extinction coefficient

chris halkides (halkidesc@uncwil.edu)
Fri, 10 Jul 1998 09:09:06 -0400

********************************************************************************
A well characterized protein with known molecular weight has a defined
extinction coefficient for quantitating protein amount by UV analysis.
However, when the protein is lyophilized, the gravimetric weight of the
lyophilized material is much higher by about 20% than the value calculated
from UV analysis. This preparation is also extremely pure which excludes
impurities contributing to the overall weight. What can be happening and
how would you test your hypothesis?

John Nakamura
********************************************************************************

John,

I have heard that lyophilized proteins contain substantial amounts
of tightly bound water molecules (more than enough to account for the 20%
difference), but I have been unable to locate a good reference to back this
up. I suppose to be really rigorous, one must also include counterions. I
am not sure how to prove this.

On the other hand, reported e(280) values for the same protein can
differ by this much as well, so we shouldn't overlook the default
hypothesis of experimental error in the extinction coefficient.

Chris Halkides

Christopher Halkides
Dept. of Chemistry, UNCW
601 S. College Road
Wilmington, NC 28403-3297
(910) 962-7427