Quantitative analysis of protein mixtures by second derivative absorption spectroscopy

A new method for the quantitative analysis of protein mixtures based on multicomponent analysis of the second derivative near uv spectra is described. Using bovine eye lens crystallins, we demonstrate that the technique can provide precise concentrations of closely related proteins within mixtures, under both native and denaturing conditions. We have also successfully used the method to analyze the subunit composition of a heteromultimeric protein aggregate. The method is more rapid and precise than alternative approaches and offers the advantage of substantially reduced interference from many extraneous solution components and light scattering. It is also nondestructive and extremely sensitive, requiring only small volumes of sample at low total protein concentrations. Prospective applications are proposed for the study of eye lens crystallins, as well as for other protein/protein and protein/nonprotein mixtures.