Purification and characterization of a trypsin inhibitor from Putranjiva roxburghii seeds
2008
Chaudhary, N.S. | Shee, C. | Islam, A. | Ahmad, F. | Yernool, D. | Kumar, P. | Sharma, A.K.
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 1.4 x 10⁻¹¹ M. The inhibitor retained the inhibitory activity over a broad range of pH (pH 2-12), temperature (20-80 °C) and in DTT (up to100 mM). The complete loss of inhibitory activity was observed above 90 °C. CD studies, at increasing temperatures, demonstrated the structural stability of inhibitor at high temperatures. The polypeptide backbone folding was retained up to 80 °C. The CD spectra of inhibitor at room temperature exhibited an α, β 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.
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