UV-Visible spectroscopy

Standalone role of optical spectroscopy in our studies has been put forth with the onset of our research on protein radicals. Direct studies on catalytic radicals have traditionally being focused on spin resonance properties of a characteristic unpaired electron as seen by EPR spectroscopy. In many cases this classical approach has yielded remarkable details on local environment of an unpaired electron. In other cases, however, protein radical radicals stay close to an odd-spin metal that generated them. Strong magnetic interactions that take place between paramagnetic radical and metal centers may render both EPR invisible, such as in the case of Cu_B and tyrosine radical in cytochrome c oxidase.

Our UV-Vis spectrometer provides best stability and sensitivity in the UV region. Its open-bench, flexible design can handle efficiently variety of samples from rotating tubes at 90^oK to to transmission electrochemical cell. Back-illuminated CCD camera gives highest quantum yield in far UV. Two narrow-band, tunable light sources allow to carry specific photolysis of samples.

Weak optical signatures of some amino acid radicals in the visible spectrum have been known for a long time, although these are often obscured by stronger, and variable, signatures of metals and other cofactors.The biggest potential, however, is in the ultraviolet region which remains unexplored in biology mostly due to past technical limitations. Not only the UV region carries more structural information than visible region, it carries information about the configuration of the radical site prior to oxidation – something neither visible absorption nor spin resonance provides. While electronic absorption is arguably less sensitive to spin properties than EPR spectroscopy, UV absorption is not upset by spin silence of strongly coupled systems.

All radicals of amino acids and their derivatives examined so far exhibit intense and characteristic UV changes. This spectral region is expected to be relatively free from absorption by larger redox cofactors such as porphyrins, flavines etc. While aromatic amino acids absorb light appreciably in the 280-nm region, their absorption result in a rather static and robust background, unless such amino acids undergo oxidations.

Currently we seek to develop a more inclusive and accessible method of detecting protein radicals in proteins. In order to identify and study radicals in protein systems we need to know specific signatures of those radicals. These signatures can be understood by generating corresponding radical or its model in a “pure” form which, ideally, has sufficient concentration and is stable long enough to study it. We use several approaches that have potential to stabilize model radicals, including photolysis of frozen glasses, immobilization on modified surfaces, imbedding in supported phospholipid bilayers, electrochemical oxidation etc.