| Proteinse inhibitors (PIs) are regulatory proteins controlling activity of proteolyticenzymes. They are grouped primarily as serine, cysteine, aspartic andmetallo-proteinse inhibitors. Of these, serine PIs are the most studied and have beenisolated from various leguminous plants. The leguminous plants contain two majorfamilies of serine PIs, the Kunitz and Bowman-Birk type inhibitors. Kunitz typeinhibitors are usually 18-22 kDa proteins, with two disulfide linkages and a singlereactive site of trypsin, whereas Bowman-Birk type inhibitors are usually 8-10 kDaproteins, with seven disulfide linkages and two reactive sites of trypsin andchymotrypsin.Serine PIs form an important group of defense proteins in plants since theseproteins inhibit serine proteases in the alkaline midgut of many herbivorouslepidopterous species. Serine PIs prevent proteolysis in the insect midgut which leadsto inadequate digestion and absorption of essential amino acid, retarded growth and,eventually, death by starvation. However, in the evolving processes, insects adapt tohost plant PIs by producing sufficient serine class proteinases and/or alteringexpression of alternative gut proteinase to overcome the native PIs produced by hostplant species. These results highlight that an alternative strategy with which toenhance plant defense systems is to screen new defensive PIs from the non-host otherthan the host plant species. The Chinese medicine Jue-ming-zi, the seed of Cassiaobtusifolia or Cassia tora, is commonly found in the southwest regions in China andhas been widely used in many Chinese medical prescriptions for the treatment ofheadache, dizziness, dysentery, and eye disease due to its pharmaceutical ingredients.Besides the utilization of this specie as a source of pharmacy, Jue-ming-zi is also usedas food and vegetables to supplement the inadequate animal protein ingestion, because of the relatively high contents of nutritional ingredients contained in theseeds.Pieris rapae (Lepidoptera, Pieridae) is an economical pest, which causes severecrucifer crop damages in several countries of Asia annually. It is a polyphagous pestof several crucifer plants (e. g. Brassica oleracea, Brassica oleracea, Brassica rapa,and Brassica napus). In this work, we reported the isolation, characterization andfunction of a trypsin inhibitor from Cassia obtusifolia and Cassia tora seedsrespectively.Cassua tora trypsin inhibitor (COTI) was isolated from Cassia tora seeds by waterextraction, thermal denaturation, 30%-50% ammonium sulfate fractionation,DEAE-Sephadex A-50 and Sephadex G-75 chromatography. SDS-PAGE underreducing conditions showed a clear band with molecular mass of 6.5 kDa.Surprisingly, a single band was observed in SDS-PAGE under nonreducing conditionwith molecular mass of 13 kDa. The irregular 13 kDa band was the dimerization ofthe CTTI, since Bowman-Birk protease inhibitors had extreme hydrophobic propertyand tendency to self-associate. The inhibitor was purified more than 19-fold with arecovery of 13% and a final inhibitory activity of 317 UI mg-1 protein. COTI showedhigh endurance in a wide pH range of 2-12 and remained around 85% of the initialactivity. The thermal stability of the inhibitor considerably decreased at temperatures＞60℃. The purified inhibitor showed a two-state transition during the heatingprocess. CTTI was stable and retained nearly 82% of its activity by heating to 60℃,but its activity sharply decreased after treatment at 70℃, causing the loss of 77% ofthe initial activity. The emission spectrum of native CTTI exhibited a peak at 337 nmthat was characteristic emission of tryptophan residues partially solvent exposed. Thefluorescence emission peak became red-shifted to 344 nm upon heat treatment,indicating exposure of the tryptophans to a more hydrophilic environment.Furthermore a quenching in the fluorescence intensity was observed. These resultssuggested that partly unfolding of CTTI occurred after treatment at 70℃. The far-UVCD spectrum of the native CTTI was observed as a strong positive peak at 189 nmand a negative peak at 203 nm, indicating the present of mainβ-sheet in the secondarystructure. Based on CD Pro analysis, the percentage of secondary structure of nativeCTTI protein was composed of 12.3%α-Helix, 30.9%β-sheet, 20.1%β-turn and36.8% random coil. Thermal treatment of CTTI results in transformation of 7.2%β-sheet and 3.7%β-turn conformation into random coil, suggesting partially unfold occurred in secondary structure of CTTI. Tryptophan fluorescence and far-UV CDspectra indicated partly unfolding for CTTI protein during thermal treatment.Cassia obtusifolia trypsin inhibitor (COTI) was isolated from by ammonium sulfateprecipitation, Sepharose 4B-trypsin affinity and Sephadex G-75 chromatography.COTI consisted of a single polypeptide chain with accurate molecular mass of 19,812.55 Da. COTI displayed high stability in a wide pH range of 2-12, since theinhibitory activity still remained above 80% of the initial activity. The thermal stabilitystudy indicated that COTI underwent a two-state transition during heating process. COTI wasstable below 60℃for 10 min treatment and maintained almost 100% of its activity.Heating at 70℃resulted in 34% loss of inhibitory activity of its initial activity.Heating at 80℃, 90℃and 100℃resulted in 50%, 56% and 70% loss ofinhibitory activity respectively. Reduction of COTI resulted in the gradual loss oftrypsin inhibitory activity as the prolonged treatment time. It took 120 min to abolishthe activity to 50% of its original value, while almost 100% loss of inhibitoryactivities occurred after 12 h incubation. The inhibitor, which inhibited trypsinactivity with an apparent Ki of 0.3 uM, had one reactive site. The native COTI wasfound to be completely resistant to proteolysis in SGE However, after heated at 70℃,reduction or lysine-modification, COTI produced some degree of susceptibility topepsin digestion. Modification of histidine, arginine and tyrosine had no effect on theCOTI activity, whereas modification of lysine resulted in 93% loss of COTI activity.Based on the modification-activity relationship of chemically modified COTI, weprovide evidence that the lysine residues are responsible for the antitryptic activity ofCOTI. The fluorescence spectrum of native COTI exhibited a major peak at 313 nm,indicating that there was no tryptophan fluorescence and the fluorescence spectrumwas only from the partially solvent exposed tyrosine residues. Complete reduction byDTT led to not only a 12% quenching in the fluorescence intensity at 313 nm, butemerging a new fluorescence peak centered at 345 nm. Modification of lysineresidues indicated no shift of the fluorescence peak except for a sharp decrease of 73% in fluorescence intensity. The Far-UV CD spectrum of the native COTI wasobserved as a strong positive peak at 187 nm, a crossover from positive to negative at190 nm and a negative peak at 200 nm. Secondary structural analysis indicated thepresence of 4%α-helix, 26.7% ofβ-sheet, 30.6% ofβ-turn and 38.2% random coil.Reduced COTI exhibited a 15% decrease ofβ-sheet and a 28% increase ofα-helix.Modification of lysine residues resulted in a 12% decrease inβ-sheet and a subsequent 10% increase inα-helix. Thus, the decrease in COTI activity can beattributed to the changes in the conformation of the protein. Furthermore, theinteractions of the DTT-treated COTI or the lysine-modified COTI with trypsin wereanalyzed by monitoring their fluorescence spectra. The fluorescence spectrum of thecomplex of trypsin with native COTI showed a fluorescence peak of 326 nm. Theemission spectrum of complex of trypsin with lysine-modified COTI indicated noshift of the peak at 326 nm, but a 76% decrease in fluorescence intensity. Thefluorescence peak of the complex of trypsin with DTT-treated COTI changed to 343nm and a 25% decrease in fluorescence intensity. Those results suggested that therewere abnormal complexes formation of the trypsin with the treated COTI due to somechange of the conformation of COTI. After trypsin digestion, three peptide fragmentsof COTI were chosen for MS/MS (Fig 7). The primary sequences were supported byMALDI and MS/MS analysis. Multiple sequence alignment of COTI and Kunitz typeinhibitors from Leguminosae family reveals variable levels of sequence similarity andmany conserved residues. Among these Kunitz type inhibitors, COTI showed thehighest homology with APTI (Adenanthera pavonina trypsin inhibitor, Leguminosae,Mimosoideae, 54%) with two conserved cysteines. Both conserved cysteines areindispensable for formation of disulfide linkages that are responsible for thefunctional stability of Kunitz type inhibitors. The inhibitory activities of COTItowards trypsin and Pieris rapae proteases were determined by in vitro enzyme assays.The results showed that COTI was highly active against trypsin, inhibiting 90% ofproteolytic activity at a concentration of 40μg ml-1. In this case, at the sameconcentration, the inhibition of Pieris rapae proteases activity by COTI was almost60%. Bioassays revealed a delayed insect growth when Pieris rapae larvae were fedon COTI-soaked diet compared with control groups. After 6 days ingestion, pupationoccurred and a reduction of about 30% in larval weight was observed in comparisonwith the control groups.
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