Purification and characterization of a Bowman-Birk proteinase inhibitor from the seeds of black gram (Vigna mungo).
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 approximately 8kDa correlating to the peak 8041.5Da 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 309.8nM) and chymotrypsin (K(i) of 10.7muM), however, with a molar ratio of 1:2 with trypsin. BgPI was stable up to a temperature of 80 degrees 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 degrees 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.
Prasad
E R
ER
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India.
Dutta-Gupta
A
A
Padmasree
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002845:Chromatography; D002942:Circular Dichroism; D004229:Dithiothreitol / Q000737:chemistry; D004586:Electrophoresis; D007887:Fabaceae / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D011480:Protease Inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D055550:Protein Stability; D012639:Seeds / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D010940:Plant Proteins; D011480:Protease Inhibitors; D020033:Protein Isoforms; D014357:Trypsin; D004229:Dithiothreitol