Photoaffinity labelling is one of the most powerful chemical modification t
echniques employed in the study of protein architecture and interactions. T
his approach utilises photolabile derivatives of ligands (substrates), term
ed photoaffinity probes, for a covalent labelling of target proteins (enzym
es). Upon UV-light photolysis, photoaffinity probes are converted to highly
reactive intermediates which al e able to modify amino acid residues of a
target protein. The goal of this technique is the identification of the pro
be binding site(s) in a probe-protein covalent complex. In this review, maj
or concepts of photoaffinity labelling are described with respect to select
ion criteria of appropriate photoaffinity probes and assessment of advantag
es and disadvantages of currently used photolabile probes. In addition, sev
eral examples of photoaffinity probe application in protein research focuse
d on the cytochrome P450 active centre are presented.