The Simultaneous Determination of Partial Specific Volumes and Molecular Weights with Microgram Quantities

Abstract A method is described for the simultaneous measurement of partial specific volumes and molecular weights of proteins and other substances by sedimentation equilibrium experiments in H2O and D2O (or D2O18) solutions. In effect, the method is a differential sedimentation equilibrium technique based upon the change produced in the equilibrium concentration distribution when the density of the solution is increased by the use of D2O (or D2O18). The equations used for the two parallel sedimentation equilibrium experiments in H2O and D2O (or in H2O and D2O18) account for deuterium exchange by the incorporation of a factor which changes only slightly for different proteins and can be estimated with sufficient accuracy from knowledge of the composition of the solute. Experiments were conducted on H2O and D2O solutions of adenosine, ferredoxin, ribonuclease, myoglobin, α-chymotrypsinogen, and bovine plasma albumin, and the resulting values of the partial specific volumes and molecular weights were in excellent agreement with currently accepted values. In addition excellent results were obtained for myoglobin and α-chymotrypsinogen from parallel sedimentation equilibrium experiments on H2O and D2O18 solutions. With D2O18 solutions the potential accuracy in the determination of partial specific volumes is substantially greater than with D2O since the density of D2O18 is so much greater and the consequent effect on the redistribution of the solute molecules is so large. The method is direct and no more laborious than a conventional sedimentation equilibrium experiment, does not depend on knowledge of the solute concentration, yields partial specific volumes accurate to better than 1.0%, and can be used in certain situations with impure samples.