Schwarz F P, Ahmed H, Bianchet M A, Amzel L M, Vasta G R
Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, Rockville, Maryland 20850, USA.
Biochemistry. 1998 Apr 28;37(17):5867-77. doi: 10.1021/bi9716478.
Isothermal titration calorimetry (ITC) measurements of the binding 1-beta carbohydrate-substituted galactopyranoside derivatives to galectin-1 from bovine spleen, a dimer with one binding site per subunit, were performed at 283-285 and 298 K. The disaccharides were lactose, methyl beta-lactoside, lactulose, 4-O-beta-D-galactopyranosyl-D-mannopyranoside, 3-O-beta-D-galactopyranosyl-D-arabinose, 2'-O-methyllactose, lacto-N-biose, N-acetyllactosamine, and thiodigalactopyranoside. The site binding enthalpies, DeltaHb, are the same at both temperatures and range from -42.2 +/- 3.3 kJ mol-1 for thiodigalactopyranoside to -24.5 +/- 0.5 kJ mol-1 for lacto-N-biose, and the site binding constants range from 4.86 +/- 0.78 x 10(3) M-1 for methyl beta-lactoside at 297.8 K to 6.54 +/- 0.97 x 10(4) M-1 for N-acetyllactosamine at 281.3 K. The binding reactions are enthalpically driven, exhibit enthalpy-entropy compensation, and, with the exception of N-acetyllactosamine, follow a van't Hoff dependence of the binding constant on temperature. The number of contacts at distances <4.0 A between the disaccharide and galectin was determined from the energy-minimized conformation of the complex derived from the X-ray crystallographic structure of the galectin-N-acetyllactosamine complex determined by Liao et al. [Liao, D. I., Kapadia, G., Ahmed, H., Vasta, G. R., and Herzberg, O. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 1428-1432]. The binding enthalpies calculated from changes in the solvent-accessible surface areas of the galectin binding site upon binding of the disaccharide were in close agreement with the experimental values for lactose, lactulose, lacto-N-biose, and N-acetyllactosamine, all of which exhibit binding enthalpies >-36 kJ mol-1. Differential scanning calorimetry measurements on solutions of galectin and its disaccharide complexes show that the galectin dimer does not dissociate upon denaturation in contrast to the legume lectins. At the denaturation temperature, the galectin in the absence of sugar exists as a tetramer, and the extent of this association is substantially reduced in the presence of a disaccharide.
在283 - 285 K和298 K温度下，采用等温滴定量热法（ITC）对1-β-碳水化合物取代的吡喃半乳糖苷衍生物与牛脾脏半乳糖凝集素-1（一种每个亚基有一个结合位点的二聚体）的结合进行了测量。二糖包括乳糖、β-甲基乳糖苷、乳果糖、4-O-β-D-吡喃半乳糖基-D-甘露吡喃糖苷、3-O-β-D-吡喃半乳糖基-D-阿拉伯糖、2'-O-甲基乳糖、乳糖-N-二糖、N-乙酰乳糖胺和硫代二吡喃半乳糖苷。在两个温度下，位点结合焓ΔHb相同，范围从硫代二吡喃半乳糖苷的-42.2±3.3 kJ·mol⁻¹到乳糖-N-二糖的-24.5±0.5 kJ·mol⁻¹，位点结合常数范围从297.8 K时β-甲基乳糖苷的4.86±0.78×10³ M⁻¹到281.3 K时N-乙酰乳糖胺的6.54±0.97×10⁴ M⁻¹。结合反应由焓驱动，表现出焓-熵补偿，并且除N-乙酰乳糖胺外，结合常数遵循范特霍夫温度依赖性。根据廖等人[廖，D. I.，卡帕迪亚，G.，艾哈迈德，H.，瓦斯塔，G. R.，和赫茨伯格，O.（1994年）美国国家科学院院刊91，1428 - 1432]测定的半乳糖凝集素-N-乙酰乳糖胺复合物的X射线晶体结构推导出的复合物能量最小化构象，确定了二糖与半乳糖凝集素之间距离<4.0 Å的接触数。根据二糖结合后半乳糖凝集素结合位点溶剂可及表面积的变化计算出的结合焓与乳糖、乳果糖、乳糖-N-二糖和N-乙酰乳糖胺的实验值密切一致，所有这些二糖的结合焓均>-36 kJ·mol⁻¹。对半乳糖凝集素及其二糖复合物溶液的差示扫描量热法测量表明，与豆科凝集素不同，半乳糖凝集素二聚体在变性时不会解离。在变性温度下，无糖存在时的半乳糖凝集素以四聚体形式存在，在二糖存在时这种缔合程度会大幅降低。
