Purification And Partial Characteristics Of A Trypsin Inhibitor From Brassica Campestris Seeds

A new trypsin inhibitor, designated BCTI, was purified from the seeds extract of oilseed rape (Brassica campestris var. sijiu) by defatting, heating shock, precipitation with (NH4)2SO4, ion-exchange chromatography on DEAE cellulose (DEAE-52) and gel filtration on Sephadex G-50. The molecular weight of BCTI is 14.4 kD by SDS-PAGE, and the isolectric point of the inhibitor is 4.9.The activity of BCTI is inhibited by low pH and the inhibition is reversible. The experiment showed that BCTI was thermal stability. It was completely inactive in DTT until the concentration of DTT reached 20 mmol/L (15 min). The activity of BCTI is inhibited partly in 8 mol/L urea, while that is completely inactive in 6 mol/L guanidine hydrochloride.Inhibition kinetic tests of the inhibitor showed that BCTI was single-headed and competitive inhibitor.The intrinsic fluorescence emission was measured with excitation at 280 run. The fluorescence spectrum of BCTI under conditions of thermal inactivation (100℃, 15 min) showed a little decrease in fluorescence intensity. In the presence of 1 mmol/L DTT or 6 mol/L guanidine hydrochloride, the emission band appeared a red shift with increased intensity. In 8 mol/L urea, the maximum fluorescence emissionshowed a blue shift with increase intensity. With the excitation at 295 nm, the BCTl fluorescence result showed 325.6 nm peak, suggesting that the tryptophan residue was in a partially hydrophobic-shielded environment. The tryptophan fluorescence spectrum of BCTI under conditions of thermal inactivation at (100℃, 15 min) showed a little decrease in intensity and a slight red shift of the emission band. In the presence of 1 mmol/L DTT and 6 mol/L guanidine hydrochloride, the results appeared a red shift with decreased intensity. In 8 mol/L urea, the maximum fluorescence emission showed a blue shift with increased intensity. Fluorescence spectrum results indicated that disulfide bonds and hydrophobic, electrostatic interactions played significant roles in maintaining the 3D structure and stability of BCTl.