Fractionation of fluorescent-labeled proteins according to the degree of labeling

Abstract

Labeling of macromolecules often facilitates the investigation of their properties and interactions. However, labeling normally leads to a heterogeneous population of labeled species which may differ significantly in their chemical and biological reactivity. This report deals with a method for separating labeled materials into fractions according to their degrees of labeling. Preparative acrylamide gel electrophoresis or gel filtration on Sephadex G-100 separate fluorescent-labeled proteins into fractions having different degrees of labeling. The dansyl and the fluorescein derivatives of ovalbumin and bovine serum albumin have been studied. Typical electrophoretic separations result in fractions differing by as little as 0.05–0.1 in the average mole ratio of dye to protein. However, the nonintegral values obtained make it clear that the preparations are still nonuniform with respect to degree of labeling. Fractions obtained by gel filtration show several bands when tested by analytical acrylamide gel electrophoresis, while those obtained by preparative gel electrophoresis show only a single band. In the electrophoretic separations, the more rapidly migrating species were found to be more heavily labeled. This finding is explicable on the basis that the introduction of a dye molecule onto a protein should alter the net charge by −3 for fluorescein, and by −1 for dansyl.