High-molecular-weight protein hydrodynamics studied with a long-lifetime metal-ligand complex

Abstract

[Ru(2,2'-bipyridine)(2)(4,4'-dicarboxy-2,2'-bipyridine)](2+) (RuBDc) is a very photostable probe that possesses favorable photophysical properties including long lifetime, high quantum yield, large Stokes' shift, and highly polarized emission. In the present study, we demonstrated the usefulness of this probe for monitoring the rotational diffusion of high-molecular-weight (MW) proteins. Using frequency-domain fluorometry with a high-intensity, blue light-emitting diode (LED) as the modulated light source, we compared the intensity and anisotropy decays of RuBDc conjugated to immunoglobulin G (IgG) and immunoglobulin M (IgM), which show a six-fold difference in MW We obtained slightly longer lifetimes for IgM (<tau>=428 ns in buffer) than IgG (<tau>=422 ns in buffer) in the absence and presence of glycerol, suggesting somewhat more efficient shielding of RuBDc from water in IgM than in IgG. The anisotropy decay data showed longer rotational correlation times for IgM (1623 and 65.7 ns in buffer) as compared to IgG (264 and 42.5 ns in buffer). Importantly, the ratio of the long rotational correlation times of IgM to IgG in buffer was 6.2, which is very close to that of MW of IgM to IgG (6.0). The shorter correlation times are most likely to be associated with domain motions within the proteins. The anisotropy decays reflect both the molecular size and shape of the immunoglobulins, as well as the viscosity. These results show that RuBDc can have numerous applications in studies of high-MW protein hydrodynamics and in fluorescence polarization immunoassays (FPI) of high-MW analytes.

Fig. 1.

Chemical structure of NHS ester of [Ru(bpy)₂(dcbpy)]²⁺.

Fig. 2.

Absorption and fluorescence spectra of [Ru(bpy)₂(dcbpy)]²⁺ conjugated to IgG in 10 mM MOPS, 0.85% (w/v) NaCl (pH 7.4) at room temperature. The spectra for [Ru(bpy)₂(dcbpy)]²⁺ conjugated to IgM are identical.

Fig. 3.

Frequency-domain intensity decays of [Ru(bpy)₂(dcbpy)]²⁺ conjugated to IgG (left) and IgM (right). The measurements were done with a modulated blue LED.

Fig. 4.

Frequency-domain anisotropy decays of [Ru(bpy)₂(dcbpy)]²⁺ conjugated to IgG (left) and IgM (right). The excitation was from a blue LED.

Fig. 5.

Time-domain representation of anisotropy decays of [Ru(bpy)₂ (dcbpy)]²⁺ conjugated to IgG and IgM.