title: Stability studies on derivatives of the bovine pancreatic trypsin inhibitor.
abstract: Gibbs energy, enthalpy, and entropy data were determined for two selectively modified analogues of bovine pancreatic trypsin inhibitor (BPTI) to provide a model free set of thermodynamic parameters that characterize (a) the energetic and entropic contributions of the 14-38 disulfide bridge and (b) the variation of the overall stability resulting from the introduction of two negative charges into the positions 14 and 38. The two BPTI analogues studied were BPTI having Cys-14 and Cys-38 carboxymethylated (BPTI-RCOM) and BPTI having Cys-14 and Cys-38 carboxamidomethylated (BPTI-RCAM). They were obtained from native BPTI by reduction, followed by modification of the sulfhydryl groups with iodoacetic acid or iodoacetamide, respectively. The temperature dependence of all thermodynamic parameters of BPTI is drastically altered in the absence of the third disulfide bridge. Even the apparently minute difference of two dissociable carboxyl groups instead of uncharged amide groups in positions 14 and 38 has surprisingly large effects on the temperature dependence of the stabilization enthalpy. The Gibbs energy of BPTI at pH 2, 25 degrees C, decreases by approximately 70% when the 14-38 disulfide bond is cleaved. BPTI-RCOM is more stable than BPTI-RCAM in the whole pH range studied. The difference of -4 kJ/mol at pH 2, 25 degrees C, is reduced to -2.7 kJ/mol at pH 5, 25 degrees C. This finding demonstrates that the presence of two negative charges reduces the higher stability of BPTI-RCOM slightly; however, the overall effect of the two charges is still a stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D007611:Aprotinin / Q000031:analogs & derivatives*; D002417:Cattle; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
chemical_list: D007611:Aprotinin