Match!

Phase diagram of a crystalline protein: Determination of the solubility of concanavalin A by a microquantitation assay

Published on Sep 1, 1989in Journal of Crystal Growth1.573
· DOI :10.1016/0022-0248(89)90213-3
Vincent Mikol7
Estimated H-index: 7
(CNRS: Centre national de la recherche scientifique),
Richard Giegé54
Estimated H-index: 54
(CNRS: Centre national de la recherche scientifique)
Abstract
Abstract A quick and miniature method has been devised for determining protein solubility and used to investigate the equilibrium solubility of concanavalin A from the Jack Bean with its crystals as a function of ammonium sulfate concentration, temperature and pH. The crystals were characterized by X-ray diffraction and their morphologies related to the corresponding solubilities. The protein solution concentration was estimated out of small crystallizing drops using a rapid and sensitive microassay. Measurements of protein quantity were carried out in 96-well microplates in an automatic spectrophotometer. The resulting phase diagram has permitted to analyse the solubility of concanavalin A, to estimate supersaturation and to devise readily new ways of crystal growth of this lectin, namely by pH and temperature variations. Moreover, the approach is proved to be a valuable tool to design crystallization experiments of new molecules and to improve and control protein crystal growth.
  • References (21)
  • Citations (45)
📖 Papers frequently viewed together
150 Citations
255 Citations
85 Citations
78% of Scinapse members use related papers. After signing in, all features are FREE.
References21
Newest
#1Vincent MikolH-Index: 7
#2Jean-Luc RodeauH-Index: 16
Last. Richard GiegéH-Index: 54
view all 3 authors...
Possible pH variations during crystallization of biological macromolecules by the vapor diffusion method have not been taken into account in most experiments so far reported. The present study demonstrates that when ammonium sulfate is used as the precipitant, pH changes occur due to ammonia transfer following ammonium/ammonia equilibrium. The pH in a crystallization droplet is shown to be controlled by that of the reservoir. The theory of the effect is given and the consequences of pH variation...
30 CitationsSource
255 Citations
#1R. Boistelle (CNRS: Centre national de la recherche scientifique)H-Index: 20
#2Jean-Pierre Astier (CNRS: Centre national de la recherche scientifique)H-Index: 13
Abstract Crystallization from solution is a sequence of events which occur more or less consecutively but are rarely completely unconnected. The present contribution is a survey of nucleation, growth, phase transition, habit modification and ripening. Its aim is to provide a theoretical basis to biochemists who intend to approach crystallization.
326 CitationsSource
Abstract Finding the proper conditions for protein crystallization involves the investigation of many possible combinations of solution parameters. Development of an effective search strategy depends on determining how parameter variations influence crystal formation and quality. The present work describes the use of successive automated grid searches (SAGS) where solution pH and precipitant concentration are varied to determine optimal conditions for protein crystal growth in hanging drop vapor...
58 CitationsSource
#1Sandra B. Howard (UAH: University of Alabama in Huntsville)H-Index: 6
#2Pamela J. Twigg (UAH: University of Alabama in Huntsville)H-Index: 3
Last. Edward J. Meehan (UAH: University of Alabama in Huntsville)H-Index: 16
view all 4 authors...
150 CitationsSource
#1Keith B. Ward (NRL: United States Naval Research Laboratory)H-Index: 12
#2Mary Ann Perozzo (NRL: United States Naval Research Laboratory)H-Index: 3
Last. William M. Zuk (NRL: United States Naval Research Laboratory)H-Index: 3
view all 3 authors...
Abstract A significant concern in the field of protein crystallography remains the routine preparation of single crystals suitable for x-ray diffraction studies. Crystallization techniques developed within the last three decades typically require accurate manipulation of microliter quantities of protein, buffer, and precipitating solutions. The accuracy and reproducibility necessary for such processes, together with the repetitiveness of the tasks, make several of the crystallization methods par...
73 CitationsSource
#1Naomi E. Chayen (Imperial College London)H-Index: 33
#2J. Akins (Imperial College London)H-Index: 1
Last. David M. Blow (Imperial College London)H-Index: 30
view all 4 authors...
Abstract In order to quantify protein crystallization techniques, a method for measuring protein solubility in high salt concentration has been developed. It is based on a sensitive protein concentration assay, using binding to Coomassie blue dye. The protein concentration in a supernatant from which glucose isomerase is crystallising has been studied as a function of time. Equilibrium is established in 3–5 weeks, and the protein concentration remaining in solution is defined as the solubility o...
29 CitationsSource
#1Marc L. Pusey (MSFC: Marshall Space Flight Center)H-Index: 26
#2Kim M. Gernert (Duke University)H-Index: 5
39 CitationsSource
Abstract A method for immobilizing protein crystals has been devised for determining face growth rates, and used to investigate the growth kinetics of hen egg white lysozyme crystals. Growth rates were determined at 22 degrees C in 0.1 M sodium acetate, 5% NaCl, pH 4.0, on the visually identified (110) face of tetragonal lysozyme crystals. Protein concentrations ranged from 13 to 57 mg/ml (saturation concentration = 1.7 mg/ml). Growth rate data were fit to the equation R = kappa sigma ri, where ...
74 Citations
#1Mitsuo AtakaH-Index: 2
#2Shoji Tanaka (UTokyo: University of Tokyo)H-Index: 31
If protein single crystals larger than those suitable for x-ray analysis are obtained, various spectroscopic, thermal, mechanical, and electrical measurements become possible. To understand the factors governing the crystal size, tetragonal lysozyme crystals were grown in batches at 15°C from solutions of different protein and salt concentrations between pH 4–7. The number and size of the crystals, and the protein concentration remaining in the supernatant, varied markedly with the initial salt ...
95 CitationsSource
Cited By45
Newest
#1John Vargas-Badilla (NU: University of Nebraska–Lincoln)H-Index: 2
#2Saumen Poddar (NU: University of Nebraska–Lincoln)H-Index: 1
Last. David S. Hage (NU: University of Nebraska–Lincoln)H-Index: 48
view all 5 authors...
Abstract Several approaches were compared for the entrapment of proteins within hydrazide-activated silica for use in affinity microcolumns and high performance affinity chromatography. Human serum albumin (HSA) and concanavalin A (Con A) were used as model proteins for this work. Items considered in this study included the role played by the solution volume, amount of added protein, and use of slurry vs. on-column entrapment on the levels of solute retention and extent of protein immobilization...
Source
#1Joseph R. Luft (Hauptman-Woodward Medical Research Institute)H-Index: 29
#2Janet Newman (CSIRO: Commonwealth Scientific and Industrial Research Organisation)H-Index: 27
Last. Edward H. Snell (UB: University at Buffalo)H-Index: 23
view all 3 authors...
While crystallization historically predates crystallography, it is a critical step for the crystallographic process. The rich history of crystallization and how that history influences current practices is described. The tremendous impact of crystallization screens on the field is discussed.
32 CitationsSource
#1Richard Giegé (CNRS: Centre national de la recherche scientifique)H-Index: 54
Protein crystallization has been known since 1840 and can prove to be straightforward but, in most cases, it constitutes a real bottleneck. This stimulated the birth of the biocrystallogenesis field with both ‘practical’ and ‘basic’ science aims. In the early years of biochemistry, crystallization was a tool for the preparation of biological substances. Today, biocrystallogenesis aims to provide efficient methods for crystal fabrication and a means to optimize crystal quality for X-ray crystallo...
66 CitationsSource
3 CitationsSource
Abstract A novel approach for the investigation of proteins or macromolecules is outlined in this conceptual study. The preparation of grapho-epitaxial layers on nanotemplated substrates is proposed as an alternative to the preparation of single crystals by vapour diffusion techniques. Crystal structure investigations of such layers may then be performed via grazing-incidence diffraction (GIXRD) in the Laue mode. Quantitative expressions for the position and intensities of XRD peaks in this geom...
5 CitationsSource
#1Kedar S. Deshpande (TU Delft: Delft University of Technology)H-Index: 3
#2Tangir Ahamed (TU Delft: Delft University of Technology)H-Index: 10
Last. Marcel Ottens (TU Delft: Delft University of Technology)H-Index: 20
view all 6 authors...
This paper reviews the basic principles of the recently developed self-interaction chromatography (SIC) technique with regard to protein solution stability and protein crystallization. It gives experimental protocols for both normal-scale and micro-scale SIC experiments and reviews recent developments and current applications of this novel technique in the biopharmaceutical area. This paper aims to be a benchmark in the further proliferation of this highly effective and fast technology for the r...
5 CitationsSource
#1Alexander E. S. Van Driessche (CSIC: Spanish National Research Council)H-Index: 15
#2Jose A. Gavira (CSIC: Spanish National Research Council)H-Index: 24
Last. Fermín Otálora (CSIC: Spanish National Research Council)H-Index: 23
view all 4 authors...
We describe a method for precisely measuring the solubility of proteins in aqueous solution using laser confocal differential interference contrast microscopy. The method is based on the in situ observation of single steps on a protein crystal surface which allows a fast and precise determination of solubility as a function of temperature. To demonstrate the effectiveness of this novel approach the solubility dependence on temperature of glucose isomerase and hen egg white lysozyme was determine...
17 CitationsSource
#1I. Shulgin (UB: University at Buffalo)H-Index: 13
#2Eli Ruckenstein (UB: University at Buffalo)H-Index: 71
Abstract A new approach regarding the solubility of various sparingly soluble solutes, such as proteins, drugs and gases, and the local structure around a solute molecule is presented. This approach is based on an expression for the activity coefficient derived through the Kirkwood–Buff fluctuation theory of solutions. First, an expression for the solubility of proteins in aqueous solutions in terms of the preferential binding parameter is derived and criteria for salting-out or salting-in by va...
5 CitationsSource
#1Eli Ruckenstein (UB: University at Buffalo)H-Index: 71
#2Ivan L Shulgin (UB: University at Buffalo)H-Index: 3
The goal of this review is to examine the effect of salts and organic additives on the solubility of proteins in aqueous mixed solvents. The focus is on the correlation between the aqueous protein solubility and the osmotic second virial coefficient or the preferential binding parameter. First, several approaches which connect the solubility and the osmotic second virial coefficient are presented. Most of the experimental and theoretical results correlate the solubility and the osmotic second vi...
49 CitationsSource
#1Matthew J. Jones (MLU: Martin Luther University of Halle-Wittenberg)H-Index: 10
#2Joachim Ulrich (MLU: Martin Luther University of Halle-Wittenberg)H-Index: 26
Die Erzeugung von industriellen Proteinen (Massenproduktion von Enzymen) gewinnt zunehmend an Bedeutung. Die technische Kristallisation (sowohl Verdrangungs- als auch Kuhlungskristallisation) ist eine geeignete Technologie zur Reindarstellung von Proteinen. Es wird aber deutlich, dass das Thema zum einen angegangen wird, ohne die Voraussetzung fur eine erfolgreiche Entwicklung eines Kristallisationsprozesses zu haben, d. h. ohne Loslichkeitsdaten. Zum anderen wird zwar mit viel Kristallisations-...
6 CitationsSource