The Conformational Properties of the Basic Pancreatic Trypsin-Inhibitor

The conformational properties of the basic pancreatic trypsin-inhibitor have been studied using both physical and chemical techniques. The structural properties of this mini-protein are quite unusual. The folded conformation remains unaltered at 77 °C and pH 2.1 or in 6 M guanidine-HC] at pH 7.5. The selective cleavage [22] of the disulfide bridge Cys14-Cys38 reduces considerably the stability of the inhibitor towards heat or guanidine-HCl. A conformational study of the carboxamidomethyl, carboxymethyl, and aminoethyl derivatives of the inhibitor has been carried out after partial reduction and modification of Cys14 and Cys38 by iodoacetamide, iodoacetic acid, and ethyleneimine. There exist three folded isomeric forms of the inhibitor between pH 1 and 11. Form I is the most stable form; it exists near neutral pH. It is extremely thermostable and remains folded in 6 M guanidine-HCl. Form II is stable below pH 1.5–2; its transition temperature is 81 °C. Form III is predominant at pH 10–11 at 20 °C. A detailed analysis of the conformational isomers I, II and III has been carried out with the carboxamidomethylated inhibitor. Both physico-chemical and chemical data indicate that the acidic conformational change I ⇌ II is dependent upon the ionisation of a buried carboxylate which may be the side-chain of Glu7. The alkaline isomerisation I ⇌ III is induced by the unmasking of the α-ammonium of the N-terminal arginine residue. The apparent pK of the group is 9.4 at 25 °C. Acetylation of the α-amino group stabilizes a form very similar to III and prevents the formation of form I at neutral pH.