Refractive Index of Concentrated Protein Solutions

Abstract

AN accurate knowledge of the specific refraction increment α, or dn/dC, of proteins is important in measurements which involve the use of the ultra-centrifuge, electrophoresis and light-scattering. Recently, it has become important in connexion with cytological methods of refractometry¹ and mass determination² of living cells. The refraction increment has hitherto usually been measured only at rather low concentrations. The highest concentration reported seems to be by Howard³ in 1920, who found that the variation of refractive index with concentration of hæmoglobin was linear up to about 17 per cent. Armstrong et al. ⁴ found that α for crystalline serum albumin was constant up to a concentration of 14.6 per cent. Since in cytology considerably higher concentrations may be encountered, and it is well known that the refractive indices of some substances, for example sugars, do not vary quite linearly with concentration, we have measured the refractive indices of bovine serum albumin solutions up to concentrations exceeding 50 per cent. In order to avoid the difficulties associated with the measurement of protein concentrations by dry-weight or nitrogen determinations, we have obtained a factor proportional to the concentration by measuring the optical density at 278 mµ, using a Hilger “Uvispek” ultra-violet spectrophotometer. Deviations from the Beer–Lambert law were avoided by diluting all samples accurately by different amounts to give approximately the same final optical density. The final concentration was about 0.2 per cent in each case, giving optical densities between 1.0 and 1.3 for a 1-cm. path-length.