> > 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.
This does not rule out other metals, though. For instance, protein kinases in eukaryotes always use magnesium, but the ones in TB - which are nearly identical in global structure (with > 30% sequence identity) and appear to be enzymatically similar - use manganese instead, as do several other TB homologs of magnesium-binding proteins. This is a quirk of TB's physiology but there are certainly other protein families (glyoxalases, dioxygenases) that vary in metal preference depending on organism and substrate preference. I don't know if Mn would cause that color (although some Mn compounds are brown or orange-brown); however, there are many ligands that could. Heme will also change color depending on iron oxidation state, and possibly local chemical environment. Endogenously purified full-length E. coli catalase, for instance, is green, but a common proteolytic fragment (from the same prep) is yellow-brown. 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. > Mass spec, obviously; if it's a metal, ICP-AES may be more effective, but I'm not sure how much protein you need. You can also use X-ray fluorescence to identify metals, but as far as I know this requires a synchrotron. Sometimes the visual light spectrum alone is enough to tell you what's there, but it can be difficult to cross-reference this with published data since (AFAIK) there is no online database of spectra.
