DETERMINATION OF HYDRATION AND PARTIAL SPECIFIC VOLUME OF PROTEINS WITH SPRING BALANCE

The buoyant weight of a macromolecular component (2) enclosed within a semipermeable membrane and kept at equilibrium with solvent (water) and diffusible solutes depends upon the partial specific volume and on the hydration of the macromolecules, as long as binding of other components besides thepure solvent can be neglected. In an aqueous solvent with a density close to the density of water or in water itself the partial specific volume of the macromolecular solute determines the buoyant weight. Increased density of the solvent (e.g., by addition of glycerol) leads to an increase of thebuoyancy attributable to the “hydration” water “bound” to the macromolecules. By the use of a quartz spring balance the buoyant weights of bovine serum albumin, tobacco mosaic virus, and tobacco mosaic virus protein in water, aqueous buffer, and glycerol-buffer mixtures were determined directly. Values for the partial specific volumes in buffer at 20° were determined independently by means of pycnometry. The results, V2 = 0.727, 0.733, and 0.734cc per g for tobacco mosaic virus, tobacco mosaic virus protein, and bovine serum albumin, respectively, are in good agreement with data in the literature. Values obtained with the spring balance agree within about 1%. Increasing glycerol concentration (c < 30% w/v) unveils a linear relationship between density and buoyant weight. The slope, —dW/dp = γ2GV1 + γ2V2, as a measure of the hydration G (γ2 being the amount of solute 2, and V1 V2, the partial specific volumes of water and the macromolecular component (2)) leads to G = 0.16, 0.20, and 0.28 g of water per g of protein for tobacco mosaic virus, tobacco mosaic virus protein, and bovine serum albumin, respectively, in fair agreement with previous data from other determinations. © 1969, American Chemical Society. All rights reserved.