Zinc Binding Motif - AAA vs. UV/Vis

[Michael S Curtis]

I have been working on the physical characterization of a zinc binding domain 
and have seen something curious. To get an accurate protein concentration I 
have been using amino acid analysis (AAA). To make protein concentration 
determinations a little quicker I wanted to use the protein concentration 
determined by AAA and using A280 measurement with Beer's Law get an 
experimentally determined extinction coefficient. When I compare the 
experimentally determined extinction coefficient with a theorhetical 
coefficient (Gill and von Hippel, Anal. Biochem., 182, 319-326), the 
experimentally determined coefficient is approximately three times the 
theorehtical. Gill and von Hippel show that in the worst case they were seeing 
a 43% deviation between experimentally and theorehtically determined 
extinction coefficients; average was about 10%, I am seeing significatnly 
larger. I'm thinking that the zinc binding with the domain is somehow 
effecting the A280 absorbance. I know zinc itself does not absorb 
significantly at 280, but I'm wondering is the zinc binding to its ligands is 
somehow effecting their abosorption. I am very comfortable with the accuracy 
of the AAA and A280 measurment. Any insight or ideas on why there may be such 
a great difference in the theorehtically determined extinction coefficient and 
the experimentally determined coeffiecient would be greatly appreciated.

Thanks,

Mike Curtis
curtis at bms.com