SYNTHESIS AND EVALUATION OF RU-COMPLEXES AS ANISOTROPY PROBES FOR PROTEIN HYDRODYNAMICS AND IMMUNOASSAYS OF HIGH-MOLECULAR-WEIGHT ANTIGENS

We investigated three unsymmetrical Ru-complexes, namely [Ru(bpy)(2)(p hen-ITC)](2+), [Ru(bpy)(2)(dcbpy)] and [Ru(bpy)(2)(mcbpy)](+) for use as probes for rotational diffusion and in immunoassays of high-molecul ar-weight antigens. For this purpose we synthesized reactive forms of these metal-ligand complexes and conjugated them to human serum albumi n (HSA). The maximal anisotropies (r(0)) for the HSA-bound forms in fr ozen solution are 0.23, 0.17 and 0.14 for the (dcbpy), (mcbpy) and (ph en-ITC) derivatives, respectively. The activated Ru metal-ligand compl exes have either one or two NHS-esters or an isothiocyanate group as t he reactive moiety. The usefulness of these complexes in immunoassays was determined by titration of the labeled HSA with polyclonal anti-HS A. The highest steady state anisotropy (r) values (0.190) were observe d for the [Ru(bpy)(2)(dcbpy)]-labeled HSA on titration with polyclonal antibody. However, a relative increase in the steady state anisotropy (r/r(0)) on titration with polyclonal antibody was found for the phen -ITC probe (96%), as compared to the dcbpy (83%) or mcbpy (79%) deriva tives. These findings were confirmed by time-resolved frequency-domain measurements. In particular the higher mean correlation times calcula ted for the phen-ITC derivative suggests reduced local probe motion fo r this probe when bound to HSA as compared to the (mcbpy) and (dcbpy) conjugates.