pH对花生凝集素寡聚平衡及糖结合特性的影响。

Effect of pH on oligomeric equilibrium and saccharide-binding properties of peanut agglutinin.

作者信息

Decastel M, De Boeck H, Goussault Y, De Bruyne C K, Loontiens F G, Frénoy J P

出版信息

Arch Biochem Biophys. 1985 Aug 1;240(2):811-9. doi: 10.1016/0003-9861(85)90090-6.

Abstract

The conformation and saccharide-binding properties of peanut agglutinin (PNA) depend on pH as studied by analytical ultracentrifugation, fluorescence, circular dichroism, equilibrium dialysis, and absorption spectroscopy. PNA is tetrameric in neutral solution and dissociates reversibly into dimers below pH 5.1. Below pH 3.4, the lectin is totally dimeric. Lowering of the pH induces reversible changes in the tertiary and secondary structures of PNA. Binding of saturating amounts of lactose to tetrameric (pH 6.9) or dimeric (pH 3.2) PNA resulted in identical ultraviolet difference spectra. Fluorescence studies of PNA as a function of pH in the presence of lactose indicated that tryptophanyl residues, present at or near the saccharide binding site, are more accessible to the ligand in dimeric than in tetrameric PNA. For solutions of dimeric PNA, containing only minor amounts of tetramers (pH 3.6), equilibrium dialysis with MeUmb-beta Gal beta(1----3)GalNac showed that the binding capacity of PNA was the same as for tetrameric PNA (one binding site per protomer) but the apparent association constant was one order of magnitude lower than for tetrameric PNA. The enhancement of MeUmb-beta Gal beta(1----3)GalNac fluorescence upon binding to PNA was pH dependent: 50% at neutrality, 16% at pH 3.7, and unobservable at pH 3.0, suggesting that the microenvironment of this PNA-bound chromophore changed progressively with pH and was dependent on ionization of an acidic amino acid residue.

摘要

通过分析超速离心、荧光、圆二色性、平衡透析和吸收光谱研究发现，花生凝集素（PNA）的构象和糖结合特性取决于pH值。PNA在中性溶液中为四聚体，在pH 5.1以下可逆地解离为二聚体。在pH 3.4以下，凝集素完全为二聚体。降低pH值会诱导PNA的三级和二级结构发生可逆变化。向四聚体（pH 6.9）或二聚体（pH 3.2）PNA中加入饱和量的乳糖，会产生相同的紫外差光谱。在乳糖存在下，对PNA随pH值变化的荧光研究表明，位于糖结合位点或其附近的色氨酸残基在二聚体PNA中比在四聚体PNA中更容易与配体接触。对于仅含有少量四聚体的二聚体PNA溶液（pH 3.6），用甲基伞形酮-β-半乳糖β(1→3)N-乙酰半乳糖胺进行平衡透析表明，PNA的结合能力与四聚体PNA相同（每个亚基一个结合位点），但表观缔合常数比四聚体PNA低一个数量级。甲基伞形酮-β-半乳糖β(1→3)N-乙酰半乳糖胺与PNA结合后荧光增强与pH值有关：在中性时为50%，在pH 3.7时为16%，在pH 3.0时不可见，这表明该与PNA结合的发色团的微环境随pH值逐渐变化，并且取决于酸性氨基酸残基的电离。