Purification and characterization of a trypsin inhibitor from Putranjiva roxburghii seeds.
A highly stable and potent trypsin inhibitor was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family by acid precipitation, cation-exchange and anion-exchange chromatography. SDS-PAGE analysis, under reducing condition, showed that protein consists of a single polypeptide chain with molecular mass of approximately 34 kDa. The purified inhibitor inhibited bovine trypsin in 1:1 molar ratio. Kinetic studies showed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 1.4x10(-11) M. The inhibitor retained the inhibitory activity over a broad range of pH (pH 2-12), temperature (20-80 degrees C) and in DTT (up to100 mM). The complete loss of inhibitory activity was observed above 90 degrees C. CD studies, at increasing temperatures, demonstrated the structural stability of inhibitor at high temperatures. The polypeptide backbone folding was retained up to 80 degrees C. The CD spectra of inhibitor at room temperature exhibited an alpha, beta pattern. N-terminal amino acid sequence of 10 residues did not show any similarities to known serine proteinase inhibitors, however, two peptides obtained by internal partial sequencing showed significant resemblance to Kunitz-type inhibitors.
Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667, India.
Navneet S
NS
Chaudhary
Chandan
C
Shee
Asimul
A
Islam
Faizan
F
Ahmad
Dinesh
D
Yernool
Pravindra
P
Kumar
Ashwani K
AK
Sharma
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D029624:Ferns / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D012639:Seeds / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013696:Temperature; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D014361:Trypsin Inhibitors