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Bioscience Reports (2010) 30, (341–349) (Printed in Great Britain)

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Original Paper

Rapid affinity-purification and physicochemical characterization of pumpkin (Cucurbita maxima) phloem exudate lectin

Akkaladevi Narahari and Musti J. Swamy¹

School of Chemistry, University of Hyderabad, Hyderabad 500046, India

Key words: affinity chromatography, carbohydrate binding protein, CD, haemagglutinin, secondary structure, thermal unfolding.

Abbreviations used: CIA, Coccinia indica agglutinin; DSC, differential scanning calorimetry; ESI-MS, electrospray ionization MS; LAA, Luffa acutangula agglutinin; MCL, Momordica charantia lectin; PBS-2ME, 20 mM sodium phosphate buffer, pH 7.4, containing 150 mM NaCl and 10 mM 2-mercaptoethanol; PPL, pumpkin phloem exudate lectin; SGSL, Trichosanthes anguina (snake gourd) seed lectin; TCSL, Trichosanthes cucumerina seed lectin.

¹To whom correspondence should be addressed (email mjssc@uohyd.ernet.in).

Synopsis

The chito-oligosaccharide-specific lectin from pumpkin (Cucurbita maxima) phloem exudate has been purified to homogeneity by affinity chromatography on chitin. After SDS/PAGE in the presence of 2-mercaptoethanol, the pumpkin phloem lectin yielded a single band corresponding to a molecular mass of 23.7 kDa, whereas ESI-MS (electrospray ionization MS) gave the molecular masses of the subunit as 24645 Da. Analysis of the CD spectrum of the protein indicated that the secondary structure of the lectin consists of 9.7% α-helix, 35.8% β-sheet, 22.5% β-turn and 32.3% unordered structure. Saccharide binding did not significantly affect the secondary and tertiary structures of the protein. The haemagglutinating activity of pumpkin phloem lectin was mostly unaffected in the temperature range 4–70 °C, but a sharp decrease was seen between 75 and 85 °C. Differential scanning calorimetric and CD spectroscopic studies suggest that the lectin undergoes a co-operative thermal unfolding process centred at approx. 81.5 °C, indicating that it is a relatively stable protein.

Received 18 August 2009; accepted 7 September 2009

Published as Immediate Publication 7 September 2009, doi:10.1042/BSR20090117

© The Authors Journal compilation © 2010 Biochemical Society