Figures & data
Figure 1. (a) Toyopearl Super-Q chromatography of acetone extract of AS (15 mg). The column (29 × 1.6 cm) was equilibrated with 0.05M Tris-HCl buffer, pH 8.0 and a linear NaCl gradient (0–0.6 M) in 0.05 M Tris-HCl buffer was used for elution. Flow rate was 0.6 mL/min. ASa: tubes 5–12, ASb: tubes 110–135 and ASc: tubes 143–155. Approximately 95% inhibition of trypsin was observed for fraction ASb and less than 10% was observed for ASa and ASc. (b) Affinity chromatography of fraction ASb using a trypsin-agarose column (1 mL). The column was equilibrated with 10 mM acetic acid and 20 mM HCl was used for elution. Flow rate was 0.5 mL/min. ASb1: tubes 2–5, ASb2: tubes 8–11. (c) RP-HPLC of fraction ASb2 (500 µg) on a Grace Vydac C-18 RP column. The column was equilibrated with 0.1% (v/v) TFA/water. Elution was performed with an acetonitrile gradient at a flow rate of 1 mL/min.
![Figure 1. (a) Toyopearl Super-Q chromatography of acetone extract of AS (15 mg). The column (29 × 1.6 cm) was equilibrated with 0.05M Tris-HCl buffer, pH 8.0 and a linear NaCl gradient (0–0.6 M) in 0.05 M Tris-HCl buffer was used for elution. Flow rate was 0.6 mL/min. ASa: tubes 5–12, ASb: tubes 110–135 and ASc: tubes 143–155. Approximately 95% inhibition of trypsin was observed for fraction ASb and less than 10% was observed for ASa and ASc. (b) Affinity chromatography of fraction ASb using a trypsin-agarose column (1 mL). The column was equilibrated with 10 mM acetic acid and 20 mM HCl was used for elution. Flow rate was 0.5 mL/min. ASb1: tubes 2–5, ASb2: tubes 8–11. (c) RP-HPLC of fraction ASb2 (500 µg) on a Grace Vydac C-18 RP column. The column was equilibrated with 0.1% (v/v) TFA/water. Elution was performed with an acetonitrile gradient at a flow rate of 1 mL/min.](/cms/asset/e229686f-e1f7-4879-b406-a6b252ee79fe/ienz_a_836642_f0001_b.jpg)
Table 1. Purification table of ASTI from 5 g starting material.
Figure 2. Reducing SDS-PAGE patterns of ASTI purified by affinity chromatography and RP-HPLC. Lane 1, MW markers; lane 2, affinity purified fraction (ASb2) and lane 3, RP-HPLC purified fraction (ASb2b). 50–10 refer to kilo Dalton values of molecular weight markers.
![Figure 2. Reducing SDS-PAGE patterns of ASTI purified by affinity chromatography and RP-HPLC. Lane 1, MW markers; lane 2, affinity purified fraction (ASb2) and lane 3, RP-HPLC purified fraction (ASb2b). 50–10 refer to kilo Dalton values of molecular weight markers.](/cms/asset/ae5ffa12-c47a-43d7-800c-47b82e88290c/ienz_a_836642_f0002_b.jpg)
Figure 3. (a) Separation of peptides generated by digestion of ASTI A-chain with S. aureus V8 protease. Peptides were separated by RP-HPLC on a TSK gel ODS 120 T column using a gradient of acetonitrile in 0.1% TFA. Flow rate was 1.0 mL/min. (b) Separation of peptides generated by digestion of ASTI A-chain with endoproteinase Arg-C. Peptides were separated by RP-HPLC on a TSK gel ODS 120 T column using a gradient of acetonitrile in 0.1% TFA. Flow rate was 1.0 mL/min. (c) Separation of peptides generated by digestion of ASTI A-chain with Achromobacter protease I. Peptides were separated by RP-HPLC on a TSK gel ODS 120 T column using a gradient of acetonitrile in 0.1% TFA. Flow rate was 1.0 mL/min.
![Figure 3. (a) Separation of peptides generated by digestion of ASTI A-chain with S. aureus V8 protease. Peptides were separated by RP-HPLC on a TSK gel ODS 120 T column using a gradient of acetonitrile in 0.1% TFA. Flow rate was 1.0 mL/min. (b) Separation of peptides generated by digestion of ASTI A-chain with endoproteinase Arg-C. Peptides were separated by RP-HPLC on a TSK gel ODS 120 T column using a gradient of acetonitrile in 0.1% TFA. Flow rate was 1.0 mL/min. (c) Separation of peptides generated by digestion of ASTI A-chain with Achromobacter protease I. Peptides were separated by RP-HPLC on a TSK gel ODS 120 T column using a gradient of acetonitrile in 0.1% TFA. Flow rate was 1.0 mL/min.](/cms/asset/b30f12b9-046d-4db9-9549-40e6b771742b/ienz_a_836642_f0003_b.jpg)
Figure 4. Amino acid sequence of the A- and B-chain of ASTI, deduced from protease digestions, RP-HPLC and sequence analysis. (E), S. aureus V8 protease digest of A-chain; (K), Achromobacter protease 1 digest of A-chain and (R), endoproteinase Arg-C digest of A-chain.
![Figure 4. Amino acid sequence of the A- and B-chain of ASTI, deduced from protease digestions, RP-HPLC and sequence analysis. (E), S. aureus V8 protease digest of A-chain; (K), Achromobacter protease 1 digest of A-chain and (R), endoproteinase Arg-C digest of A-chain.](/cms/asset/2c9503da-846e-41db-aeda-e9eba4e0fbbe/ienz_a_836642_f0004_b.jpg)
Figure 5. (a) Alignment between the sequence of the A-chain of ASTI with other comparative plant seed inhibitor sequences obtained from ClustalW search. Comparative sequences are obtained with Kunitz-type trypsin inhibitor A chain, ACTI-A (A. confusa), gi|299509|; Kunitz-type trypsin inhibitor A-chain, LLTI-A (L. leucocephala), gi|18202442|; trypsin inhibitor A-chain, BVTI-A (Bauhinia variegata), gi|15082208| and KTI A-chain, KTI-A gi|162138868|. (*), Identical sequences; (:), similarities of three or four amino acids. (b) Alignment between the sequence of the B-chain of ASTI with other comparative plant seed inhibitor sequences obtained from ClustalW search. Comparative sequences are obtained with Kunitz-type trypsin inhibitor B-chain, ACTI-B (A. confusa), gi|299508|; SBTI and Kunitz-type trypsin inhibitor B-chain, LLTI-B (L. leucocephala), gi|18202443|. (*), Identical sequences; (:), similarities of three or four amino acids.
![Figure 5. (a) Alignment between the sequence of the A-chain of ASTI with other comparative plant seed inhibitor sequences obtained from ClustalW search. Comparative sequences are obtained with Kunitz-type trypsin inhibitor A chain, ACTI-A (A. confusa), gi|299509|; Kunitz-type trypsin inhibitor A-chain, LLTI-A (L. leucocephala), gi|18202442|; trypsin inhibitor A-chain, BVTI-A (Bauhinia variegata), gi|15082208| and KTI A-chain, KTI-A gi|162138868|. (*), Identical sequences; (:), similarities of three or four amino acids. (b) Alignment between the sequence of the B-chain of ASTI with other comparative plant seed inhibitor sequences obtained from ClustalW search. Comparative sequences are obtained with Kunitz-type trypsin inhibitor B-chain, ACTI-B (A. confusa), gi|299508|; SBTI and Kunitz-type trypsin inhibitor B-chain, LLTI-B (L. leucocephala), gi|18202443|. (*), Identical sequences; (:), similarities of three or four amino acids.](/cms/asset/7bd00034-7777-4eb7-8641-b79f28877aea/ienz_a_836642_f0005_b.jpg)
Figure 6. A phylogenetic tree analysis of ASTI with other inhibitors. Protein sequences were obtained from http://blast.ncbi.nlm.nih.gov/ by a BLAST search of ASTI. Accession numbers for the sequences used are as follows: trypsin isoinhibitor (Adenanthera pavonina), APTI, gi|225058|; LLTI, gi|18202442|; Prosopis juliflora trypsin inhibitor, PJTI, gi|417176|; ACTI, gi|299509|; Tamarindus indica trypsin inhibitor, TITI, gi|308756025|; BVTI, gi|32363181|; Bauhinia ungulate Factor X inhibitor, BUXI, gi|32363179|; Psophocarpus tetragonolobus trypsin inhibitor, PTTI, gi|86450987 and chymotrypsin inhibitor (Erythrina variegate), gi|265716|. The scale bar shows a branch length of 0.1 (i.e. a 10% difference in amino acids).
![Figure 6. A phylogenetic tree analysis of ASTI with other inhibitors. Protein sequences were obtained from http://blast.ncbi.nlm.nih.gov/ by a BLAST search of ASTI. Accession numbers for the sequences used are as follows: trypsin isoinhibitor (Adenanthera pavonina), APTI, gi|225058|; LLTI, gi|18202442|; Prosopis juliflora trypsin inhibitor, PJTI, gi|417176|; ACTI, gi|299509|; Tamarindus indica trypsin inhibitor, TITI, gi|308756025|; BVTI, gi|32363181|; Bauhinia ungulate Factor X inhibitor, BUXI, gi|32363179|; Psophocarpus tetragonolobus trypsin inhibitor, PTTI, gi|86450987 and chymotrypsin inhibitor (Erythrina variegate), gi|265716|. The scale bar shows a branch length of 0.1 (i.e. a 10% difference in amino acids).](/cms/asset/3dca1fa8-2dc3-4dd6-ba39-29f5c5e6cac6/ienz_a_836642_f0006_b.jpg)