Quaternary association and reactivation of dimeric concanavalin A.
The reconstitution of dimeric concanavalin A (ConA) in terms of quaternary association and reactivation, after denaturation in urea, has been investigated using intrinsic fluorescence, 8-anilino-1-naphthalenesulfonate (ANS) binding, far-UV circular dichroism (CD), and an activity assay developed through a combination of affinity binding and the o-phthalaldehyde (OPA) procedure of protein estimation. The equilibrium denaturation of dimeric ConA in urea exhibits a biphasic unfolding pathway involving an intermediate with hydrophobic exposure, and the overall free energy of stabilization for the dimeric protein is obtained as 16.3 kcal mol(-1). The time course of reassociation and regain of activity during reconstitution reveals that the reactivation of ConA runs almost parallel to the process of subunit association. The reactivation reaction follows second-order kinetics, with a rate constant (k) of 2.6 x 10(2) M(-1) s(-1). These results may provide insight into the relationship between quaternary association and function of legume lectins.
Chatterjee
Anindya
A
Department of Chemistry, Presidency College, 86/1 College Street, Kolkata 700073, India.
Mandal
Dipak K
DK
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D003208:Concanavalin A / Q000737:chemistry*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D007887:Fabaceae; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D037102:Lectins / Q000737:chemistry; D011485:Protein Binding; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D011506:Proteins / Q000737:chemistry; D012639:Seeds / Q000378:metabolism; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014466:Ultraviolet Rays; D014508:Urea / Q000494:pharmacology; D009764:o-Phthalaldehyde / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D037102:Lectins; D011506:Proteins; D003208:Concanavalin A; C027132:1-anilino-8-naphthalenesulfonate; D009764:o-Phthalaldehyde; D014508:Urea