Purification and Characterization of a Kunitz-Type Trypsin Inhibitor from Acacia victoriae Bentham Seeds

Abstract

An Acacia victoriae trypsin inhibitor (AvTI) was purified from the seeds of prickly wattle (A. victoriae Bentham) by salt precipitation, ion exchange, and gel filtration chromatography and then characterized by electrophoresis and N-terminal amino acid sequencing. AvTI had a specific activity of 138.99 trypsin inhibitor units per milligram (TIU mg(-1)), which was 21-fold higher than that of the salt precipitate. A molecular mass of 13 kDa was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, which also indicated that AvTI may consist of two polypeptide chains linked by at least one disulfide bond. Although only a single peak was resolved by ion exchange and reverse phase high-performance liquid chromatography (RP-HPLC), native-PAGE and isoelectric focusing revealed the presence of three isoforms possessing acidic pI values of 5.13, 4.76, and 4.27, respectively. N-Terminal amino acid sequencing analysis of native and reduced AvTI showed two sequences with a high degree of homology with a typical Kunitz-type trypsin inhibitor. All isoforms had considerable trypsin inhibitory activity but showed relatively very low inhibition against alpha-chymotrypsin.

Glycosylation, amino acid analysis and kinetic properties of a major Kunitz-type trypsin inhibitor from Acacia victoriae Bentham seeds

“…Wattle soluble proteins were extracted and purified using AKTA fast protein liquid chromatography (FPLC) according to the procedures described in a previous study with the purification stages being monitored by continuous assay of trypsin activity (Ee et al, 2009b). The fraction precipitated with 25-50% (w/v) ammonium sulphate, which had the highest trypsin inhibition activity of the salt fractions, was designated as AS-2.…”

“…AvTI (13 kDa) was prepared to achieve final concentrations of: 0.00, 0.02, 0.04 and 0.06 lM. The molecular mass of AvTI was calculated by SDS-PAGE analysis under reducing conditions (Ee et al, 2009b). A mixture of the assay buffer (2.6 ml), TAME (0.3 ml) and 0.1 ml of 1 mM HCl, without enzyme, was used as a reference blank.…”

“…We also documented the isolation and partial characterisation of the major Kunitz-type trypsin inhibitor, which was reported to consist of two polypeptide chains with a molecular mass of $18.3 kDa (Ee, Zhao, Rehman, & Agboola, 2009b). We observed that the A. victoriae trypsin inhibitor (AvTI) was of the Kunitz type, existing in at least three isoforms with a narrow range of molecular masses, which suggested the possibility of glycosylation in its native form although the archetypical Kunitz-type inhibitor, soybean trypsin inhibitor, is not a glycoprotein (Kunitz, 1947).…”

Pathogenesis-related genes and proteins in forest tree species

“…Seeds of Acacia confuse (ACT1) and A. victoriae (AvT1) contained a kunitz-type PI and inhibited trypsin and alpha-chymotrypsin (Lin et al 1991;Ee et al 2008Ee et al , 2009). In A. plumose seeds, three highly heat-stable serine protease inhibitor isoforms were purified and designated as ApTIA, ApTIB, and ApTIC, and their toxicity was demonstrated on Aspergillus niger, Thielaviopsis paradoxa and Colletotrichum sp.…”

Affinity purification of urinary trypsin inhibitor from human urine

“…As previously reported, an Apios americana trypsin inhibitor was purified from Apios tubers by ionexchange chromatography on DEAE Cellulofine A-500 and by gel-filtration chromatography on Sephadex G-50 [9]. A trypsin inhibitor termed KPHTI was purified from the skin secretions of frog Kaloula pulchra hainana by ion-exchange chromatography on DEAE-Sephadex A-50, gel-filtration chromatography on Sephadex G-75 and ion-exchange chromatography on Q-Sepharose [10]; an Acacia victoriae trypsin inhibitor was purified from the seeds of prickly wattle (A. victoriae Bentham) by salt precipitation, ion exchange chromatography on Q-Sepharose Fast Flow and gel filtration chromatography on Superdex 200 [11]; a trypsin inhibitor from Putranjiva roxburghii seeds was purified by ion-exchange chromatography on CM sepharose, ionexchange chromatography on DEAE-sepharose and gelfiltration chromatography [12]. Two rat UTIs were purified from pooled urine by salting out, affinity chromatography [13]; a trypsin inhibitor was isolated from Plathymenia foliolosa (Benth.)…”