Purification And Partial Characterization Of A Low-molecular Trypsin Inhibitor From Oilrape

A low-molecular-mass trypsin inhibitor named BNTIa was purified from the seeds extract of oilrape by defatting, heating, precipitation with (NH4)2SO4, ion-exchange chromatography on DEAE cellulose (DEAE-52). Another trypsin inhibitor named BNTIb also existed in the extract of oilrape. The rupture of disulfide bonds will alter the inhibitory activity of BNTIa and BNTIb. Both isolectric potint of the two inhibitor are 5.20.A series studies of BNTIa showed that the inhibitor has a relative molecular weight of 8700 by SDS-PAGE. The inhibitor is composed of one peptide chain and thermal stablization. The inhibitor is thermal stability, and is tetramer on the native condition. The maximum absorption is at 268nm according to the ultraviolet sqictrum.The intrinsic fluorescence emission was measured with excitation at 280nm. At the wave length the BNTI fluorescence results evidenced a red shift, suggesting that the tyrosine residue is in a partially solvent-shielded environment. The tyrosine fluorescence spectrum of BNTI under conditions of thermal inactivation at 80 ? 60min, showed a decrease in fluorescence intensity and a blue shift of the emission band. At 8mol/L Urea, the maximum fluorescence emission showed a blue shift with decrease intensity. In the presence of 1mmol/L DTT the results evidenced two emission band with decreased intensity . At 6mol /L GlnHCl, the emission band appeared a red shift with increased intensity. This red shift is consistent with atyrosine environment totally exposed to the solventWith the excitation at 295nm. At the wave length the BNTI fluorescence results evidenced a blue shift, suggesting that the tryptophan residue is in a partially hydrophobic-shielded environment. The tryptophan fluorescence spectrum of BNTI under conditions of thermal inactivation at 80 癈 60min, showed a decrease in fluorescence intensity and a red shift of the emission band. At 8mol/L Urea, the maximum fluorescence emission showed a red shift with decreased intensity. In the presence of 1mmol/L DTT the results evidenced a blue shift with decreased intensity . At 6mol/L GlnHCl, the emission band appeared a red shift with increased intensity. This red shift is consistent with a tryptophan environment totally exposed to the solvent. Fluorescence spectrum results showed that disulfide bonds and hydrophobic, electrostatic interactions play significant roles in maintaining the stability of BNTI.