Protocol to determine accurate absorption coefficients for iron-containing transferrins.

An accurate protein concentration is an essential component of most biochemical experiments. The simplest method to determine a protein concentration is by measuring the A(280) using an absorption coefficient (epsilon) and applying the Beer-Lambert law. For some metalloproteins (including all transferrin family members), difficulties arise because metal binding contributes to the A(280) in a nonlinear manner. The Edelhoch method is based on the assumption that the epsilon of a denatured protein in 6 M guanidine-HCl can be calculated from the number of the tryptophan, tyrosine, and cystine residues. We extend this method to derive epsilon values for both apo- and iron-bound transferrins. The absorbance of an identical amount of iron-containing protein is measured in (i) 6 M guanidine-HCl (denatured, no iron), (ii) pH 7.4 buffer (nondenatured with iron), and (iii) pH 5.6 (or lower) buffer with a chelator (nondenatured without iron). Because the iron-free apoprotein has an identical A(280) under nondenaturing conditions, the difference between the reading at pH 7.4 and the lower pH directly reports the contribution of the iron. The method is fast and consumes approximately 1mg of sample. The ability to determine accurate epsilon values for transferrin mutants that bind iron with a wide range of affinities has proven to be very useful; furthermore, a similar approach could easily be followed to determine epsilon values for other metalloproteins in which metal binding contributes to the A(280).