Studies of interactions of carbonic anhydrase B with water and urea: II. Spin diffusion of associates of protein intermediate state at 4.2 m of urea

The formation of carbonic anhydrase B associates (pH 5.7, urea concentration 4.2 M, 297 K) was studied as a function of protein concentration and time by nuclear magnetic resonance spectroscopy (spin diffusion method). It was found that the association process proceeds in two steps. The first step is relatively fast and cannot be controlled by our methods. During this step, persistent units are built. These consist of protein molecules that are able to interact with solvent molecules and with each other when protein solution contains 4.2 M of urea. Persistent units are relatively small (two, three protein molecules), and their mobility matches one of a single protein. The second step is slower, and throughout this step large structures are formed from persistent units. The parameters G* and S*, which characterize spin diffusion in a protein and a solvent, respectively (when spin diffusion excitation happens away from NMR spectral signals) are related to the probable size distribution of protein-solvent associates and are determined by their collective properties.