LONG-LIFETIME RU(II) COMPLEXES FOR THE MEASUREMENT OF HIGH-MOLECULAR-WEIGHT PROTEIN HYDRODYNAMICS

We describe the synthesis and characterization of two asymmetrical rut henium(II) complexes, [Ru(dpp)(2)(dcbpy)](2+) and [Ru(dpp)(2)(mcbpy)]( 2+), as well as the water soluble sulfonated derivatives [Ru(dpp(SO3Na )(2))(2)(dcbpy)](2+) and [Ru(dpp(SO3Na)(2))(2)(mcbpy)](2+) (dpp is 4,7 -diphenyl-1,10-phenanthroline, dcbpy is 4,4'-dicarboxylic acid-2,2'-bi pyridine, mcbpy is 4-methyl,4'-carboxylic acid-2,2'-bipyridine, and dp p(SO3Na)(2) is the disulfonated derivative of dpp) as probes for the m easurement of the rotational motions of proteins. The spectral (at?sor ption, emission, and anisotropy) and photophysical (time-resolved inte nsity and anisotropy decays) properties of these metal-ligand complexe s were determined in solution, in both the presence and absence of hum an serum albumin (HSA). These complexes display lifetimes ranging from 345 ns to 3.8 mu s in deoxygenated aqueous solutions under a variety of conditions. The carboxylic acid groups on these complexes were acti vated to form N-hydroxysuccinimide (NHS) esters which were used to cov alently label HSA, and were characterized spectroscopically in the sam e manner as above. Time-resolved anisotropy measurements were performe d to demonstrate the utility of these complexes in measuring long rota tional correlation times of bioconjugates between HSA and antibody to HSA. The potential usefulness of these probes in fluorescence polariza tion immunoassays was demonstrated by an association assay of the Ru(I I)-labeled HSA with polyclonal antibody. (C) 1998 Elsevier Science B.V .