Qualitative and quantitative insights into the capacity and association constant for the binding of chymotrypsin to polyacrylate-coated gold nanoparticles is determined using fluorescence quenching, optical absorption and circular dichroism spectroscopy, isothermal calorimetry, and gel electrophoresis. The collective data reveal a binding capacity and constant for this particular system of similar to 7 and similar to 2 x 10(6) M-1, respectively. These values vary among the individual techniques, and not all techniques are able to provide quantitative information. The present study demonstrates that accurately quantifying the association between nanoparticles and biological materials requires using multiple approaches to ensure consistency among the binding parameters determined.