Chapter 8 Density Labeling of Proteins

Abstract

Publisher Summary This chapter discusses the density labeling of proteins. A simple way of demonstrating enzyme synthesis is the method of density labeling of the enzyme protein with heavy isotopes followed by equilibrium density gradient sedimentation. This method is based on the same rationale as the classic method of density labeling and isopycnic centrifugation of nucleic acids and is the same method by which the semiconservative reduplication of DNA was demonstrated. If a protein is centrifuged for sufficient time in a density gradient, it will form a band at the point in the gradient where the density of the gradient corresponds to its own buoyant density. After centrifugation, the content of the centrifuge tube is divided into fractions of 3–5 drops. The density slope of the gradient can be conveniently determined by measuring the refractive index in some fractions. The other fractions are assayed for enzymatic activity. Density labeling can be combined with radioactive tracer methods to determine the turnover of proteins in general or in individual enzymes.