Physical-chemical characterization and stability study of alpha-trypsin at pH 3.0 by differential scanning calorimetry.
alpha-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 ss-sheet secondary structure and it has two domains with similar structures. In the present work, a stability study of alpha-trypsin in the acid pH range was performed and some physical-chemical denaturation parameters were measured by using differential scanning calorimetry (DSC). The alpha-trypsin has a shelf-life (t(95%)) of about 10 months at pH 3.0 and 4 degrees C and its hydrolysis into the psi-trypsin isoform is negligible during 6 months. The observed ratio DeltaH(cal)/DeltaH(vH) is close to unity, which suggests the occurrence of a two-state transition. At pH 3.0, alpha-trypsin unfolded with T(m) = 325.9 K and DeltaH = 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 alpha-trypsin calculated at 298 K was DeltaG(U)=6.10 kcal mol(-1) at pH 3.0. These values are in the range expected for a small globular protein. These results show that the thermodynamic parameters of unfolding of beta-trypsin do not change substantially after its conversion to alpha-trypsin.
Santos
A M C
AM
Department of Biochemistry and Immunology, Biological Sciences Institute, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, MG, Brazil. alexandremcs@yahyoo.com.br
Santana
M A
MA
Gomide
F T F
FT
Miranda
A A C
AA
Oliveira
J S
JS
Boas
F A S Vilas
FA
Vasconcelos
A B
AB
Bemquerer
M P
MP
Santoro
M M
MM
D000818:Animals; D002152:Calorimetry, Differential Scanning / Q000379:methods; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry* / Q000302:isolation & purification
D007527:Isoenzymes; D014357:Trypsin