Kinetic Analysis of Subunit Oligomerization of the Legume Lectin Soybean Agglutinin

• Published in: Biochemistry (Easton) Vol. 42; no. 42; pp. 12217 - 12222
• Main Authors: Chatterjee, Manjeer, Mandal, Dipak K
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 28.10.2003
• Subjects: Biopolymers - chemistry; Circular Dichroism; Cross-Linking Reagents - chemistry; Electrophoresis, Polyacrylamide Gel; Glycine max - chemistry; Kinetics; Models, Molecular; Plant Lectins - chemistry; Protein Denaturation; Soybean Proteins - chemistry; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet
• ISSN: 0006-2960; 1520-4995
• 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 × 104 M-1 s-1 at 37 °C) is involved in the rate-determining step of the oligomerization reaction.