Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor

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.

Table 1. Purification table of ASTI from 5 g starting material.

Step (fraction)	Total protein (mg)	Total activity (U)*	Specificactivity (U/mg)	Purification (fold)	Yield (%)
Acetone (AS)	90	12.42	0.14	1	100
IEC (ASb)	15.9	4.20	0.26	1.91	33.8
Affinity (ASb2)	2.1	1.22	0.58	4.22	9.8
RP-HPLC (ASb2b)	1	1.00	1.00	7.22	8.0

*Inhibitory activity: one unit was defined as the amount of protein needed to inhibit one unit of trypsin activity. One unit of trypsin activity was defined as the enzyme activity that hydrolyses 25 mmol of Z-Gly-Pro-Arg-pNA/min under specified conditions.

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 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 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 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 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 7. Double–Dixon plot to determine the inhibition constant (K_i) of ASTI interacting with bovine trypsin. Substrate concentrations of 25 and 5 mM were used. Bovine trypsin concentration was 1 nM. 25 mM (♦) (R² = 0.913) and 5 mM (R² = 0.920) (▴).