MONOMER CONCENTRATIONS AND DIMERIZATION CONSTANTS IN CRYSTALLIZING LYSOZYME SOLUTIONS BY DIALYSIS KINETICS

Dialysis kinetics measurements have been made to study the effect of i onic strength on the dimerization of lysozyme in acidic solutions that lead to the growth of tetragonal lysozyme crystals, Using glutaraldeh yde cross-linked dimers of lysozyme, we have determined that both mono mers and dimers can escape from 25,000 molecular weight cutoff dialysi s membranes with velocity constants of 5.1 x 10(-7) and 1.9 x 10(-7) s (-1) for the monomer and dimer species, respectively. The flux from 25 K MWCO membranes has been measured for lysozyme in pH 4.0 buffered sol utions of 1, 3, 4, 5, and 7% NaCl over a wide range of protein concent rations. Assuming that dimerization is the first step in crystallizati on, a simple monomer to dimer equilibrium was used to model the flux r ates, Dimerization constants calculated at low protein concentrations were 265, 750, 1212, and 7879 M(-1) for 3, 4, 5, and 7% NaCl, respecti vely, These values indicate that dimerization increases with the ionic strength of the solution suggesting that aggregation is moderated by electrostatic interactions, At high protein concentrations and high su persaturation, the dimerization model does not describe the data well, However, the Li model that uses a pathway of monomer <-> dimer <-> te tramer <-> octamer <-> 16-mer fits the measured flux data remarkably w ell suggesting the presence of higher order aggregates in crystallizin g solutions.