Application of electrophoresis-convection to the fractionation of bovine gamma-globulin.

A method of fractionation of proteins in solution in which an electrophoretic adaptation of the principles of the Clusius column is utilized was suggested by Kirkwood in 1941 (1) and tested experimentally by Nielsen and Kirkwood (2) several years later. Recently an electrophoresis-convection apparatus of improved design has been described and successfully used in the fractionation of the pseudoglobulin of horse diphtheria antitoxin (3) and bovine serum proteins (4). Fractionation occurs in a narrow vertical channel between two semipermeable membranes, connecting an upper and lower reservoir. Separation depends upon the superposition of diflerential horizontal electrophoretic transport of the components on vertical convective transport of the solution as a whole. The vertical convective transport is controlled by the horizontal densit,y gradient produced by the electrophoretic migration of the proteins across the channel. The result of the superposition of horizontal electrophoretic transport and vertical convective transport is movement of the mobile components from the top reservoir to the bottom reservoir at rates depending on their mobilities, with a relative enrichment of the top reservoir with respect to the slow components and the bottom reservoir with respect to the fast components. The separation of a protein mixture possessing discrete mobility and isoelectric point spectra, e.g. serum, into its constituent proteins is accomplished by successive immobilization of the components at their respective isoelectric points and transport of the mobile components from the top reservoir of the apparatus. In the case of a protein which migrates as a single boundary in an electric field but possesses a specified mobility distribution as revealed by reversible electrophoretic boundary spreading, e.g. y-globulin, fractionation is accomplished by means of a modified isoelectric procedure in which the fractionation is carried out at a pH displaced by an arbitrary amount from the mean isoelectric point of the heterogeneous protein. Transport in the apparatus leads to a redistribution of the protein ions, such that the fractions withdrawn from the top and bottom reservoirs possess mobility dis-