Side-chain selective and covalent labelling of proteins by organometallic complexes of heavy transition metals--possible application in radio-crystallography of proteins.

Organo-rhenium and organo-tungsten N-succinimidyl and N-sulfosuccinimidyl esters react at amino groups of proteins to form stable amide bonds between the protein and the heavy transition metal complexes. We show here that factors such as pH of the reaction medium and nature of the reagent (coordinating metal, surrounding ligands, type of ester) had a marked influence on the final number of coupled organometallic groups per protein molecule, defined as the coupling ratio. By operating with stoichiometric quantities of reagent with respect to the number of aqino groups, up to 30 organometallic groups were introduced into the model protein, bovine serum albumin (BSA). BSA conjugates were characterized by gel electrophoresis in non-denaturating conditions. Hen egg-white lysozyme organo-tungsten conjugates were prepared in the same manner and the peptides resulting from their tryptic hydrolysis were analyzed by reverse-phase HPLC. A tentative assignment of the preferential amino sites of conjugation is suggested from the latter results.