Khan M I, Sastry M V, Surolia A
J Biol Chem. 1986 Mar 5;261(7):3013-9.
A basic lectin (pI approximately 10.0) was purified to homogeneity from the seeds of winged bean (Psophocarpus tetragonolobus) by affinity chromatography on Sepharose 6-aminocaproyl-D-galactosamine. The lectin agglutinated trypsinized rabbit erythrocytes and had a relative molecular mass of 58,000 consisting of two subunits of Mr 29,000. The lectin binds to N-dansylgalactosamine, leading to a 15-fold increase in dansyl fluorescence with a concomitant 25-nm blue shift in the emission maximum. The lectin has two binding sites/dimer for this sugar and an association constant of 4.17 X 10(5) M-1 at 25 degrees C. The strong binding to N-dansylgalactosamine is due to a relatively positive entropic contribution as revealed by the thermodynamic parameters: delta H = -33.62 kJ mol-1 and delta S0 = -5.24 J mol-1 K-1. Binding of this sugar to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent in alpha-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are critical for sugar binding to this lectin. Lectin difference absorption spectra in the presence of N-acetylgalactosamine indicate perturbation of tryptophan residues on sugar binding. The results of stopped flow kinetics with N-dansylgalactosamine and the lectin are consistent with a simple one-step mechanism for which k+1 = 1.33 X 10(4) M-1 s-1 and k-1 = 3.2 X 10(-2) s-1 at 25 degrees C. This k-1 is slower than any reported for a lectin-monosaccharide complex so far. The activation parameters indicate an enthalpically controlled association process.
通过在琼脂糖6-氨基己酰-D-半乳糖胺上进行亲和层析，从四棱豆（Psophocarpus tetragonolobus）种子中纯化出一种碱性凝集素（pI约为10.0），使其达到均一性。该凝集素能凝集经胰蛋白酶处理的兔红细胞，相对分子质量为58,000，由两个Mr为29,000的亚基组成。该凝集素与N-丹磺酰半乳糖胺结合，导致丹磺酰荧光增加15倍，同时发射最大值发生25纳米的蓝移。该凝集素每个二聚体有两个该糖的结合位点，在25℃时的缔合常数为4.17×10⁵ M⁻¹。对N-丹磺酰半乳糖胺的强结合归因于热力学参数所揭示的相对正的熵贡献：ΔH = -33.62 kJ mol⁻¹，ΔS⁰ = -5.24 J mol⁻¹ K⁻¹。这种糖与凝集素的结合表明它能容纳D-半乳糖C-2碳上的一个大的疏水取代基。对其他糖类的研究表明，在异头位置处于α-构象的疏水取代基会增加结合亲和力。C-4和C-6羟基对于糖类与该凝集素的结合至关重要。在N-乙酰半乳糖胺存在下的凝集素差异吸收光谱表明，糖类结合时色氨酸残基受到扰动。用N-丹磺酰半乳糖胺和凝集素进行的停流动力学结果与一个简单的单步机制一致，在25℃时k₊₁ = 1.33×10⁴ M⁻¹ s⁻¹，k₋₁ = 3.2×10⁻² s⁻¹。这个k₋₁比迄今为止报道的任何凝集素-单糖复合物的都要慢。活化参数表明这是一个由焓控制的缔合过程。
