Long-lifetime Ru(II) complexes for the measurement of high molecular weight protein hydrodynamics

We describe the synthesis and characterization of two asymmetrical ruthenium(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'-bipyridine, mcbpy is 4-methyl,4'-carboxylic acid-2,2'-bipyridine, and dpp(SO3Na)2 is the disulfonated derivative of dpp) as probes for the measurement of the rotational motions of proteins. The spectral (absorption, emission, and anisotropy) and photophysical (time-resolved intensity and anisotropy decays) properties of these metal-ligand complexes were determined in solution, in both the presence and absence of human serum albumin (HSA). These complexes display lifetimes ranging from 345 ns to 3.8 microseconds in deoxygenated aqueous solutions under a variety of conditions. The carboxylic acid groups on these complexes were activated to form N-hydroxysuccinimide (NHS) esters which were used to covalently lable HSA, and were characterized spectroscopically in the same manner as above. Time-resolved anisotropy measurements were performed to demonstrate the utility of these complexes in measuring long rotational correlation times of bioconjugates between HSA and antibody to HSA. The potential usefulness of these probes in fluorescence polarization immunoassays was demonstrated by an association assay of the Ru(II)-labeled HSA with polyclonal antibody.