Kinetic analyses for bindings of concanavalin A to dispersed and condensed mannose surfaces on a quartz crystal microbalance.
A biotinylated mannotriose (Man3-bio) was dispersively immobilized in the matrix of biotinylated lactose (Gal-Glc-bio) on a streptavidin-covered, 27-MHz quartz crystal microbalance (QCM), and binding kinetics of concanavalin A (Con A) to Man3-bio in the Gal-Glc-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. Association constants (K(a)) and binding and dissociation rate constants (k(on) and k(off)) could be determined separately as the 1:1 and 1:2 bindings of Con A to Man3-bio on the surface. When Man3-bio was immobilized with content of 1 to 5 mol% in the matrix, the 1:1 binding of Con A to Man3-bio was obtained as K(a)=(4+/-1)x10(6) M(-1), k(on)=(4+/-1)x10(4) M(-1) s(-1), and k(off)=(12+/-2)x10(-3) s(-1). On the contrary, when Man3-bio was immobilized with content of 20 to 100 mol% in the matrix, the 1:2 binding of Con A to Man3-bio was obtained as K(a)=(14+/-2)x10(6) M(-1), k(on)=(14+/-2)x10(4) M(-1) s(-1), and k(off)=(7+/-2)x10(-3) s(-1). Thus, K(a) for the 1:2 binding was 10 times larger than that for the 1:1 binding, with a three times larger binding rate constant (k(on)) and a three times smaller dissociation rate constant (k(off)). This is the first example to obtain separate kinetic parameters for the 1:1 and 1:2 bindings of lectins to carbohydrates on the surface.
Mori
Toshiaki
T
Department of Biomolecular Engineering, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan.
Toyoda
Momoko
M
Ohtsuka
Tatsuro
T
Okahata
Yoshio
Y
D019921:Biotinylation; D003208:Concanavalin A / Q000378:metabolism*; D007700:Kinetics; D037102:Lectins / Q000378:metabolism; D011485:Protein Binding; D011791:Quartz / Q000737:chemistry*; D014312:Trisaccharides / Q000737:chemistry* / Q000378:metabolism
D037102:Lectins; D014312:Trisaccharides; D003208:Concanavalin A; D011791:Quartz; C023872:mannotriose