Quantitative Fluorimetric Determination of Cell-Surface Glycoconjugates with Fluorescein-Substituted Lectins

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Abstract
Fluorescein-substituted lectins, which can be used to visualize cell surface glycoconjugates, are shown to be usable in the quantitative determination of the number of receptor sites and of their association constant. The fluorescence measurements of the fluorescein-substituted lectins released from the cell surface with the related inhibitor, give quantitative data in a large range of fluorescein-substituted lectin concentration (0.1 to 100 μg/ml). Using fluorescein-substituted concanavalin A or [3H]acetyl-concanavalin A, it was found that baby hamster kidney cells (BHK 21, wild-type) bind 10 ± 2 × 106 lectin molecules per cell with an apparent association constant of 1.8 or 1.7 × 106 1 × mol−1, respectively. Using the fluoresceinyl and [3H]acetyl-substituted wheat germ agglutinin, we found 40 ± 5 × 106 sites per cell with an apparent binding constant of 1 and 1.3 ± 0.3 × 106 1 × mol−1, respectively. When fluorescein-substituted succinyl wheat germ agglutinin was used instead of the unsuccinylated wheat germ agglutinin derivatives, the number of binding sites was reduced 7 times, while the binding constant was very slightly lowered. Concanavalin A derivatives gave monotonic Scatchard plots; on the opposite, wheat germ agglutinin derivatives gave biphasic Scatchard plots suggesting that wheat germ agglutinin binds to two classes of receptors.