The Simultaneous Determination of Partial Specific Volumes and Molecular Weights with Microgram Quantities*78

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 Hz0 and D20 (or D2018) 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 DzO (or D2018). The equations used for the two parallel sedimentation equilibrium experiments in Hz0 and D20 (or in H20 and D2018) 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 H20 and D20 solutions of adenosine, ferredoxin, ribonuclease, myoglobin, ar-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 cr-chymotrypsinogen from parallel sedimentation equilibrium experiments on Hz0 and D2018 solutions. With D2018 solutions the potential accuracy in the determination of partial specific volumes is substantially greater than with Dr.0 since the density of D2018 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 l.O%, and can be used in certain situations with impure samples.