Turbidimetric Analysis

Turbidimetric analysis is useful for the study of multivalent interactions that result in the formation of large protein aggregates. The method is typically employed to evaluate the ability of multivalent saccharide derivatives to promote lectin cross-linking (Fig. 7). To conduct the assay, multivalent ligand and lectin are mixed and allowed to form cross-linked complexes; the formation of large protein aggregates is monitored by changes in the optical density of the solution [55-57]. The abilities of various multivalent ligands to cross-link lectin is correlated with their relative affinities. As a measure of monovalent ligand function, inhibitory potencies of these species can be obtained by evaluating their abilities to inhibit lectin cross-linking by a specific multivalent ligand. This analysis is complicated, however, by the possibility

Figure 7 Multivalent ligand-induced lectin cross-linking. Multivalent ligands, which are able to simultaneously span two lectin molecules, can form cross-linked aggregates. These aggregates are detectable by spectroscopic means in a turbidimetric assay.

that the monovalent ligands could initially contribute to optical density increases. In principle, they could occupy binding sites on the lectin that do not directly result in cross-linking, thereby releasing epitopes on the multivalent ligand that then attract more lectin to the aggregate.

The complexities of comparing monovalent and multivalent ligands by turbidimetric analysis are not the only disadvantages of this method. One potential problem is that it does not effectively report on the activities of multivalent ligands that act by simultaneously binding to multiple sites within a lectin oligomer. In addition, the relative activities of a multivalent ligand for two related lectins cannot be determined because the inherent solubilities of the lectins and their cross-linked complexes will differ. Importantly, because precipitation of the lectin-ligand complex occurs, the process is generally under kinetic control; consequently, the relative potencies of ligands may not correlate with their intrinsic affinities for the lectin. As with most assays of multivalent ligand function, the species being detected is not the initial protein-ligand complexation event. Still this assay is widely used because it can be conveniently and rapidly performed.

C. Enzyme-Linked Immunosorbant Assays

A wide range of variations on the enzyme-linked immunosorbant assay (ELISA) have been used to evaluate monovalent and multivalent inhibitors owing to the convenience and utility of these methods (Fig. 8) [58,59]. Generally, one binding partner is immobilized on the bottom of a microtiter plate, and a binding counterpart, labeled with a reporter, is allowed to adhere to the immobilized partner. Monovalent and multivalent compounds can be tested for their ability to inhibit the binding interaction. The reporter group can be a radiolabel, fluorophore, biotin, conjugated antibody, peroxidase, or other enzyme conjugate. In the case of a protein-saccharide interaction, either the lectin or the ligand can be immobilized. Inhibitory potencies obtained from ELISA and related assays provide an estimate of the functional affinity of various ligands, although confounding parameters can complicate the analysis. The immobilized partner is often displayed in a random mixture of orientations, some of which may not be optimal for binding. All soluble ligands will encounter these various receptor orientations; therefore a comparison of ligand activity generally affords the proper qualitative relative ranking. One problem with the variety of orientations observed is that they may not reflect those found in natural settings. Consequently, the artificial ligand spacing inherent in the ELISA can introduce a bias to

Figure 8 An example of an ELISA. Monomeric lectin is immobilized in the bottom of a microtiter well. Labeled multivalent ligand binds to the lectin. The assay can also be set up to observe lectin binding to immobilized ligand.

the assay. ELISAs, however, are simple to perform and can be used for rapid screening of multiple inhibitors.

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