J. P. Munch

Effect of macromolecular impurities on lysozyme solubility and crystallizability: dynamic light scattering, phase diagram, and crystal growth studies

Abstract The effects of macromolecular impurities on protein solubility and crystallizability were investigated by dynamic light scattering and crystal growth experiments using hen egg-white lysozyme as the model protein. In the presence of traces of protein impurities, representing no more than 2% (w/w) of the total protein, the average diffusion coefficients of the macromolecular particles found in undersaturated lysozyme solutions are significantly lower than those measured with purest lysozyme preparations. This fact is explained by the simultaneous existence of individual molecules and of large size aggregates in contaminated solutions, as indicated by the bimodal light scattering autocorrelation function. Controlled contamination experiments in which ovalbumin or conalbumin were added to purest lysozyme indicate that aggregates result from heterogeneous association of lysozyme molecules with the structurally unrelated proteins. These aggregates might become starting points for heterogeneous nucleation leading to the growth of ill-shaped microcrystals. Aggregates in under- or supersaturated lysozyme solutions containing NaCl can be eliminated by filtration over microporous membranes. As a result the number of ill-shaped crystals diminishes drastically; that of well-shaped tetragonal crystals decreases also but their size increases.

The influence of impurities on protein crystallization ; the case of lysozyme

Several batches of hen egg white lysozyme were compared on the basis of their biochemical purity and homogeneity as well as of their ability to crystallize in the tetragonal space group in the presence of sodium chloride and sodium acetate at pH 4.5. Trace amounts (

Dynamic light scattering studies of the aggregation of lysozyme under crystallization conditions

The intensity autocorrelation functions of light scattered by lysozyme solutions under pre‐crystallization conditions in NaCl‐containing media were recorded at scattering angles from 20° to 90°. The measurements, conducted on freshly prepared protein solutions supersaturated more than 3‐fold, indicate the simultaneous presence of two scatterer populations which can be assigned to individual protein molecules and to large particles. When solutions are undersaturated, or slightly supersaturated, light scattering only reveals the presence of the small scatterers. In the supersaturated medium, where aggregates were detected, lysozyme crystals grew in a time‐span of 1–3 days after the scattering experiments. These results are correlated with the nucleation step during protein crystallization.

Interactions between lysozyme molecules under precrystallization conditions studied by light scattering

Abstract Light scattering experiments were undertaken on lysozyme under various solvent conditions. When the protein is undersaturated, attractive interparticular interactions are detected. They are enhanced when the temperature is decreased, but are much weaker in NaCl solutions in which the protein crystallizes than in ammonium sulfate solutions in which it forms amorphous precipitates. When the protein in a NaCl solution is brought to supersaturation by a temperature decrease, light scattering measurements indicate the simultaneous presence of two scatter populations which can be assimilated to individual lysozyme molecules and to large particles. Kinetic experiments in which the temperature is quenched rapidly indicate that the apparent hydrodynamic radius of the large particles increases regularly with time up to a plateau value of about 2600A˚.

Elastic behaviour of salt-free polyelectrolyte gels

Poly(acrylic acid) gels ionized after preparation for different preparation concentrations and degrees of cross-linking and ionization, have been studied as a function of the swelling ratio and at swelling equilibrium. Measurements performed as a function of the polymer concentration indicated that for low swelling ratios ( < 100) the shear modulus follows a scaling law with an exponent in agreement with classical theories. For a given concentration the shear modulus has been found to decrease with the degree of ionization as qualitatively predicted by a recent model. At swelling equilibrium the shear modulus varies linearly with the osmotic pressure of the counter-ions in a large concentration domain and for various cross-linking densities. The osmotic prefactor values, found both by mechanical and by light-scattering experiments, were in agreement with various independent measurements performed on poly(acrylic acid) solutions.

Frozen-in Intensity Fluctuations of Light Scattered by Partially Ionized Gels

We report results of static light scattering for partially ionized gels at different cross-link densities and different swelling degrees and for the corresponding semi-dilute solutions. The r.m.s. fluctuations of the scattered intensity obtained when scanning through various positions in the gels is proportional to the excess of scattering of the gel with respect to the semi-dilute solution, whatever the method used to vary the ratio of the actual swelling degree to its equilibrium value, i.e. varying the cross-link density or the swelling extent. The excess of scattering is attributed to a frozen-in component associated with scattering domains with a spatial extent less than ~ 300 A.

Ordering phenomena of polymeric clusters near the sol-gel transition : light scattering experiments

Light scattering has been performed on solutions of polymeric clusters of poly(vinyl chloride) near the sol-gel transition where a peak in the scattering function is observed. This effect is analysed here in terms of a liquidlike order of polydisperse clusters. Clusters distributions were quenched to T = 20 °C for solutions prepared at high temperature at different concentrations ΦQ below the critical gelation concentration ΦQ*. For a system diluted after quenching, the weight average molecular weight Mw (ΦQ) and the peak position q* are shown to scale as Mw ∝ Rz1.6±0.1 and q* ∝ Φ-1/3 Mw1/3, respectively, in which Rz (ΦQ) is the z-average radius of gyration and Φ(ΦQ) is the polymer volume fraction.

Flexibility of short ds-DNA intercalated by a dipyridophenazine ligand.

We use Förster Resonant Energy Transfer (FRET) in order to measure the increase of flexibility of short ds-DNA induced by the intercalation of dipyridophenazine (dppz) ligand in between DNA base pairs. By using a DNA double strand fluorescently labeled at its extremities, it is shown that the end-to-end length increase of DNA due to the intercalation of one dppz ligand is smaller than the DNA base pair interdistance. This may be explained either by a local bending of the DNA or by an increase of its flexibility. The persistence length of the formed DNA/ligand is evaluated. The described structure may have implications in the photophysical damages induced by the complexation of DNA by organometallic molecules.