Investigating Protein Structure on a Shoestring: Solvent Exposure of Tryptophan and Tyrosine Side Chains from Differential UV Absorption

A forgotten version of differential UV spectroscopy is resumed to form a simple and low-budget laboratory experiment for Chemistry, Biology, and Biotechnology undergraduate students. The shifts in the UV absorption of the solvent-exposed chromophoric side chains of a protein that are produced by a change in the solvent nature, from water to 4:1 water:ethylene glycol, produce an overall change in the UV absorption spectrum of the protein. Analysis of the resulting differential spectrum offers a straightforward way to estimate the number of exposed tyrosines and tryptophans. The experiment also includes an attempt to validate both the methodology and its specific implementation that consists of repeating the practical operations and the spectral analysis in conditions in which the side chains of all tyrosines and tryptophans in the protein should be exposed to solvent and, thus, contribute to the generated differential spectrum, namely, at a high concentration of a chaotropic agent. Students critically analyze the latter hypothesis also on the basis of the results of a parallel laboratory experiment in which they investigate fluorometrically the unfolding of the same protein caused by the same chaotropic agent.