title: The D-galactose specific lectin of field bean (Dolichos lablab) seed binds sugars with extreme negative cooperativity and half-of-the-sites binding.
abstract: The field bean (Dolichos lablab) lectin designated as PPO-haemagglutinin (DLL-II) is bifunctional, exhibiting both polyphenol oxidase and haemagglutinating activity. The lectin is unusual in that it binds galactose (Gal), lactose (Lac) and N-acetylgalactosamine (GalNAc) only in the presence of (NH₄)₂SO₄ and exhibits negative cooperativity and half-of-the-sites binding. Circular dichroism, isothermal titration calorimetry and fluorescence quenching were used to assess the sugar binding in the presence of (NH₄)₂O₄. Comparison of the near-UV CD spectra with and without bound sugar revealed ligand induced conformational changes. The intrinsic fluorescence quenching data indicate that DLL-II exhibits weak binding to Gal in the presence of (NH₄)₂SO₄ with a stoichiometry of one bound ligand per dimer. ITC data fitted using a two sets of sites binding model presented a similar picture. The K(a)'s for Gal, Lac and GalNAc in the presence of (NH₄)₂SO₄ were 0.16±0.002, 0.21±0.004 and 8.45±0.78 (×10⁻³) M⁻¹ respectively. The Hill plot for the binding of these sugars to DLL-II was curvilinear with a tangent slope <1.0 indicating negative cooperativity. DLL-II thus exhibits half-of-the-site binding, an extreme form of negative cooperativity in which the second ligand does not bind at all. This is the first report of a legume lectin, exhibiting half-of-the-sites binding.
authors.affiliation: Department of Protein Chemistry and Technology, CSIR-Central Food Technological Research Institute, Mysore 570020, India.
authors.forename: Devavratha H
authors.initials: DH
authors.lastname: Rao
authors.forename: Lalitha R
authors.initials: LR
authors.lastname: Gowda
mesh_terms: D001665:Binding Sites; D005690:Galactose / Q000378:metabolism*; D008024:Ligands; D037121:Plant Lectins / Q000378:metabolism*; D011485:Protein Binding; D012639:Seeds / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D008024:Ligands; D037121:Plant Lectins; C442499:dolichos Lablab lectin; D005690:Galactose