pos-Graduate at Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, MG, 31270901, Brazil.
Jamil Silvano
JS
de Oliveira
Pos-Graduate at Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, MG, 31270901, Brazil.
Marcelo Matos
MM
Santoro
Pos-Graduate Program at Biochemistry and Pharmacology, Federal University of Espírito Santo, Vitória, ES, 29043090, Brazil.
Juliana Barbosa
JB
Coitinho
Pos-Graduate Program at Biochemistry and Pharmacology, Federal University of Espírito Santo, Vitória, ES, 29043090, Brazil. Electronic address: alexandre.santos@ufes.br.
Alexandre Martins Costa
AMC
Santos
D000818:Animals; D055162:Biocatalysis; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004795:Enzyme Stability / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism*; D014867:Water / Q000737:chemistry*
D007527:Isoenzymes; D014867:Water; D000431:Ethanol; D014357:Trypsin
Enzymology; Isoforms; Organic solvent; Structure; Thermodynamic; Trypsin