pH dependence thermal stability of a chymotrypsin inhibitor from Schizolobium parahyba seeds.
The thermal stability of a Schizolobium parahyba chymotrypsin inhibitor (SPCI) as a function of pH has been investigated using fluorescence, circular dichroism, and differential scanning calorimetry (DSC). The thermodynamic parameters derived from all methods are remarkably similar and strongly suggest the high stability of SPCI under a wide range of pH. The transition temperature (T(m)) values ranging from 57 to 85.3 degrees C at acidic, neutral, and alkaline pH are in good agreement with proteins from mesophilic and thermophilic organisms and corroborate previous data regarding the thermal stability of SPCI. All methods gave transitions curves adequately fitted to a two-state model of the unfolding process as judged by the cooperative ratio between the van't Hoff and the calorimetric enthalpy energies close to unity in all of the pH conditions analyzed, except at pH 3.0. Thermodynamic analysis using all these methods reveals that SPCI is thermally a highly stable protein, over the wide range of pH from 3.0 to 8.8, exhibiting high stability in the pH region of 5.0-7.0. The corresponding maximum stabilities, DeltaG(25), were obtained at pH 7.0 with values of 15.4 kcal mol(-1) (combined fluorescence and circular dichroism data), and 15.1 kcal mol(-1) (DSC), considering a DeltaC(p) of 1.72 +/- 0.24 kcal mol(-1) K(-1). The low histidine content ( approximately 1.7%) and the high acidic residue content ( approximately 22.5%) suggests a flat pH dependence of thermal stability in the region 2.0-8.8 and that the decrease in thermal stability at low pH can be due to the differences in pK values of the acidic groups.
Teles
Rozeni C L
RC
Universidade de Brasília, Depto Biologia Celular, Laboratório de Biofísica, Brasilia DF, Brazil, 70910-900.
Calderon
Leonardo de A
Lde A
Medrano
Francisco J
FJ
Barbosa
João A R G
JA
Guimarães
Beatriz G
BG
Santoro
Marcelo M
MM
de Freitas
Sonia M
SM
D002152:Calorimetry, Differential Scanning; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D002942:Circular Dichroism; D006639:Histidine / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D010940:Plant Proteins / Q000737:chemistry*; D010944:Plants / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D012639:Seeds; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014466:Ultraviolet Rays
C096591:Chymotrypsin inhibitor protein, Schizolobium parahybum; D010940:Plant Proteins; D006639:Histidine; D002918:Chymotrypsin