Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

MALLS and Sedimentation Equilibrium 135
Mx is the molar extinction coefficient, ℘ is the path length of the centerpiece
in cm, and n is the stoichiometry of the larger association species.
K conc = K fringe × (dn/dc × ℘ / ⊕) n– 1 × (M 1) n– 1 / n
where K conc is the association constant in molar concentration terms, K fringe is
the signal association constant, dn/dc is the specific refractive increment, ℘ is
the path length of the centerpiece in cm, ⊕ is the lightsource wavelength in cm,
M 1 is the monomer molecular weight, and n is the stoichiometry of the larger
association species.
The Sednterp program (5) is employed to calculate the partial specific volume
of the proteins from the amino acid compositions using the method of
Cohn and Edsall (8). The program also calculates the solvent density using
known values from physical tables.
4. Notes
1. Sample cell leakage is one of the more common problems encountered with the
analytical ultracentrifuge. This can be remedied by careful assembly of sample
cell components, the use of Spinkote grease on the cell housing gasket and screw
ring, tightening of the cell components to recommended torque pressures, and
avoiding the mixing of cell components from different sample cell sets. Care
should be taken not to apply excessive amounts of Spinkote grease because it will
end up being sprayed onto the walls of the vacuum chamber at high rotor speeds
and possibly coat the lenses of the optical systems.
2. The rotor vacuum chamber should be wiped clean with a lint-free cloth between
each sample run and the lenses checked and cleaned if necessary.
3. The flash lamp for the Absorbance Optical System gets a coating on it over time
and intensity checks should be performed periodically as detailed in the XLI
instruction manual and the lamp cleaned if necessary.
4. To reduce problems with systematic fringe distortion in the Interference Optical
System, which will adversely affect the fitting of the data, blank scans should be
performed at the same speeds as the sample scans using the same sample cells
filled with just buffer solution. These blank scans should then be subtracted from
the sample scans prior to fitting.
References
1. (1990) Instruction Manual for the Dawn Model F, Wyatt Technology Corp., Santa
Barbara, California.
2. (1994) Wyatt Optilab 903 Interferometric Refractometer Instruction Manual and
Software Guide, Wyatt Technology Corp., Santa Barbara, California.
3. Wyatt, Philip J. (1993) Light scattering and the absolute characterization of macromolecules.
Anal. Chimi. Acta. 272, 1–40.
4. (1997) Optima XL-I Analytical Ultracentrifuge Instruction Manual, Spinco Business
Center of Beckman Instruments, Palo Alto, California.