Boundary centrifugation in isovolumetric and isokinetic cesium sulfate density gradients: application to cartilage proteoglycans and other macromolecules.
A boundary sedimentation methodology is described that avoids plateau dilution and simplifies the calculation of centrifugal parameters. The technique is designed for the preparative ultracentrifuge and uses a newly developed sectorial cell. It is based on previous developments of the transport method and depends on isokinetic or isovolumetric Cs2SO4 density and viscosity gradients. These gradients are prepared with a single-chamber mixing device, and the only two parameters required for their calculations are presented in a tabulated form for general use with most available rotors and cell sizes. Conditions are specified (1) to assure that the density and shape of the sedimenting molecules remain invariant through the selected electrolytic gradient, (2) to monitor the gradient profiles, and (3) to verify attainment of isokinetic or isovolumetric sedimentations. A set of equations is presented to calculate the average and transport sedimentation coefficients and the differential sedimentation coefficient distribution for both the isokinetic and isovolumetric centrifugal regimes. The method was applied to slowly diffusing polydisperse proteoglycan monomers, to a paucidisperse DNA from bacteriophage PM2, and to a diffusible monodisperse system (purified bovine serum albumin). In all cases, the expected results were obtained.
D000818:Animals; D002356:Cartilage; D002417:Cattle; D002499:Centrifugation, Density Gradient / Q000295:instrumentation / Q000379:methods; D002586:Cesium; D003090:Coliphages; D004279:DNA, Viral / Q000302:isolation & purification*; D011509:Proteoglycans / Q000302:isolation & purification*; D014461:Ultracentrifugation / Q000295:instrumentation / Q000379:methods; D014783:Viscosity
D004279:DNA, Viral; D011509:Proteoglycans; D002586:Cesium