Protocol to determine accurate absorption coefficients for iron-containing transferrins

Abstract

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 (ε) 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 ε 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 ε 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 1 mg of sample. The ability to determine accurate ε 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 ε values for other metalloproteins in which metal binding contributes to the A 280.