Analytical Ultracentrifugation (AUC) experiments are a direct measurement of basic hydrodynamic and thermodynamic properties of macromolecules in solution. Because sedimentation relies on the principal property of mass and the fundamental laws of hydrodynamics, AUC is a valuable technique for a wide variety of solution conditions and the information obtained does not depend upon assumed relationships to measurements of standard molecules. It is a biophysical method that does not depend on the interaction of the sample with a matrix or surface. The direct determination of boundary sedimentation or gradient formation can be further analyzed using curve fitting methods to rigorously determine sample purity, choose between binding models, detect and characterize conformational changes, measure equilibrium constants for self and hetero-associating systems, and determine thermodynamic parameters such as the second virial coefficient.
Methods available;a) Sedimentation velocity experiments will be carried out on a Beckman Instruments Optima XL-I Analytical Ultracentrifuge equipped with a real-time video-based data acquisition system and Rayleigh optics. Apparent sedimentation coefficient distribution patterns will be computed by the time derivative method using signal averaging (Stafford 1992; Stafford 1994; Stafford 1994; Liu and Stafford 1995; Stafford 1997; Stafford 2000; Stafford and Sherwood 2004) Sedimentation boundaries will be analyzed using time derivative g(s*) analysis as described previously (Stafford 1992; Stafford 1994; Stafford 1994) and with the recently developed SEDANAL software for the analysis of interacting and non-interacting systems (Stafford and Sherwood 2004). Protein concentrations will be typically in the range 0.1 to 1.0 mg/ml. All protein solutions will be dialyzed against their respective buffers and dialysate used for all dilutions.
Using SEDANAL, molecular weights will be computed from sedimentation velocity profiles by non-linear least squares curve fitting algorithm employing the simplex directed search method (Nelder and Mead 1965) using the finite element method of Claverie (Claverie et al. 1975; Claverie 1976). Values of s and D obtained from the fitting procedure are substituted into the Svedberg equation to obtain the molecular weight. Values of the partial specific volume and hydration are computed from the amino acid sequence data for the proteins using the consensus partial volumes of Perkins (Perkins 1986) and the hydration data of Kuntz and Kauzman (Kuntz and Kauzman 1974) respectively. Combination of the hydration data with frictional coefficient measurements allows shape analysis according to various hydrodynamic theories including Perrin's equations for ellipsoids of revolution and bead modeling as described by Terrak et al. (Terrak et al. 2003)
b) Sedimentation equilibrium analysis will be carried out according to the methods of Yphantis and Roark (Yphantis 1964; Roark and Yphantis 1969; Roark and Yphantis 1971; Yphantis and Roark 1972). The data will be analyzed by global fitting of combined data sets from multiple loading concentrations and speeds, as described previously (Mukhopadhyay et al. 2005) using the global equilibrium fitter included in the SEDANAL package .
For more details or consultation on the application of Analytical Ultracentrifugation to a specific application please contact auc@analyticalbiotechservices.com for a price quotation and availability of this service.
