Polyethylene glycol: Catalytic effect on the crystallization of phosphoglucomutase at high salt concentration☆

Published on Aug 1, 1986in Journal of Crystal Growth1.573
· DOI :10.1016/0022-0248(86)90173-9
William J. Ray3
Estimated H-index: 3
(Purdue University),
William J. Ray13
Estimated H-index: 13
(Purdue University),
Charles E. Bracker27
Estimated H-index: 27
(Purdue University)
A cold, aqueous solution containing (NH4)2SO4 at 53% of saturation and 5.9% w/v polyethylene glycol-400 (PEG) produces PEG-rich coacervate droplets (16%(NH4)2SO4 and 37% PEG) when warmed to 25°C. In partition experiments conducted at low protein concentration, phosphoglucomutase and several other common proteins concentrate at least 20-fold in the PEG-rich phase. A temperature-induced phase separation similar to that above, but conducted in the presence of 5 mg/ml of phosphoglucomutase, can produce coacervate droplets in which the concentration of protein is about 500 mg/ml and thus approaches that in the crystal phase. The nucleation and subsequent conversion of such droplets into micrometer-size crystals of phosphoglucomutase were studied by light microscopy. Nucleation usually occurs in the periphery of these droplets, and neither phase nucleates efficiently by itself, although both support growth. Most droplets do not nucleate and subsequently dissolve as the protein concentration in the surrounding medium is depleted by incorporation into growing srystals. A major role of PEG in the nucleation/crystallization process is to repress the formation of salt-induced, disordered aggregates whose non-lattice protein-protein interactions presumably are less mobile than those in the droplet phase. In this sense, PEG acts as a nucleation catalyst. Such a mode of action is supported by studies on the effect of PEG in the conversion of salt-induced aggregates of phosphoglucomutase into protein crystals. An analogous role in the nucleation and slow growth of much larger crystals under somewhat different conditions is inferred. Such a PEG-induced effect may be general for proteins that crystallize from concentrated salt solutions.
  • References (9)
  • Citations (30)
📖 Papers frequently viewed together
981 Citations
179 Citations
7,072 Citations
78% of Scinapse members use related papers. After signing in, all features are FREE.
Abstract A suspension of microcrystals of phosphoglucomutase in 60% ammonium sulfate exhibits a maximal catalytic activity in substrate-velocity studies that is about 0.2 of that obtained with the soluble enzyme under the same conditions. The apparent Michaelis constants for the reaction in the crystal phase are altered to an even smaller extent, relative to that in solution, although the parameters for the monophosphate and bisphosphate are increased more than 3 and more than 5 orders of magnit...
8 Citations
#1Zheng-Jiong LinH-Index: 1
#2M. KonnoH-Index: 1
Last. Michael G. RossmannH-Index: 110
view all 7 authors...
Abstract The three-dimensional structure of rabbit phosphoglucomutase has been determined to 2.7 A resolution by a combination of isomorphous and molecular replacement techniques. Heavy atom positions were found by using vector search and difference Fourier methods. The two molecules in the asymmetric unit form a dimer with its 2-fold axis perpendicular to and intersecting with a crystallographic 4(1) axis. Thus, the dimers are arranged so that they form fibers that are coincident with the 4(1) ...
18 Citations
#1William J. Ray (Purdue University)H-Index: 13
#1William J. Ray (Purdue University)H-Index: 3
Last. Joseph M. Puvathingal (Purdue University)H-Index: 2
view all 2 authors...
Abstract Commercial samples of polyethylene glycol as well as detergents incorporating the polyoxyethylene linkage may contain substantial concentrations of peroxides and aldehydes, as well as aldehyde precursors. Facile procedures for essentially eliminating either the first or all three of these contaminants from concentrated aqueous stock solutions are described. Under some conditions, the purified solutions can be stored at room temperature under aerobic conditions for time periods of weeks ...
63 CitationsSource
#1Christina ThallerH-Index: 35
#2Gregor EicheleH-Index: 57
Last. Johan N. JansoniusH-Index: 42
view all 6 authors...
38 CitationsSource
120 CitationsSource
#1W J RayH-Index: 1
#2M A HermodsonH-Index: 1
Last. W C MahoneyH-Index: 1
view all 4 authors...
Abstract The complete amino acid sequence of rabbit muscle phosphoglucomutase has been determined by isolating the 11 peptide fragments produced by the cyanogen bromide cleavage reaction and subjecting these to automated sequencing procedures. Products produced by treatment of some of these fragments with hydroxylamine, iodosobenzoic acid, mild acid, cyanogen bromide in formic and heptafluorobutyric acids, Staphylococcus aureus V8 protease, and trypsin (with or without blocking at lysine residue...
65 Citations
89 Citations
#1Alexander McPhersonH-Index: 51
981 Citations
Publisher Summary This chapter discusses the phenomenon of phase separation in polymer–polymer–water mixtures and describes the general behavior of particles and macromolecules in polymer two phase systems. A general problem with all partition methods is that they are highly empirical. Many factors govern the partition behavior. Thus, the physical and chemical nature of the phase polymers, the ionic composition of the phases, the nature of the partitioned substance all influence partition in a c...
171 CitationsSource
Cited By30
#1Nafisa Begam (University of Tübingen)H-Index: 5
#2Olga Matsarskaia (University of Tübingen)H-Index: 4
Last. Frank Schreiber (University of Tübingen)H-Index: 49
view all 5 authors...
In globular protein systems, upper critical solution temperature (UCST) behavior is common, but lower critical solution temperature (LCST) phase transitions are rare. In addition, the temperature sensitivity of such systems is usually difficult to tune. Here we demonstrate that the charge state of globular proteins in aqueous solutions can alter their temperature-dependent phase behavior. We show a universal way to tune the effective protein interactions and induce both UCST and LCST-type transi...
#1Chengqian Yuan (CAS: Chinese Academy of Sciences)H-Index: 15
#2Aviad Levin (University of Cambridge)H-Index: 10
Last. Xuehai Yan (CAS: Chinese Academy of Sciences)H-Index: 51
view all 9 authors...
The transition of peptides and proteins from the solution phase into fibrillar structures is a general phenomenon encountered in functional and aberrant biology and is increasingly exploited in soft materials science. However, the fundamental molecular events underpinning the early stages of their assembly and subsequent growth have remained challenging to elucidate. Here, we show that liquid-liquid phase separation into solute-rich and solute-poor phases is a fundamental step leading to the nuc...
23 CitationsSource
#1D. A. BarlowH-Index: 1
#2Jan Gregus (Abraham Baldwin Agricultural College)H-Index: 1
2 CitationsSource
: Classical nucleation theory (CNT), which was established about 90 years ago, has been very successful in many research fields, and continues to be the most commonly used theory in describing the nucleation process. For a fluid-to-solid phase transition, CNT states that the solute molecules in a supersaturated solution reversibly form small clusters. Once the cluster size reaches a critical value, it becomes thermodynamically stable and favored for further growth. One of the most important assu...
10 CitationsSource
#1Andrea Sauter (University of Tübingen)H-Index: 8
#2Felix Roosen-RungeH-Index: 18
Last. Frank SchreiberH-Index: 49
view all 6 authors...
We present a real-time study of protein crystallization of bovine β-lactoglobulin in the presence of CdCl2 using small-angle X-ray scattering and optical microscopy. From observing the crystallization kinetics, we propose the following multistep crystallization mechanism that is consistent with our data. In the first step, an intermediate phase is formed, followed by the nucleation of crystals within the intermediate phase. During this period, the number of crystals increases with time, but the ...
49 CitationsSource
#1Fajun Zhang (University of Tübingen)H-Index: 33
#2Felix Roosen-Runge (University of Tübingen)H-Index: 18
Last. Frank Schreiber (University of Tübingen)H-Index: 49
view all 6 authors...
We briefly summarize the recent progress in tuning protein interactions as well as phase behavior in protein solutions using multivalent metal ions. We focus on the influence of control parameters and the mechanism of reentrant condensation, the metastable liquid"liquid phase separation and classical vs. nonclassical pathways of protein crystallization.
27 CitationsSource
#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...
69 CitationsSource
#1Xing WangH-Index: 3
#2Jason M. GillianH-Index: 2
Last. Donald J. KirwanH-Index: 16
view all 3 authors...
Induction time and crystal morphology changed with agitation during the precipitation of salicylic acid in viscous, aqueous low molecular weight poly(ethylene glycol) 300 (PEG300) solutions with water as the antisolvent. Further, if antisolvent precipitations of either salicylic acid or methylparaben in aqueous PEG300 were conducted by equal volume mixing in a confined impinging jet, the exiting solution was optically clear and exhibited induction times as long as days. Significant mechanical ag...
23 CitationsSource
Formation behavior of liquid droplets in aqueous solutions of insulin was studied by varying pH values and Zn 2 + concentrations of the mother solutions. The nucleation and growth of insulin crystals that preferentially occurred on the droplet surface were first observed.
4 CitationsSource