ABSTRACT
Spectroscopy methods deal with the interaction between matter and electromagnetic radiation of varying wavelengths. The most common spectroscopy deals with UV and visible light absorption and can be used to characterize or quantify, for example, an aqueous solution of a protein. Aromatic amino acids typically absorb with a maximum wavelength around 260-280 nm due to their aromatic side chains. Trp has the largest ring system, and its relatively strong absorbance maximum is around 280 nm. Tyr and Phe have weaker absorbances, and their maxima are shifted toward shorter wavelengths. Absorbance measurements at 280 nm are often used for protein or peptide quantitation. Table 11.1 gives typical absorbance indices for Trp, Tyr, and Phe that can be used for this purpose. The peptide bond in a protein absorbs UV light at shorter wavelengths. In a wavelength range between 185 and 250 nm, the absorbance of a protein or peptide solution is dominated by peptide bond absorption. Studies of light absorption in this range therefore carry information about the peptide bond, its chirality, and its structure. Certain proteins also absorb light in the visible region, such as, for example, heme proteins, due to their conjugated prosthetic groups.
