Proteins crosslinking using imidoesters

The amidine bond formed is quite stable at acid pH however, it is susceptible to hydrolysis and cleavage at high pH. A typical reaction condition for using imidate crosslinkers is a buffer system consisting of 0.2 M triethanolamine in 0.1 M sodium borate, pH 8.2. After conjugating two proteins with a bifunctional imidoester crosslinker, excess imidoester functional groups may be blocked with ethanolamine. 

The following list of homobifunctional imidoesters represent compounds that are commonly used for protein crosslinking and are currently available from commercial sources. 

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... 

Bz s-imidoesters like DMS may be used to couple proteins to PE-containing liposomes by crosslinking with the amines on both molecules (Figure 22.24). However, single-step crosslinking procedures using homobifunctional reagents are particularly subject to uncontrollable polymerization of protein in solution. Polymerization is possible because the procedure is done with the liposomes, protein, and crosslinker all in solution at the same time. 

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