Re: metal analysis

Carol Beach (cmbeach@pop.uky.edu)
Wed, 15 Jul 1998 14:59:05 -0400 (EDT)

Dear Chris,
I was waiting, hoping to see a reply to your question about a state-of-
the-art method for metal quantitation in proteins. Nothing has appeared on
the bulletin board, so maybe you won't mind a suggestion of an old technique.
I spent (too much) time in grad school trying to directly measure
the percent incorporation of 5-bromouracil in E.coli DNA and also the amount
of bromide released after the DNA was exposed to x- or gamma-rays. I won't
bore you with what didn't work well (neutron activaton was almost okay).
What got me out of school was inheriting a homemade X-ray fluorescence
spectrometer.
Low energy X-rays can knock an inner shell electron out of its shell, and
the cascading of electrons from higher energy levels to fill spaces in the
lower energy levels results in the emission of characteristic X-rays,
characteristic for the atomic number of the atom as well as for the
particular electron transition. I think this may be worth a try.
You'll need an appropriate internal standard for calibration, and
the sensitivity depends upon how long you can wait for data collection.
You'll want at least 10,000 counts net for good counting statistics. Is
there anything in the protein solution with higher Z than the metal of
interest? If so, it's possible that the characteristic radiation from the
high-Z element might have sufficient energy to ionize the element of
interest. Be careful.
Surely there's an X-ray fluorescence spectrometer at UCSF. If not,
there has to be one at Berkeley. When my Army Surplus transformer died a
month before I was finished collecting data, I found another (authentic)
X-ray fluorescence spectrometer across the street at the Kansas Geological
Survey. Check with the rock people. Or I can send you tips on how to build
your own!
Good luck.

Carol

At 10:26 AM 7/8/98 -0700, you wrote:
> We want to analyze a protein for metal content. Does anybody know
>where we might have this done? Also, what is the sensitivity of this
>technique? We could express the protein in bacteria and make milligrams if
>necessary.
>
>Chris (turck@itsa.ucsf.edu)

Carol Manley Beach, Ph.D.
Protein Sequence Analysis
Macromolecular Structure Analysis Facility
232 Combs Cancer Research Building
University of Kentucky
Lexington, KY 40536-0096

cmbeach@pop.uky.edu
(606)257-4932 (office)
(606)257-4475 (lab)
(606)323-3909 (FAX)