Physical-chemical characterization and stability study of alpha-trypsin at ph 3.0 by differential scanning calorimetry

Full text: α-Trypsin is a serine-protease with a polypeptide chain of 223 amino acid residues and six disulfide bridges. It is a globular protein with predominance of antiparallel β-sheet secondary structure and it has two domains with similar structures. In the present work, a stability study of α-trypsin in the acid pH range was performed and physical-chemical denaturation parameters were measured by using differential scanning calorimetry (DSC). The α-trypsin has a shelf-life (t95%) of about ten months at pH 3.0 and 4 deg C and its hydrolysis into the Ψ-trypsin isoform is negligible during six months as monitored by mass spectrometry (Micromass Q-ToF). The observed ΔHcal/ΔHvH ratio is close to unity for α-trypsin denaturation, which suggests the occurrence of a two-state transition, devoid of molten-globule intermediates. At pH 3.0, α-trypsin unfolded with Tm 325.9 K and ΔH= 99.10 kcal mol-1, and the change in heat capacity between the native and unfolded forms of the protein was estimated to be 1.96 ± 0.18 kcal mol-1 K-1. The stability of α-trypsin calculated at 298 K and at pH 3.0 was ΔGU = 6.10 kcal mol-1. These values are in the range expected for a small globular protein. These results show that the thermodynamic parameters for unfolding of β-trypsin do not change substantially after its conversion to α-trypsin