Photochemical crosslinking reagents

Hydrophobic crosslinking reagents each with two photochemically ac-tivatable groups have been synthesized by Mikkelson and Wallach (1976). Their reagents (Fig. 5.1) were designed to bind to the lipid bilayer of... 

Photochemical, heterobifunctional reagents for crosslinking and for the synthesis of photoaffinity labels... 

Photochemical reagents have been devised for crosslinking both soluble and particulate proteins (Chapter 5). In a recent study, Johnson et al. (1981) were able to crosslink radiolabeled glucagon to its receptor, merely by adding a bifunctional reagent that first reacted with amino groups and could subsequently be induced to form crosslinks by photolysis. 

The reagents in current use for protein crosslinking have one chemically reactive and one photochemically reactive arm, often connected by a cleavable bridge for subsequent two-dimensional electrophoretic analyses. Chemical attachment is carried out first, and the crosslink is completed by photolysis. Many of these so-called heterobifunctional molecules are listed in Table 5.1 and some of them, conforming to the criteria outlined below,... 

In a recent instructive example Rinke et al. (1980) used azidoaiylimidates to form RNA-protein crosslinks in E. coli ribosomes. Five photochemical reagents were tested and only one, methyI-4-azidophenylacetimidate, was found to give a useful extent of crosslinking. Ribosomes (A260 20 U/ml) were reacted with the imidoester (5 mM) in the dark, at pH 8 to 9, for 30 min at 37 °C. The ribosomes were then precipitated with ethanol and redissolved in buffer at a somewhat lower concentration (5 U/ml) before irradiation. The analysis was greatly aided by the use of biosynthetically... 

A further experiment was performed that demonstrated the possibilities of the photochemical approach. The reagent was attached in the dark to 30 S ribosomal subunits which were then combined with radiolabeled 50 S subunits to form complete ribosomes. Photolysis failed to reveal extensive intersubunit protein-RNA crosslinking. 

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