An automated method for determination of the molecular weight of macromolecules via sedimentation equilibrium in a preparative ultracentrifuge.

An automated method for quantitating the concentration gradient of a macromolecule brought to sedimentation equilibrium in a preparative ultracentrifuge is described. Between 30 and 80 microliter of macromolecular solution are spun in a combination centrifuge tube and optical cell fabricated from quartz. Immediately following the conclusion of centrifugation the tube is optically scanned along its length using a spectrophotometer sample cell modified for this purpose. Successive scans of the same tube containing first solution and then solvent permit the differential absorbance due to macromolecule to be measured precisely as a function of position within the centrifuge tube, and hence the radial position within the rotor. Molecular weights calculated from data so obtained from 32 centrifugations of eight proteins ranging in Mr from 12K to 340K using either swinging-bucket or fixed-angle rotors agree with previously published values with a standard deviation of 5%.