Quantitative assays of the amount of diethylenetriaminepentaacetic acid conjugated to water-soluble polymers using isothermal titration calorimetry and colorimetry

The level of conjugation of diethylenetriaminepentaacetic acid (DTPA) to the polysaccharide sodium hyaluronan (HA) has been measured by a colorimetric assay, isothermal titration calorimetry (ITC), and H-1 NMR spectroscopy The colorimetric assay is based on the red shift, upon complexation with gadolinium ion (Gd3+), of the wavelength of maximum absorption of the dye arsenate III. It can be performed in a few minutes using as little as 10 mug of polymer with a detection limit of similar to0.03 mmol of DTPA (gram of polymer)(-1). The ITC measurements yield values of the amount of DTPA linked to HA identical to those obtained by colorimetry. The levels of DTPA conjugation calculated by integration of signals at 3.1-3.2 ppm (DTPA protons) and at 2.0 ppm (HA acetamide protons) in the H-1 NMR spectrum of HA-DTPA are consistently overestimated by a factor of similar to2, compared to the data obtained by ITC and colorimetry. The longer relaxation times of protons of the polymer backbone, compared to those of protons attached to the freely moving DTPA side-chains may account for the discrepancy.