Chatterjee Manjeer, Mandal Dipak K
Department of Chemistry, Presidency College, Calcutta 700 073, India.
Biochemistry. 2003 Oct 28;42(42):12217-22. doi: 10.1021/bi034642l.
The reconstitution of soybean agglutinin (SBA), a tetrameric GalNAc/Gal-specific legume lectin, after denaturation in urea has been studied using fluorescence, far-UV CD, a hemagglutination assay, and chemical cross-linking with glutaraldehyde as a bifunctional reagent. The reconstituted protein exhibits similar quaternary structure and activity as of native lectin. The kinetics of subunit oligomerization has been determined from the cross-linking reaction of the reconstituting protein followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Monomers and tetramers could be quantitatively analyzed during reconstitution. Dimers are not detectable. The reassociation reaction follows second-order kinetics. The results are described by a kinetic mechanism in which the monomer-to-dimer association (characterized by a second-order rate constant (k(1)) of 1.4 x 10(4) M(-1) s(-1) at 37 degrees C) is involved in the rate-determining step of the oligomerization reaction.
利用荧光、远紫外圆二色光谱、血细胞凝集试验以及用戊二醛作为双功能试剂进行化学交联，研究了大豆凝集素（SBA，一种四聚体GalNAc/Gal特异性豆科植物凝集素）在尿素中变性后的复性过程。复性后的蛋白质呈现出与天然凝集素相似的四级结构和活性。通过对复性蛋白进行交联反应，随后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），测定了亚基寡聚化的动力学。在复性过程中可以对单体和四聚体进行定量分析。未检测到二聚体。重新缔合反应遵循二级动力学。结果由一种动力学机制描述，其中单体到二聚体的缔合（在37℃下由二级速率常数（k(1)）为1.4×10(4) M(-1) s(-1)表征）参与寡聚化反应的速率决定步骤。
