UV and Visible Absorbance Spectroscopy

Macromolecules of biological interest have a variety of intrinsic UV chromophores in addition to frequently being associated with other UV and visible chromophores in the form of cofactors, prosthetic groups, and substrates. A protein generally, depending on its amino acid composition, has a broad, featureless UV absorption spectrum with peaks in the 275 nm to 290 nm region. Changes in the environment of aromatic residues in proteins as a result of conformational changes induced by ligand binding have been studied by UV difference spectroscopy. UV spectroscopy has been extensively used in establishing the stoichiometry of interaction of synthetic polynucleotides. The temperature-induced absorbance changes are attributable to the existence of a hydrogen-bonded solvent — solute complex at low temperatures resulting in a lowering of the ground state energy level of the n electrons in nitrogen-1 of the base. The flavoenzymes are characterized by atypical flavin absorption spectra, due to the absorbance contribution of the metals.