title: Stability and physicochemical properties of a trypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L)DC).
abstract: The stability and physicochemical properties of the major trypsin inhibitor from the winged bean seed (designated trypsin inhibitor 2) have been studied in solution. The purified inhibitor, which stoichiometrically inhibits bovine trypsin in the molar ratio 1:1, is stable over the pH range 3-11 at ambient temperatures. Only a slight decrease in inhibitory activity occurs down to pH 2, but a sharper decrease occurs at pH values above 11. The inhibitor is stable to heat up to 60 degrees C, but at higher temperatures (60-90 degrees C) it is more stable at pH than at pH 5.5 or pH 8.0. Trypsin inhibitor 2 retains its inhibitory activity in 8 M urea at pH 8.0, but is more susceptible to 8 M urea at pH 4.0. The stronger denaturant 6 M guanidine hydrochloride, however, abolishes the inhibitory activity at both pH 4.0 and pH 8.0. The inhibitor was not inactivated in 0.14 M beta-mercaptoethanol at either pH; however, reduction in the presence of 8 M urea or 6 M guanidine hydrochloride results in a loss of inhibitory activity. Circular dichroism and optical rotatory dispersion studies indicate that the inhibitor structure is characterized by beta-sheet and unordered forms and the absence of alpha-helix. The positive CD band centered at 227 nm has been used to follow conformation change as a function of temperature. In line with the stability studies, the inhibitor conformation was thermally most stable at pH 3.0 and changed increasingly as the pH was raised. This band showed little change at neutral pH up to 8 M guanidine hydrochloride. Tyrosine titration in aqueous solution indicates that 1 or 2 of the 11 tyrosines are difficult to titrate even at pH 13. A more normal titration curve is obtained in 6 M guanidine hydrochloride, although at least one tyrosine side-chain appears to be buried in the protein interior and resists complete titration at pH values in excess of 12. These data show that this inhibitor has a high degree of stability, typical of other known protein proteinase inhibitors.
mesh_terms: D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D011487:Protein Conformation; D012639:Seeds / Q000032:analysis*; D013816:Thermodynamics; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000302:isolation & purification*
chemical_list: D014361:Trypsin Inhibitors; D014357:Trypsin