Rapid affinity-purification and physicochemical characterization of pumpkin (Cucurbita maxima) phloem exudate lectin.
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% alpha-helix, 35.8% beta-sheet, 22.5% beta-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 degrees C, but a sharp decrease was seen between 75 and 85 degrees C. Differential scanning calorimetric and CD spectroscopic studies suggest that the lectin undergoes a co-operative thermal unfolding process centred at approx. 81.5 degrees C, indicating that it is a relatively stable protein.
Narahari
Akkaladevi
A
University of Hyderabad, India.
Swamy
Musti J
MJ
D000818:Animals; D002686:Chitin / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D002942:Circular Dichroism; D028464:Cucurbita / Q000737:chemistry*; D006801:Humans; D037121:Plant Lectins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013696:Temperature
D037121:Plant Lectins; D002686:Chitin; C091028:phloem lectin