No abstract

The solubility of chicken egg white lysozyme crystallized in the tetragonal form was measured in sodium chloride solutions from 1.6 to 30.7 °C, using a miniature column solubility apparatus. Sodium chloride solution concentrations ranged from 1 to 7% (w/v). The solutions were buffered with 0.1 M sodium acetate buffer, with the solubility being measured at pH values in 0.2 pH unit increments in the pH range 4.0−5.4, with data also included at pH 4.5. Lysozyme solubility was found to increase with increasing in temperature and decreasing salt concentration. Solution pH has a varied and unpredictable effect on solubility.

Part of the challenge of macromolecular crystal growth for structure determination is obtaining crystals with a volume suitable for x-ray analysis. In this respect an understanding of the effect of solution conditions on macromolecule nucleation rates is advantageous. This study investigated the effects of supersaturation, temperature, and pH on the nucleation rate of tetragonal lysozyme crystals. Batch crystallization plates were prepared at given solution concentrations and incubated at set temperatures over 1 week. The number of crystals per well with their size and axial ratios were recorded and correlated with solution conditions. Crystal numbers were found to increase with increasing supersaturation and temperature. The most significant variable, however, was pH; crystal numbers changed by two orders of magnitude over the pH range 4.0-5.2. Crystal size also varied with solution conditions, with the largest crystals obtained at pH 5.2. Having optimized the crystallization conditions, we prepared a batch of crystals under the same initial conditions, and 50 of these crystals were analyzed by x-ray diffraction techniques. The results indicate that even under the same crystallization conditions, a marked variation in crystal properties exists.

The kinetics and hydrodynamic properties of factor V-membrane interaction were characterized. Factor V bound to membranes containing acidic phospholipids with a high collisional efficiency. For membranes of 20% phosphatidyl-serine-80% phosphatidylcholine, an association rate constant of (1.13 +/- 0.10) X 10(8) M-1 s-1 was obtained. These membranes contained about 20 factor V binding sites per vesicle of 3.6 X 10(6) daltons. This association rate represented about a 30% collisional efficiency. Dissociation of factor V was measured by a fluorescence energy transfer method with a dissociation rate constant of 0.0055 s-1 at 10 degrees C. The equilibrium dissociation constant for binding to these membranes at 10 degrees C and 0.14 M ionic strength was 5 X 10(-11) M. Ionic strength, pH, calcium, and charge density in the membrane had large effects on the rate of factor V-membrane dissociation, indicating a strongly ionic interaction between protein and membrane. In contrast, the association rate was nearly insensitive to ionic strength. The membrane-binding properties were relatively unchanged after thrombin digestion of factor V or after long-term protein storage which resulted in loss of procoagulant activity. Other proteins of the prothrombinase reaction greatly decreased the rate of factor Va-membrane dissociation. At protein saturation, factor V increased the hydrodynamic radius of phospholipid vesicles by 11.4 nm. In contrast, factor Va increased the hydrodynamic vesicle radius by only about 5 nm. The mass of membrane-bound protein was comparable for both proteins.