Kinetic analysis of subunit oligomerization of the legume lectin soybean agglutinin.
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.
Chatterjee
Manjeer
M
Department of Chemistry, Presidency College, Calcutta 700 073, India.
Mandal
Dipak K
DK
D001704:Biopolymers / Q000737:chemistry*; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008958:Models, Molecular; D037121:Plant Lectins / Q000737:chemistry*; D011489:Protein Denaturation; D030262:Soybean Proteins / Q000737:chemistry*; D013025:Glycine max / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D010835:Phytohemagglutinins
D001704:Biopolymers; D003432:Cross-Linking Reagents; D037121:Plant Lectins; D030262:Soybean Proteins; C028824:soybean lectin; D010835:Phytohemagglutinins