Chromatographic and Electrophoretic Fractionation of Soluble Proteins of Brain and Liver

Abstract

The aim of the work the first stage of which is described here is to fractionate proteins of nervous tissue in order to compare these proteins with those of other organs to find specific qualitative differences, and to compare nervous tissue proteins of different species to find similarities. If unique proteins can be found which are common to nervous tissue of all species, then they may have some relation to function in the nervous system. This paper describes methods for fractionating proteins which are water-soluble. The general plan is to use the gentlest method of extraction first, followed by more drastic methods on the water-insoluble proteins. In a previous paper (1) a method was described for fractionation of water-soluble proteins from liver by chromatography on diethylaminoethyl cellulose by elution with a parabolic gradient to 1 M chloride. The validity of the method was shown in several ways. (a) Total protein recovery was high, and the protein chromatographic patterns were consistent from liver to liver. (5) A number of enzyme activities appeared as sharp peaks with high recoveries, indicating that different proteins were separated from each other with little loss. (c) Separate fractions, when rechromatographed under the same conditions, gave peaks of protein and enzyme activities at the expected positions with high recoveries. It is recognized that the efficiency of fractionation of a protein mixture by chromatography depends on the shape of the gradient used for elution. Since a given protein is adsorbed or desorbed on a cellulose ion exchange agent within a narrow range of pH or ionic strength, separation of similarly bound proteins is particularly sensitive to changes in slope of gradient. A defect of the parabolic gradient was that much of the protein appeared as a large, broad peak and a number of enzyme activities overlapped in the region of 0.1 M chloride during the elution. It would seem reasonable that a gradient more efficient in separating individual proteins should have a lower slope in the 0.1 M region than that in the parabolic gradient. We describe here a method for fractionation of soluble proteins from liver and brain with an improved gradient elution. The degree of resolution was indicated by the fact that a given protein during the chromatography was eluted in l/15 to l/25 of the