Characterization of ß-trypsin at acid pH by differential scanning calorimetry

Abstract

Trypsin is a serino-protease with a polypeptide chain of 223 amino acid residues and contains six disulfide bridges. It is a globular protein with a predominance of antiparallel ß-sheet and helix in its secondary structure and has two domains with similar structures. We assessed the stability of ß-trypsin in the acid pH range using microcalorimetric (differential scanning calorimetry) techniques. Protein concentrations varied in the range of 0.05 to 2.30 mg/ml. Buffer solutions of 50.0 mM ß-alanine and 20.0 mM CaCl 2 at different pH values (from 2.0 to 4.2) and concentrations of sorbitol (1.0 and 2.0 M), urea (0.5 M) or guanidinium hydrochloride (0.5 and 1.0 M) were used. The data suggest that we are studying the same conformational transition of the protein in all experimental situations using pH, sorbitol, urea and guanidinium hydrochloride as perturbing agents. The observed van't Hoff ratios (∆H cal /∆H vH ) of 1.0 to 0.5 in the pH range of 3.2 to 4.2 suggest protein aggregation. In contrast, ∆H cal /∆H vH ratios equal to one in the pH range of 2.0 to 3.2 suggest that the protein unfolds as a monomer. At pH 3.00, ß-trypsin unfolded with Tm = 54 º C and ∆H = 101.8 kcal/mol, and the change in heat capacity between the native and unfolded forms of the protein (∆Cp) was estimated to be 2.50 ± 0.07 kcal mol -1 K -1 . The stability of ß-trypsin calculated at 298 K was ∆G D = 5.7 kcal/mol at pH 3.00 and ∆G D = 15.2 kcal/mol at pH 7.00, values in the range expected for a small globular protein.

Amyloid-like Fibril Formation by Trypsin in Aqueous Ethanol. Inhibition of Fibrillation by PEG

“…Trypsin is a globular protein with a predominance of antiparallel -sheet and -helix in its secondary structure. Trypsin is composed of two domains, in both of which 6 strands form an anti-parallel betabarrel in the native structure of trypsin [25][26][27]. Trypsin forms different secondary structures in organic solvents, depending on the nature and concentration of the solvent, circular dichroism (CD) measurements indicating that the extent of -sheet structure formation varies in the range 31-61% at pH 3.0 [28,29].…”

Efeito da composição do solvente sobre a estabilidade de proteínas em soluções aquosas

“…A estabilização e a desnaturação de proteínas são temas de grande interesse fisiológico 5 , terapêutico 6,7 e biotecnológico 8,9 , onde o controle da estabilidade de uma proteína representa uma importante estratégia para manutenção de sua atividade biológica. Além disso, atualmente a análise da estabilidade se tornou um instrumento poderoso para caracterização de proteínas [10][11][12] , cada vez mais utilizado na literatura com este propósito [13][14][15][16] . A estabilização e desnaturação de proteínas têm sido revistas em trabalhos clássi-cos, que mostram a natureza complexa desses processos [17][18][19][20][21][22] .…”

Thermodynamic evaluation and modeling of proton and water exchange associated with benzamidine and berenil binding to ß-trypsin

“…After zymogen cleavage, the active ß-trypsin form acquires higher plasticity, maintaining the spatial distribution of the several segments practically intact (12). Differential scanning calorimetry characterization at acid pH plus the pH titration of native and unfolded forms allowed the estimation of the stability of this enzyme at acid and neutral pH (13).…”