A Method for Determining the Sedimentation Behavior of Enzymes: Application to Protein Mixtures

Abstract

During an investigation of the gene-enzyme relationships in histidine biosynthesis in Xalmonella typhimurium, it became desirable to determine, in crude extracts, the approximate molecular weights of several enzymes. We have found sucrose gradient centrifugation to be a suitable method for determining sedimentation coefficients of enzymes in protein mixtures. A variety of enzymes of known properties have been studied in the development of the method. Although the separation cell of Yphantis and Waugh (1) has been demonstrated to be applicable to the determination of molecular weights when multiprotein solutions are used, the present method has several advantages over that system and these advantages will be discussed. Sucrose gradient centrifugation, using the swinging bucket head of the preparative ultracentrifuge, has been used extensively in the determination of sedimentation constants of viruses, mitochondria, microsomes, and ribosomes (2, 3). The adaptation of this method to relatively low molecular weight substances is reported here. In the sucrose gradient technique the sample to be studied is layered on a gradient and materials of different sedimentation properties separate from each other during the centrifugation. A hole is then punched in the bottom of the centrifuge tube and fractions are collected and analyzed. With slight modifications in the design of the apparatus for gradient production and fractionation, we have found the method to be simple and accurate. Sedimentation coefficients have been determined for a number of well characterized enzymes as well as a sample of ribonucleic acid, and the results are in good agreement with the values reported by others. The same values have been obtained whether the enzymes were analyzed as pure proteins or mixed with crude extracts. Although sedimentation coefficients were directly calculated for a variety of substances in order to determine the accuracy of the method, in general use the procedure may be simplified. The sedimentation coefficient (or approximate molecular weight) of an unknown enzyme may be determined by a simple ratio of mobilities when a standard well characterized enzyme has been added to the protein mixture. With the use of this technique the sedimentation behavior of several of the enzymes in the pathway of histidine biosynthesis in S. typhimurium has been determined.