Determination of lectin-sugar dissociation constants by agarose affinity electrophoresis

Agarose crossed affinity electrophoresis (aff-EP) was employed for the determination of lectin-sugar dissociation constants (Ki). In the first dimension of the aff-EP increasing amounts of sugar (alpha-methyl-D-mannoside) were added to a given concentration of lectin (concanavalin A). Then the electrophoresis was run with alpha 1-acid glycoprotein, alpha 1-antitrypsin and alpha-fetoprotein as markers of lectin-sugar interactions. Mathematical equations for determination of the mechanisms and constants of lectin-sugar-glycoprotein interactions were developed. The mean value of the concanavalin A-alpha-methyl-D-mannoside dissociation constant calculated according to the introduced equations was 0.28 mM. In this system it was also possible to determine lectin-glycoprotein dissociation constants (K). The observed influence of the sugar on lectin-glycoprotein binding might be due to hydrophobic interactions since the addition of nonionic detergent caused reversal of this phenomenon.