Employment of Ultraviolet Spectroscopy in Analytical Chemistry of Proteins

Abstract

Methods of direct spectroscopic analysis of protein concentration using the linear relationship between extinction and concentration (Eq. (1.2)) are frequently employed in the examination of highly purified protein preparations and frac-tional distribution of proteins. Such methods have the important advantage that the sample remains intact and can be used for further investigations. Spectrophotometric analysis of proteins can be carried out under different experimental conditions (temperature, pH, composition, and molarity of buffer systems) using different wavelengths. Not unfrequently, the choice of these conditions is arbitrary and investigators are often doubtful as to whether they should strictly follow them. Using the data presented above on the analysis of absorption spectra of chromophore groups that compose the protein molecule, and considering the effect on these spectra of various environmental factors as well as spectral changes resulting from conformational transitions of proteins, we shall attempt to give some recommendations with respect to the experimental conditions necessary for the spectroscopic determination of protein concentration.