A dialysis technique for determining aggregate concentrations in crystallizing protein solutions

Wilson, Lori J.; Adcock, L D; Pusey, Marc L.

doi:10.1088/0022-3727/26/8b/017

Cited by 14 publications

(12 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Currently in the field of protein crystal growth there is experimental evidence both supporting and disputing the presence of structured aggregates in crystalline solutions. For lysozyme, evidence supporting the aggregation theory comes from dialysis kinetics (Wilson, Adcock & Pusey, 1993), neutron scattering (Boue, Lefaucheux, Robert & Rosenman, 1993), sedimentation equilibrium (Sophianopoulos & Van Holde, 1961, 1964, nuclear magnetic resonance (Shindo, Cohen & Rupley, 1977), light scattering intensity (Pusey, 1991) and calorimetry (Banerjee, Pogolotti & Rupley, 1975). Some of these studies were carried out prior to 1980 when the conditions used for studying lysozyme aggregation were close to physiological ionic strength and neutral pH.…”

Section: Introductionmentioning

confidence: 99%

Salt-Induced Aggregation of Lysozyme Studied by Cross-Linking with Glutaraldehyde: Implications for Crystal Growth

Wang

Hayter²,

Wilson³

1996

Acta Cryst D

View full text Add to dashboard Cite

Glutaraldehyde cross-linking followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis has been used to detect aggregates of isozyme in solutions which lead to crystals. In solutions of varying NaCl content, the number of aggregates was found to be related to the ionic strength of the solution. Solutions of 1% NaCl, pH 4.0 were monomeric while those containing 7-15% NaCl, pH 4.0 were shown to be as much as 36% aggregated and 64% monomeric. The aggregates detected at the highest salt and protein concentration studied were composed of dimers, trimers and tetramers. The aggregates increased by addition of single units suggesting the aggregation pathway to be that of monomer addition. The kinetics of the cross-linking reaction were slow preventing a study of either the time dependence of aggregation or the effect of temperature on aggregate distributions. Comparison of the total aggregate concentrations for NaCl and Na(2)SO(4) showed that the concentration of aggregates was related to the ionic strength of the solution suggesting that in both crystallization and precipitation, electrostatic shielding of like-charged protein molecules is necessary in order for aggregation to occur.

show abstract

Section: Introductionmentioning

confidence: 99%

Salt-Induced Aggregation of Lysozyme Studied by Cross-Linking with Glutaraldehyde: Implications for Crystal Growth

Wang

Hayter²,

Wilson³

1996

Acta Cryst D

View full text Add to dashboard Cite

show abstract

“…It remains an open question whether lysozyme forms large aggregates when undersaturated or when in the prenucleation phase of crystallization. Recent data that support aggregation are from small-angle neutron scattering (SANS) experiments (Niimura et al, 1995;Boue et al, 1993) and dialysis kinetics (Wang et al, 1996;Wilson et al, 1993Wilson et al, , 1996. In contrast, some dynamic light-scattering (DLS) studies have concluded that scattering data that may suggest, at first examination, the formation of aggregates, are more accurately explained in terms of interparticle interactions (Muschol and Rosenberger, 1996;Eberstein et al, 1994).…”

mentioning

confidence: 99%

Interactions of lysozyme in concentrated electrolyte solutions from dynamic light-scattering measurements

Kuehner

Heyer

Rämsch

et al. 1997

Biophysical Journal

115

125

View full text Add to dashboard Cite

The diffusion of hen egg-white lysozyme has been studied by dynamic light scattering in aqueous solutions of ammonium sulfate as a function of protein concentration to 30 g/liter. Experiments were conducted under the following conditions: pH 4-7 and ionic strength 0.05-5.0 M. Diffusivity data for ionic strengths up to 0.5 M were interpreted in the context of a two-body interaction model for monomers. From this analysis, two potential-of-mean-force parameters, the effective monomer charge, and the Hamaker constant were obtained. At higher ionic strength, the data were analyzed using a model that describes the diffusion coefficient of a polydisperse system of interacting protein aggregates in terms of an isodesmic, indefinite aggregation equilibrium constant. Data analysis incorporated multicomponent virial and hydrodynamic effects. The resulting equilibrium constants indicate that lysozyme does not aggregate significantly as ionic strength increases, even at salt concentrations near the point of salting-out precipitation.

show abstract

“…Evidence of oligomers in supersaturated lysozyme solutions comes from many sources including sedimentation equilibrium (Sophianopoulos & Van Holde, 1961, 1964, calorimetry (Banerjee et al, 1975), nuclear magnetic resonance (Shindo et al, 1977), light-scattering intensity (Pusey, 1991), dialysis kinetics (Wilson et al, 1993) and neutron scattering (Boue et al, 1993). Some of these studies were performed prior to 1980 when the conditions used for studying lysozyme aggregation were close to physiological ionic strength and neutral pH.…”

Section: Introductionmentioning

confidence: 99%

Supersaturated lysozyme solution structure studied by chemical cross-linking

Hall

Clemens

Brown

et al. 2005

Acta Crystallogr D Biol Cryst

View full text Add to dashboard Cite

Glutaraldehyde cross-linking followed by separation has been used to detect aggregates of chicken egg-white lysozyme (CEWL) in supersaturated solutions. In solutions of varying NaCl content, the number of aggregates was found to be related to the ionic strength of the solution. Separation by SDS-PAGE showed that percentage of dimer in solution ranged from 25.3% for no NaCl to 27.1% at 15% NaCl, and the aggregates larger than dimer increased from 1.9% for no NaCl to 36.8% at 15% NaCl. Conversely, the percentage of monomers decreased from 72.8% without NaCl to 36.1% at 15% NaCl. Molecular weights by capillary electrophoresis (SDS-CE) were found to be multiples of the monomer molecular weights, with the exception of trimer, which indicates a very compact structure. Native separation was accomplished using size-exclusion chromatography (SEC) and gave a lower monomer concentration and higher aggregate concentration than SDS-CE, which is a denaturing separation method. Most noticeably, trimers were absent in the SEC separation. The number of aggregates did not change with increased time between addition of NaCl and addition of cross-linking agent when separated by gel electrophoresis (SDS-PAGE). The results suggest that high ionic strength CEWL solutions are highly aggregated and that denaturing separation methods disrupt cross-linked products.

show abstract

A dialysis technique for determining aggregate concentrations in crystallizing protein solutions

Cited by 14 publications

References 19 publications

Salt-Induced Aggregation of Lysozyme Studied by Cross-Linking with Glutaraldehyde: Implications for Crystal Growth

Salt-Induced Aggregation of Lysozyme Studied by Cross-Linking with Glutaraldehyde: Implications for Crystal Growth

Interactions of lysozyme in concentrated electrolyte solutions from dynamic light-scattering measurements

Supersaturated lysozyme solution structure studied by chemical cross-linking

Contact Info

Product

Resources

About