Purification and characterization of a Kunitz-type trypsin inhibitor from Acacia victoriae Bentham seeds.
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.
Ee
Kah Y
KY
E. H. Graham Centre for Innovative Agriculture and School of Agricultural and Wine Sciences, Charles Sturt University, Private Bag 588, Wagga Wagga, NSW 2678, Australia.
Zhao
Jian
J
Rehman
Ata-Ur
AU
Agboola
Samson O
SO
D000045:Acacia / Q000737:chemistry*; D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D012639:Seeds / Q000737:chemistry; D016415:Sequence Alignment; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000302:isolation & purification*
D010936:Plant Extracts; D010940:Plant Proteins; D014361:Trypsin Inhibitors