Instrument: Applied Photophysics PiStar (with Stopped Flow system)
Circular dichroism is a technique that measures the absorption of photons of light that are oriented in a specific way - circularly polarised light - by molecules. The extent to which light polarisedin this manner is absorbed allows us to deduce the presence of different stereoisomers - chemically identical molecules that are mirror images of each other.
Molecule that exist as sterioisomers include many small biological molecules and drugs, as well as biomacromolecules such as proteins. In biology, which of the two sterioisomers is present is crucially important, since one might have beneficial effects whilst the other might be toxic.
For proteins, circular dichroism can be used to determine how well folded protein molecules are.
Some molecules possess a chiral centre, that is, an atom bound to four different groups. A chiral centre can exist in two forms, termed stereoisomers (labelled D for dextrorotatory or L for levorotatory), which are chemically identical but can behave differently mechanistically during chemical and biochemical reactions due to differing binding affinities.
CD allows the chirality of a sample to understood which allows the identification of a chiral centre of interest e.g. for downstream pharmo-kinetic study. The essence of the CD effect is that right-handed and left-handed circularly polarised light are absorbed differently by stereo-isomers,hence measuring the different absorbacnes allows stereochemistry to be investigated.
Biological macromolecules also have an overall chirality imparted by the constituent chiral amino acids - naturally occurring amino acids are all L-stereoisomers. Changes in the biomolecules, for example during protein folding/denaturing, can be assessed by performing time-resolved CD using the Stopped Flow attachment.
CD works well for investigation of the effect of environment on protein folding, for instance the effect of heat, pH or denaturation of protein conformation.
The best CD results are obtained with relatively high concentrations, but that are below the level where aggregation may occur.