Hydrodynamic properties of bovine cardiac troponin-I and troponin-T

Bovine cardiac troponin-I (TN-I) and troponin-T (TN-T) have been examined in solution using ultracentrifugation, gel filtration, and viscosity. A new method of purifying TN-T, employing hydroxylapatite chromatography in 6 M urea, is reported. Cardiac TN-T (M(r) = 36,000) undergoes a reversible, concentration-dependent association in nondenaturing buffers, probably to a tetramer. The Stokes radius (R(s)) of aggregated TN-T, determined by sedimentation velocity and gel chromatography on Sephacryl S-300, is 80 Å and the reduced viscosity of the subunit ranges from 20 to 25 ml/g at protein concentrations between 0.5 and 2.5 mg/ml. These data suggest that TN-T forms highly asymmetric aggregates in solution. Bovine cardiac TN-I also has a tendency toward self-association, but is essentially monomeric (M(r) = 23,000) at protein concentrations below 1 mg/ml. The presence of reducing agent is necessary to avoid intermolecular disulfide bond formation. From gel filtration experiments, the value of R(s) is 29 Å indicating that TN-I is a moderately asymmetric protein (frictional ratio = 1.5). Similar properties are observed when both sulfhydryl groups of TN-I are modified by carboxamidomethylation.