Titration of carbohydrate residues with calcofluor

in addition to the shift in the emission maximum, we observe an increase in the fluorescence intensity (Fig. 8.15). 

The intensity decrease is clearly hyperbolic and therefore a mathematical binding analysis can be performed using the following quadratic equation obtained from the definition of the equilibrium constant (assuming all binding sites have the same dissociation constant Ka)  

Titration experiments are usually canied out by following the intensity variation. In the above-described experiments, we show that it is possible to perform titration experiments by following not only the fluorescence intensity variation but also the shift in the position of the emission maximum of calcofluor. This shift is sensitive to sialic acid residues only when they are bound to ai-acid glycoprotein (Fig. 8.14). Therefore, sialic acids of a i-acid glycoprotein possess a spatial conformation that can be detected by calcofluor. 

Addition of free sialic acid to calcofluor decreases the fluorescence intensity of the fluorophore (Fig. 8.17). Thus, the type of interaction between free sialic acid residues and calcofluor is different from that observed when sialic acids are bound to a i-acid glycoprotein. 

The fact tliat the interactions between calcofluor and sialic acids differ whether the latter are on the protein or free in solution, indicates that the sialic acids on a i-acid glycoprotein possesses a defined spatial conformation. Also, since titration curves witli calcofluor are not the same whether the sialic acid residues are present or not, one may conclude that the presence of the sialic acid confers to the carbohydrate residues backbone a spatial conformation that changes upon desialylation of the protein. 

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