Matthew Alan Bratkowski wrote:
> Hello.
>
> I am working with a protein that turns a yellowish-brown color when it is
> concentrated to around 2 mg/ml or higher in a small volume (a few hundred
> uL).  I was wondering if the protein bound a metal or other prosthetic
> group that would give it this color?  The protein's color somewhat
> resembles iron binding proteins, but there is no peak in the 400 nm range
> that would suggest heme, and an iron sulfur cluster is not that likely
> since there are only five cysteines in the protein.  Proteins with
> structures homologous to the one I am studying bind magnesium, but are not
> know to bind other metals.  Any information about what this color might
> suggest about the protein or how I could analyze possible bound metals or
> prosthetic groups using only a small amount of protein would be helpful.
>
> Matt
>   
Many proteins will turn yellowish if concentrated enough, due to the UV 
absorption tail. However, it usually takes >>10 mg/mL of homogeneous, 
cofactor-free protein to see this, so your supposition there may be a 
cofactor or metal involved warrants further investigation. The most 
straightforward way to analyze proteins for metal is to use ICP-OES, and 
it will not require a large amount of protein to do this. We routinely 
quantify zinc-metalloenzymes (and metal-substituted enzymes) using 
ICP-OES. For most first-row transition elements, ICP-OES sensitivies are 
such that you can get reasonable signal for 0.1 ppm (100 ppb) metal ion. 
We normally take protein that is 100-500 uM and dilute it in 
high-quality (18 Mohm/cm2) deionized water 30-100X, depending on the 
starting protein concentration. Using our ICP-OES (Perkin-Elmer 3000) we 
can get very reasonable readings from 1.5 mL of diluted solution (2 
replicates). If you can spare more protein, you can get more accuracy 
and S/N. Calibration can be done with commercial standards diluted to 1 
ppm. We use a mixed metal standard so we can screen for many metals at 
once. Dilution of your sample is important, as large viscosity 
differences between samples will dramatically affect the nebulizer 
efficiency of the ICP, and your results. In practice, we have found that 
a dilution of 20X or more is usually sufficient to make the viscosity 
differences between the calibration standard and the sample negligible 
in practice. (If you are a real stickler, you should do a standard 
addition to a diluted protein sample, but this is not usually necessary, 
and consumes double the protein.)

You should be aware that all dilution operations for preparing samples 
should be carried out in glass, preferably acid-washed glass, and NOT in 
plastic tubes. Samples should be analyzed immediately. Glass can slowly 
leach metal ions into your sample, including Fe and the ubiquitious Zn. 
Plastic is even worse. At  low protein concentrations, plastic eppendorf 
tubes can rapidly and irreversibly adsorb some of all of your sample. In 
addition, nearly all plasticware notoriously leaches Fe into the 
solution over time, and is thus especially unsuitable for this metal. 
Even clean, non-acid-washed glass is better than plastic. Dialysis of 
your protein against metal-free buffer or distilled water (if it can 
tolerate it) may be required to remove adventitious metal ions. 
(However, loosely protein-bound metals may be removed by this process, 
too.) Running the dialysis buffer through the ICP at the same dilution 
as the sample is advised to assess your background level of contamination.

Cheers,


-- 
------------------------------------------------------------------------
Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
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