Difference sedimentation velocity adapted to low molecular weight proteins

The difference sedimentation velocity technique reported by Kirschner and Schachman (1971, Biochemistry10, 1900–1919) has been modified to eliminate the need for a supernatant region. The method is now applicable to the measurement of small changes in sedimentation coefficient for low molecular weight proteins and other small macromolecules. Procedural changes necessary to overcome the absence of a supernatant region until late in the run have been devised and tested. A modified double-sector centerpiece was used to match the radial positions of the two menisci. The integration of the moment of the concentration difference was carried out from the meniscus to the plateau region, rather than over the peak only. The interference baseline was measured on photographs at the start of each run and after remixing. Some instability of baseline height was noted. The calculation method adjusted the baseline height to correspond with the concentration difference in the plateau region arising from unequal radial dilution. Tests of the method have been made using D₂O to retard the sedimentation of lysozyme. The interference results at low D₂O concentration (small values of Δs) are in agreement with schlieren results at high D₂O concentrations. Changes of 0.005 S have been detected.