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Clinical Chemistry 37: 87-90, 1991;
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Clinical Chemistry, Vol 37, 87-90, Copyright © 1991 by American Association for Clinical Chemistry

Isoelectric focusing electrophoresis of protein-ligand conjugates: effect of the degree of substitution

MS Barbarakis and LG Bachas
Department of Chemistry, University of Kentucky, Lexington 40506-0055.

The degree of substitution of protein-ligand conjugates can be determined from the change of the isoelectric point (pI) of the protein when ligand molecules are attached to its surface. Specifically, the pI values of conjugates with known degrees of substitution are obtained by isoelectric focusing electrophoresis and are used to generate a calibration curve that relates these two variables. The shape of the curve is sigmoidal and can be predicted by a theoretical model that takes into account the degree of substitution and the amino acid composition of the protein. By using such a calibration curve, one may estimate the degree of substitution of a given protein-ligand conjugate from its pI value. The applicability of the method is demonstrated with conjugates of pyridoxal 5-phosphate and avidin.




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Copyright © 1991 by the American Association for Clinical Chemistry.