Reaction of Hg2+ Insertion into Cysteine Pairs Within Bovine Insulin Crystals Followed via Raman Spectroscopy

Chemical modifications of protein crystals may be achieved via soaking of reactants from their precipitating solution, through the solvent channel, into the protein matrix. We describe a Raman microscopy approach to follow mercury insertion into cysteine pairs within protein single crystals, via soaking in an aqueous Hg2? solution. The method has been developed using bovine insulin as the model system. Applying an efficient mercuration protocol, consisting of a first step of disulphide bridge TCEP-induced reduction within the crystal, followed by overnight reaction with a HgCl2 solution, we obtained Hg-derivative crystals. Raman spectra collected on these derivative crystals, kept in the mother liquor, reveal a characteristic Raman band at 335 cm-1, which has been assigned to a –S–Hg–S– bridge. The analysis provides Raman-based markers of mercury binding to cysteines, and thus of mercury intoxication