A Chito-specific, Adenine Binding Agglutinin from Benincasa hispida Shows High Structural and Functional Stability

摘要

Aims: To purify and characterize a novel chito-specific lectin from Benincasa hispida fruit. Study Design: Protein purification and characterization using biochemical and biophysical techniques under various denaturing conditions. Place and Duration of Study: Biochemical Sciences Division, CSIR-National Chemical Laboratory, Pune, India, from July 2013 to Jan 2015. Methodology: Benincasa hispida fruit was used as lectin source and purified using chitin affinity and gel filtration chromatographic techniques. The hemagglutinating activity was detected with rabbit erythrocytes. Conformational studies were carried out using steady-state fluorescence, Circular dichroism and FTIR spectroscopic methods. Results: The protein exists as a homodimer of 34 kDa as determined using gel filtration chromatography and MALDI-TOF/TOF and the subunit was 17 kDa as estimated from SDS-PAGE. Circular dichroism spectroscopic studies showed that Bh L has high content of β-sheets (40%). The fluorimetric titrations with chitobiose and chitotriose sugars yielded the association constants K_a as 1.2 x102 M-1 and 1x104 M-1 at 37oC, respectively, indicating high affinity of the lectin towards the latter sugar. Lectin also bound adenine with estimated binding constant 1 x 104 M-1 implying its physiological role in plants. The native protein has total six Trp residues out of which, two are exposed on the surface and are in electropositive environment as revealed by fluorescence quenching with KI. The lectin exhibited thermostability upto 80°C retaining 50% of hemagglutination activity and showed fairly compact and stable structure as observed in the far-UV CD spectra. Also, the lectin remained active even after incubating in 6M GDn-HCl for 24 h, or at extreme pH or in 50% organic solvents. Conclusion: The present study revealed that the chito-specific lectin purified from B. hispida has novel structural and functional stability at higher temperature, in a broad pH range, and also in the presence of higher concentrations of chemical denaturants. Binding to adenine indicates its importance in plant physiology.