Photoaffinity labeling experiments

To induce the reader to proceed further, the remainder of this short chapter gives a brief account of some applications of photogenerated reagents. Five classes of photoaffinity labeling experiments are described followed by three examples of the related methods that are discussed in the later chapters of this book. 

It was concluded that dithiols should not be used in photoaffinity labeling experiments with aryl azides and monothiols should be used with caution. The reduction reaction can often be monitored spectrophotome-trically, e.g. 2-nitro-4-azidoanilines which are red-orange (Xmax 480 nm in H20) are converted to purple diamines (Xmax 550 nm). TLC and IR spectroscopy are also useful (Staros et al., 1978). 

The reaction products from aryl azides in photoaffinity labeling experiments have not been fully characterized. Indeed the reactions of aryl nitrenes in solution remain a topic of current research. 

In Westheimer s original photoaffinity labeling experiment, diazoacetyl-chymotrypsin was photolysed. Later, Westheimer s group made the improved diazo reagents listed in Table 3.1. 

The first step in an actual photoaffinity labeling experiment is to allow the ligand to bind to its receptor. In most cases the experimenter wishes to... 

Several controls related to those required in photoaffinity labeling experiments with radiolabeled ligands (see Section 4.7) must also be performed in studies of photoinactivation. In particular inactivation should not occur on irradiation in the absence of the photoaffinity label, and the receptor site should be protected against photoinactivation if it is first blocked with a photochemically inert molecule. 

In discussing photoaffinity labeling experiments with radioactive reagents, I shall focus mainly on the labeling of protein receptors with small photola-bile ligands. The same principles may be applied to related experiments. The general approach to identifying a receptor in a mixture of proteins is first described. The mixture may be extremely complex, e.g. whole cells or a membrane preparation, or relatively simple, e.g. a purified multisubunit protein. Second, experiments to locate a label within a protein are described. This may be at the level of a low resolution peptide map or in favorable cases the identification of a labeled amino acid residue. 

Although no definitive study of scavengers in photoaffinity labeling experiments has yet been made, the present indications are that thiols are the most suitable reagents. It is expected that as strong nucleophiles they should react with most photogenerated species which are electrophiles as well as with free radicals which can abstract hydrogen atoms from the... 

Conceptually similar photoaffinity labeling experiments have been reported by Schraders laboratory quite recently (70). These workers have sought to label the cytosolic progesterone receptor of chick oviduct using the polyunsaturated synthetic progestin, R5020, Compound XIII. 

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