EXPERIMENTAL-DETERMINATION OF WATER EQUILIBRATION RATES IN THE HANGING DROP METHOD OF PROTEIN CRYSTALLIZATION

The hanging drop method for protein crystallization consists of equilibrating a water droplet containing the protein and a precipitant against a reservoir solution at a higher precipitant concentration. The time for water equilibration-which affects the kinetics of crystallization-to reach 90% of completion is shown to vary between about 25 h and more than 25 days, depending on experimental conditions. Experiments were performed with three of the most widely used precipitants (ammonium sulfate, polyethylene glycol, 2-methyl-2,4-pentanediol), combining various parameters expected to affect the rate of water evaporation. The most dramatic effects were obtained when varying temperature and initial drop volume. A simple empirical equation for estimating the kinetics of water equilibration under given crystallization conditions is proposed. © 1990.