[RASMB] Multi-wavelength velocity

[Borries Demeler]

> Do you have trouble with the wavelength re-setting when you use
> multiple wavelengths in velocity experiments?

We have the same problem on all of our machines. If you measure at
230 (steep absorbance shoulder), the problem is worse because just
a single nanometer wavelength shift causes significant change in
absorbance. We sometimes see shifts as large as 2-3 nm.

One way we deal with this problem is to do wavelength scans at multiple
concentrations of the sample and fit the spectrum to a sum of Gaussians
to calculate an intrinsic extinction spectrum.  The software can then
take the extinction coefficient from any wavelength and correct the
absorbance to a unified concentration. If adjusted for molar extinction,
for example by normalizing the gaussian fit at 280 with a calculated
extinction coefficient from peptide sequence, the concentration can be
expressed in molar units. This is implemented in the UltraScan software
for use in equilibrium experiments where we frequently use multiple
wavelengths for measurements in the same experiment. This method
is described in:

Thomas R. Russell, Borries Demeler, and Shiao-Chun Tu. Kinetic Mechanism
and Quaternary Structure of Aminobacter aminovorans NADH:Flavin
Oxidoreductase: An Unusual Flavin Reductase with Bound Flavin
Biochemistry. (2004) 43(6) pp 1580 - 1590.

-Borries