Measurement of the physical and chemical properties of a purified protein requires a method for determination of the protein concentrations of the solutions employed. Spectrophotometric measurements are widely used as a convenient, sensitive, and precise method for the determination of protein concentration. However, spectrophotometric measurements require a knowledge of an extinction coefficient for each protein, since the relative number of chromophoric amino acid residues varies from protein to protein. Determination of an extinction coefficient requires an independent and precise method of measuring protein concentration. The limited supply of many purified enzymes precludes the use of dry weight measurements in determining the extinction coefficient. Although chemical methods for protein determination, such as the Kjeldahl, Folin-Ciocalteu, or biuret procedures, require much less protein, such measurements must be related to a standard reference protein.
Since each of these chemical methods is sensitive to the relative abundance of specific amino acid residues in proteins (l-3)) an arbitrary standard protein such as bovine serum albumin may be inappropriate for a given protein of unknown composition.
Although the molar refractions of the amino acid residues also exhibit considerable variation (4)) the refractive indices of proteins vary by less than 62% (4,5). Accordingly, refractometric measurements can be used to determine protein concentration. This communication describes the use of an analytical ultracentrifuge as a differential refractometer to determine the concentration of small volumes of dilute protein solutions.