Purification And Some Properties Of A Trypsin Inhibitor From Spinacia Oleracea L Seeds

A trypsin inhibitor named SOTI was purified from the seeds of Spinacia oleracea L by defatting, extracting with acidic buffer, heating, precipitation, ion-exchange chromatography, affinity chromatography and gel filtration. A series study of SOTI showed that the inhibitor has a relative molecular weight of 22kD by SDS-PAGE.The maximum absorption is at 276nm according to the ultraviolet spectrum.Inhibition kinetic tests of the inhibitor show that SOTI is single-headed and competitive inhibitor.The intrinsic fluorescence emission was measured with excitation at 280nm. At the wave length the SOTI fluorescence results evidenced a red shift, suggesting that the tyrosine residue is in a partially solvent-shielded environment. 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 the emission band appeared a red shift with decrease intensity. At 6mol /L GlnHCl, the emission band appeared a red shift with decrease intensity. This red shift is consistent with a tyrosine environment totally exposed to the solvent.With the excitation at 295nm, at the wave length the SOTI fluorescence results evidenced a red shift, suggesting that the tryptophan residue is in a partially hydrophobic environment. At 8mol/L Urea, the maximum fluorescence emissionshowed a red shift with decreased intensity. In the presence of lmmol/L DTT the results evidenced a red shift with decreased intensity. At 6mol/L GlnHCl, the emission band appeared a red shift with decreased 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 SOTI.