Purification and characterization of a Bowman-Birk proteinase inhibitor from the seeds of black gram ( Vigna mungo)

• Published in: Phytochemistry (Oxford) 2010-03, Vol.71 (4), p.363-372
• Main Authors: Prasad, E.R., Dutta-Gupta, A., Padmasree, K.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Animals; Biological and medical sciences; Bowman-Birk inhibitor; Cattle; CD spectroscopy; Chemical constitution; Chromatography; chymotrypsin; Circular Dichroism; disulfide bonds; Dithiothreitol - chemistry; Electrophoresis; enzyme inhibition; Fabaceae - chemistry; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; MALDI-TOF; matrix-assisted laser desorption-ionization mass spectrometry; Molecular Sequence Data; Plant physiology and development; plant proteins; Plant Proteins - chemistry; Plant Proteins - isolation & purification; Plant Proteins - metabolism; Plant Proteins - pharmacology; Protease Inhibitors - chemistry; Protease Inhibitors - isolation & purification; Protease Inhibitors - metabolism; Protease Inhibitors - pharmacology; protein isoforms; Protein Isoforms - chemistry; Protein Isoforms - isolation & purification; Protein Isoforms - metabolism; Protein Isoforms - pharmacology; Protein purification; Protein Stability; Proteinase inhibitor; reduction; seeds; Seeds - chemistry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; structure-activity relationships; Temperature; thermal stability; trypsin; Trypsin - metabolism; Vigna mungo
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2009.11.006

A proteinase inhibitor was purified from seeds of Vigna mungo (cv. TAU-1) using ion-exchange and affinity chromatography followed by gel-filtration chromatography. Biochemical characterization indicated that it belonged to Bowman-Birk inhibitor family. A proteinase inhibitor (BgPI) was purified from black gram, Vigna mungo (cv. TAU-1) seeds by using ammonium sulfate fractionation, followed by ion-exchange, affinity and gel-filtration chromatography. BgPI showed a single band in SDS–PAGE under non-reducing condition with an apparent molecular mass of ∼8 kDa correlating to the peak 8041.5 Da in matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrum. BgPI existed in different isoinhibitor forms with pI values ranging from 4.3 to 6.0. The internal sequence “SIPPQCHCADIR” of a peak 1453.7 m/z, obtained from MALDI-TOF-TOF showed 100% similarity with Bowman-Birk inhibitor (BBI) family. BgPI exhibited non-competitive-type inhibitory activity against both bovine pancreatic trypsin ( K i of 309.8 nM) and chymotrypsin ( K i of 10.7 μM), however, with a molar ratio of 1:2 with trypsin. BgPI was stable up to a temperature of 80 °C and active over a wide pH range between 2 and 12. The temperature-induced conformational changes in secondary structure are reversed when BgPI was cooled from 90 to 25 °C. Further, upon reduction with dithiothreitol, BgPI lost both its inhibitory activity as well as secondary structural conformation. Lysine residue(s) present in the reactive site of BgPI play an important role in inhibiting the bovine trypsin activity. The present study provides detailed biochemical characteristic features of a BBI type serine proteinase inhibitor isolated from V. mungo.