Proteindrug Binding Assays

It is well known that drugs bind to plasma proteins, particularly to serum albumin and a-acid glycoprotein, and that only the unbound, or free, fraction is responsible for any pharmacological effect. For protein-drug binding studies size-exclusion chromatography in one of three variants—namely, the Hum-mel-Dreyer method (1962), the vacancy peak method (Sebille, et al., 1979), and frontal analysis (Cooper and Wood, 1968)—is the traditional method of choice. Kraak et al. (1992) tested all three variants in a CZE version and showed that the frontal analysis procedure represents the preferred approach.

Size-exclusion chromatography methods exploit the difference in the exclusion of the drug and drug-protein complex from the column packing. In CE, separation of bound and unbound drug is based on charge and size differences. Because the protein molecule is much larger and carries more charge than a drug molecule, it is reasonable to assume that if the drug is bound to the protein, neither the protein's charge nor its molecular mass will be significantly altered. Consequently, both the drug and the drug-protein complex will have the same electrophoretic mobility, and methods similar to those developed for size-exclusion chromatography can be used with capillary electrophoresis, provided the protein and the unbound drug have different electrophoretic mobilities.

When the Hummel-Dreyer method is applied in CZE, the capillary is filled with the background electrolyte containing the drug, which causes a large detector background. Then a small sample containing the drug, buffer, and the protein is injected. The total concentration of the drug in the sample is the same as in the background electrolyte, but a part of it is bound to the protein. If potential is switched on, the protein-drug complex starts to move to the cathode, leaving a local deficiency in the drug concentration in the direction to the anode. This deficiency causes a negative peak, which moves with the mobility of the drug, and its size corresponds to the amount of the bound drug. During the migration, the protein-drug complex is in equilibrium with the free drug in the buffer. Thus, the protein-drug complex will give a positive peak. As stressed by Kraak et al. (1992), if at the detection wavelength the absorbance of the binding protein is zero, and if the molar absorptivities of the drug and drug-protein complex are the same, then the areas of the positive and negative peaks must be equal. This situation, however, rarely occurs in practice (Fig. 8.11).

To apply the vacancy peak method, the capillary is filled with the background buffer containing the protein as well as the drug. The situation is similar to the Hummel-Dreyer method described above, and the background signal in the detector is quite high. Next a small plug of the buffer only is applied and the power is switched on. Assuming that the mobilities of the protein and the protein-drug complex are higher than the mobility of the drug itself, the following effects are seen at the front and rear edge of the buffer plug:

At the front edge the drug is migrating more slowly than the protein and therefore it stays behind.

At the rear end the protein migrates faster than the plug.

This process progresses until both fronts reach one another. Then, since the whole process is reversible, in the middle of the plug the protein starts to ln|. plug: BSA





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