Site-specific photoaffinity labeling

Photoaffinity labeling of specific sites on the Rubisco SSu For the isolation of SSu peptides, spinach Rubisco holoenzyme was photoaffinity labeled and the labeled subunits separated by gel-filtration chromatography in urea. Photolabeled SSu tryptic peptides were isolated by ion-exclusion and reverse phase HPLC ... 

Photoaffinity labeling can be particularly useful when dealing with noncompetitive inhibitors, where the site of binding cannot be inferred from competition with specific substrate or cofactor molecules. 

Photochemically reactive molecules have often been used as labels for specific sites in proteins and nucleic acids. Psoralen derivatives serve as relatively nonspecific photochemically activated crosslinking agents for DNA and double-stranded RNA.195 Aryl azides are converted by light to aryl nitrenes, which react in a variety of ways including insertion into C-H bonds (Eq. 23-27).200 201 In some cases UV irradiation can be used to join natural substrates to enzymes or hormones to receptors. For example, progesterone, testosterone, and other steroids have been used for direct photoaffinity labeling of their receptors.202 Synthetic benzophenones have also been used widely as photoactivated probes.203... 

Another test for specific labeling is to determine whether the ligand binding site is blocked. But, as 1 pointed out in the discussion of photoinactivation experiments, there are several other possible causes of apparent binding site occupation besides the covalent attachment of a ligand. It has also been noted that specific labeling as defined by a protection experiment may not always yield a blocked receptor when the photoaffinity label is a macromolecule. When sodium channels in tissue culture cells were labeled... 

Compounds which exhibit specific interactions with a particular site of an enzyme other than an active site are called cofactors and allosteric effectors. These compounds are not considered in this review. Specific compounds which interact with the active site itself will be classified into two types. One of them include simple competitive inhibitors and photoaffinity labeling reagents. Compounds of this type exhibit a specific interaction only with the binding site (specificity site) of the enzyme. 

Mills, J. S., Miettinen, H. M., Barnidge, D., Vlases, M. J., Wimer-Mackin, S., Dratz, E. A., Sunner, J., andjesaitis, A. J. (1998). Identification of a ligand binding site in the human neutrophil formyl peptide receptor using a site-specific fluorescent photoaffinity label and mass spectrometry./. Biol. Chem. 273, 10428-10435. 

---