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Changes of pH during Biomacromolecule Crystallization by Vapor Diffusion using Ammonium Sulfate as the Precipitant

Published on Apr 1, 1989in Journal of Applied Crystallography2.867
· DOI :10.1107/S0021889888013433
Vincent Mikol7
Estimated H-index: 7
,
Jean-Luc Rodeau16
Estimated H-index: 16
,
Richard Giegé56
Estimated H-index: 56
Abstract
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 variations during crystallization are discussed in terms of reproducibility of experiments. An application, the crystallization of concanavalin A induced by pH variation, is presented.
  • References (1)
  • Citations (30)
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A complete guide to techniques and procedures for the preparation of proteins for crystallographic studies. Describes methods for protein crystallization; formation of isomorphous heavy atoms; x-ray diffraction and analysis; and photographic and computerbased data collection methods and instrumentation.
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Abstract Salt/polyethylene glycol (PEG) mixtures are employed as precipitants for biological macromolecules. The dependence of precipitation curves (PCs) on salt species was investigated for integral membrane protein/detergent particles. By relating this dependence to properties of ions dissociated from added salts, the following roles and effects of various ions were clarified. In the presence of ions whose interaction with water is stronger than water–water interaction, the coordination of sol...
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#1Rui-Qing Chen (NPU: Northwestern Polytechnical University)H-Index: 5
#2Qing-Di Cheng (NPU: Northwestern Polytechnical University)H-Index: 4
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Protein crystallization occurs when the solution conditions are suitable for nucleation and growth of the crystals. pH, as one of the most important parameters that governs the protein crystallization process, can affect the conformation, activity, electrostatic interactions and solubility of protein in the solution. Hence, manipulating the pH is an important path to success in crystallizing proteins. In this paper, we show that varying pH levels over the incubation time during the crystallizati...
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The Protein Data Bank (PDB) is the largest available repository of solved protein structures and contains a wealth of information on successful crystallization. Many centres have used their own experimental data to draw conclusions about proteins and the conditions in which they crystallize. Here, data from the PDB were used to reanalyse some of these results. The most successful crystallization reagents were identified, the link between solution pH and the isoelectric point of the protein was i...
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The crystallization of proteins is dependent on the careful control of numerous parameters, one of these being pH. The pH of crystallization is generally reported as that of the buffer; however, the true pH has been found to be as many as four pH units away. Measurement of pH with a meter is time-consuming and requires the reformatting of the crystallization solution. To overcome this, a high-throughput method for pH determination of buffered solutions has been developed with results comparable ...
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#1Richard Giegé (CNRS: Centre national de la recherche scientifique)H-Index: 56
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...
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#2Roger A. SayleH-Index: 15
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In protein crystallization, as well as in many other fields, it is known that the pH at which experiments are performed is often the key factor in the success or failure of the trials. With the trend towards plate-based high-throughput experimental techniques, measuring the pH values of solutions one by one becomes prohibitively time- and reagent-expensive. As part of an HT crystallization facility, a colour-based pH assay that is rapid, uses very little reagent and is suitable for 96-well or hi...
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In general, the crystallization of proteins is a very complex process. Experiences of many scientists point out that majority of proteins is difficult to crystallize and even if a protein tends to crystallize relatively easily there are many parameters that must be taken into account. There are multiple reasons that point out the difficulty of protein crystal growth. Apparently, protein molecules are very complex (large, flexible molecules often composed of several subunits), relatively chemical...
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#1Lindsay A. Matthews (McMaster University)H-Index: 4
#2Andrew Duong (McMaster University)H-Index: 9
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