Examples of photochemical crosslinking

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. 

In another case in which a number of exemplary control experiments were done, Markwell and Fox (1980) crosslinked the outer membranes of enveloped viruses with methyl 3-(p-azidophenyl)dithio]propionimidate. Virus (4 mg protein/ml) was reacted with the imidate (0.1 to 0.5 mM) at 0°C for 30 min at pH 8.5. The reaction was quenched with 50 mM ammonium acetate, 50 mM NEM (30 min, 25 °C), and the vims recovered by centrifugation. After irradiation the crosslinked polypeptides were examined in a two-dimensional SDS-polyacrylamide gel. One complication was that the crosslinking pattern had to be compared with a native pattern of disulfide linkages, and a reagent with a different cleavable crosslink may have been a better choice. As mentioned above, the analysis was simplified by the use of surface labeling. 

Control experiments showed that all the crosslinks were cleavable. No crosslinks were formed if the reagent was omitted, if photolysis was omitted, or if the reagent was first inactivated by treatment with ammonium acetate. These experiments proved that neither monofunctional cross-linking, nor disulfide interchange to give crosslinking in the dark occurred. If the membranes were solubilized in SDS before irradiation no cross- 

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It will be noticed that in this, and in all other examples of photochemical crosslinking, that crosslinking is far from complete and it seems that a substantial proportion of the photogenerated intermediates must react with buffer components or water, or form highly unstable bonds. Intramolecular crosslinks may be reflected in spots above the diagonal or worse as streaks in the first dimension (Huang and Richards, 1977). 

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