Abstract
Proper interpretation of analytical ultracentrifugation (AUC) data for purified proteins requires ancillary information and calculations to account for factors such as buoyancy, buffer viscosity, hydration, and temperature. The utility program SEDNTERP has been widely used by the AUC community for this purpose since its introduction in the mid-1990s. Recent extensions to this program (1) allow it to incorporate data from diffusion as well as AUC experiments; and (2) allow it to calculate the refractive index of buffer solutions (based on the solute composition of the buffer), as well as the specific refractive increment (dn/dc) of proteins based on their composition. These two extensions should be quite useful to the light scattering community as well as helpful for AUC users. The latest version also adds new terms to the partial specific volume calculations which should improve the accuracy, particularly for smaller proteins and peptides, and can calculate the viscosity of buffers containing heavy isotopes of water. It also uses newer, more accurate equations for the density of water and for the hydrodynamic properties of rods and disks. This article will summarize and review all the equations used in the current program version and the scientific background behind them. It will tabulate the values used to calculate the partial specific volume and dn/dc, as well as the polynomial coefficients used in calculating the buffer density and viscosity (most of which have not been previously published), as well as the new ones used in calculating the buffer refractive index.
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Program and source code availability
The program and its source code are in the public domain; it can be redistributed or modified under the terms of version 3 of the GNU General Public License as published by the Free Software Foundation (see http://www.gnu.org/licenses/). The program installer can be downloaded from the author’s website (http://www.jphilo.mailway.com/download.htm); both the program installer and the source code are available at the RASMB software depot (http://philo.rasmb.org/software/sednterp/).
Notes
In this article, the term “buffer” means the solution in which the protein or other macromolecule is dissolved (whether or not that solution contains a buffering agent). That is, the buffer represents all solution components other than the macromolecule.
Do not confuse the s* symbol or the term “apparent sedimentation coefficient” as used here with the same symbol and terminology used in the context of the sedimentation coefficient distributions g(s*), ls-g(s*), or G(s*). In that context, the “apparent” adjective and the ‘*’ symbol relate to the spreading of sedimentation boundaries by diffusion, not to the fact that the value depends on buffer density, viscosity, or temperature.
It should probably be stated explicitly that standardizing s* to “conditions of pure water” only means correcting for differences in buffer density and viscosity relative to pure water, and not that one should actually measure the value in pure water (which would give highly distorted values for any protein due to strong electrostatic solution non-ideality effects at low ionic strength).
Note that the corresponding equation in Laue et al. (1992) was incorrect.
The tables in the Handbook of Chemistry and Physics are based on n = 1.3330 for pure water (relative to air).
Note that although in this article we are distinguishing between the f/fp and f/f0 ratios in order to document all calculation details, in a publication the f/fp ratio would normally be described as “the f/f0 ratio”. In other words, outside the context of SEDNTERP, the symbol f0 normally means the frictional coefficient calculated from the equivalent radius, no matter how that equivalent radius was calculated.
These coefficients differ from, and provide better accuracy than, those for corresponding Eqs. 23 and 24 in Laue et al. (1992).
Note that in Zhao et al. 2011 the molar refractivity value listed for cysteine is actually that for cystine.
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Acknowledgements
The author wishes to thank Thomas Laue and David Hayes for their major contributions to creating versions 1 and 2 of SEDNTERP, for ongoing support, and for useful discussions regarding new features for version 3. He also thanks Mattia Rocco for encouraging adding the dn/dc, refractive index, and improved \(\overline{v}\) calculations (and also testing those features), as well as John Correia and Walter Stafford for beta testing.
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Philo, J.S. SEDNTERP: a calculation and database utility to aid interpretation of analytical ultracentrifugation and light scattering data. Eur Biophys J 52, 233–266 (2023). https://doi.org/10.1007/s00249-023-01629-0
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DOI: https://doi.org/10.1007/s00249-023-01629-0