scholarly journals The association behaviour of β-lactamases. Sedimentation equilibrium studies in ammonium sulphate solutions

1986 ◽  
Vol 237 (2) ◽  
pp. 511-517 ◽  
Author(s):  
E H Braswell ◽  
J R Knox ◽  
J M Frère
Keyword(s):  
Equilibrium Constants ◽  
Solubility Limit ◽  
Sedimentation Equilibrium ◽  
Association Model ◽  
Sodium Cacodylate ◽  
Dimerization Constant ◽  
Equilibrium Studies ◽  
Crystal Forms ◽  
Beta Lactamases ◽  
Equilibrium Sedimentation

The beta-lactamases (EC 3.5.2.6) from TEM plasmid RP4, Bacillus licheniformis 749/C and Enterobacter cloacae P99 were studied in solution over a wide concentration range by equilibrium sedimentation. Though crystal symmetries indicate that all three enzymes are potentially dimeric in their crystal forms, in 50 mM-sodium cacodylate at pH 6.5 the enzymes show only a small tendency to associate, indicated by a weight-average Mr (Mw) at 3% (w/v) concentration about 9% greater than that of the monomer. Although the mode of association could not be determined, this extent of association corresponded to a dimerization constant of about 2 × 10(2) M-1. In 2.1 M-(NH4)2SO4 the B. licheniformis enzyme shows some association at concentrations over 1%, displaying an Mw value at 7% concentration about 60% more than the monomer. Under the same conditions Mw for the Entero. P99 enzyme is about 60% greater than the monomer near the solubility limit of about 2%. However, the Mw for the TEM enzyme is over twice that of the monomer at its solubility limit (3%) in 1.7 M-(NH4)2SO4. Fitting the sedimentation data of the TEM enzyme in 1.7 M-(NH4)2SO4 with a dimerization model and an indefinite-isodesmic-association model yielded equilibrium constants of 1.5 × 10(4) and 3.3 × 10(2) M-1 respectively, with the indefinite-isodesmic model giving the better fit. Fitting the data for the other two enzymes yielded values of 1.4 × 10(3) and 1.7 × 10(2) M-1 respectively for the Entero. P99 enzyme and 4.5 × 10(2) and 45 M-1 respectively for the B. licheniformis enzyme. It could not be determined which model was the better fit for these two enzymes. Since none of the beta-lactamases studied here showed strong evidence of the terminal aggregate being a dimer, we conclude that crystalline dimers, if they exist, will not be tightly associated or physiologically significant.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from sheep liver

1969 ◽  
Vol 115 (4) ◽  
pp. 639-643 ◽  
Author(s):  
R. H. Villet ◽  
K. Dalziel
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sedimentation Velocity ◽  
Sedimentation Equilibrium ◽  
Equilibrium Studies ◽  
Phosphogluconate Dehydrogenase ◽  
Sheep Liver ◽  
Equilibrium Sedimentation ◽  
Purification And Properties

A method is described for the isolation of 6-phosphogluconate dehydrogenase from sheep liver. The product appears to be homogeneous in polyacrylamide-gel electrophoresis and in sedimentation-velocity and sedimentation-equilibrium studies in the ultracentrifuge. The molecular weight is estimated as 129000 from equilibrium sedimentation.

scholarly journals Role of dimerization in the control of the functioning of the human haemoglobin mutant haemoglobin Howick (β37 Trp→Gly)

1994 ◽  
Vol 300 (2) ◽  
pp. 553-556 ◽  
Author(s):  
T Brittain
Keyword(s):  
Equilibrium Constant ◽  
Equilibrium Constants ◽  
Oxygen Affinity ◽  
Amino Acid Side Chain ◽  
Oxygen Binding ◽  
Dimerization Constant ◽  
Equilibrium Studies ◽  
Human Haemoglobin ◽  
Dissociation Equilibrium ◽  
Wide Range

Haemoglobin Howick shows a high oxygen affinity (p50 = 1 mmHg) and a low co-operativity (n = 1.3). Equilibrium studies show the protein to be essentially totally dimeric in the oxygenated form. A wide range of rapid kinetic experiments indicate that the deoxygenated form of the protein exists in a tetramer<-->dimer equilibrium with an associated equilibrium constant of 3 microM. These kinetic data also indicate that the oxygenated form of the protein exists in a tetramer<-->dimer equilibrium with an associated equilibrium constant of 35 mM, and furthermore clearly identifies a large increase in the rate of the tetramer-to-dimer dissociation process as the origin of the vastly increased dissociation equilibrium constants. Simulations of the protein-concentration-dependence of the oxygen-binding properties of haemoglobin Howick, based on the measured equilibrium parameters, closely fits the experimental data. The change in dimerization constant for the deoxygenated form of the protein corresponds remarkably well to the free-energy change predicted for the simple transfer of the amino acid side chain at position beta 37 from a hydrophobic to a hydrophilic environment during the dimerization process.

EVIDENCE FOR SELF-ASSOCIATION OF PROTHROMBIN FRAGMENT 1 IN THE ABSENCE OF CALCIUM IONS: IMPLICATIONS FOR THE INTERPRETATION OF COOPERATIVITY OF CALCIUM BINDING

1987 ◽  
Author(s):  
Craig M Jackson ◽  
George M Brenckle ◽  
Philip J Hogg ◽  
Donald J Winzor
Keyword(s):  
Calcium Binding ◽  
Metal Ion ◽  
Equilibrium Constants ◽  
Binding Constants ◽  
Sedimentation Equilibrium ◽  
Equilibrium Studies ◽  
Prothrombin Fragment ◽  
Preferential Binding ◽  
Exclusion Chromatography ◽  
Self Association

The necessity to consider the binding of metal ion by prothrombin and its fragment 1 in terms of two entirely different mechanistic models has been removed. Cooperativity of Ca+T binding to prothrombin and prothrombin fragment 1 may reflect isomerization and/or self association of the protein. Sedimentation equilibrium studies have demonstrated that both prothrombin and its fragment 1 reversibly dimerize in the absence of Ca++.Based on this pre-existing equilibrium, a model for preferential binding of Ca++ to the dimer has been found capable of accounting quantitatively for the interaction of Ca2+ with fragment 1. This phenomenon is described by the relationship r ={pkA[A][S] (1 + kA[S])p-1 + qkc X [A]2[S](1 + kc [S])q-1}/[A] in which X (1,000 M-1) denotes the association constant for the pre-existing monomer-dimer equilibrium, and p, kA (10, 100 M-1) and q, kc (20, 2,000 M-1) are the respective stoichiometries and intrinsic binding constants for the interactions of Ca++ with monomeric and dimeric fragment 1, A. There is also evidence from exclusion chromatography and sedimentation velocity experiments that different isomeric states of prothrombin and its fragment 1 exist. It is therefore proposed that a model based on co-existence of isomeric and dimeric protein states will enable quantitative differences in the Ca++-mediated responses of fragment 1 and prothrombin to be rationalized solely in terms of differences in the relative magnitudes of equilibrium constants for the same interactions in the two systems.

scholarly journals Sedimentation Equilibrium Studies on Protein From Kookaburra Beak

1976 ◽  
Vol 29 (6) ◽  
pp. 481
Author(s):  
EF Woods
Keyword(s):  
Molecular Weight ◽  
Optical System ◽  
Optical Rotatory Dispersion ◽  
Rotatory Dispersion ◽  
Sedimentation Equilibrium ◽  
Optical Rotatory ◽  
Equilibrium Studies ◽  
Aqueous Buffer ◽  
Random Coils ◽  
Equilibrium Sedimentation

Fractionated samples of the soluble S-carboxymethyl proteins from kookaburra beak (Frenkel and Gillespie 1976) were examined by equilibrium sedimentation. The molecular weight was found to be 11 300 when the photoelectric scanning absorption optical system was employed and 13 700 when Rayleigh interference optics were used. Possible explanations for this difference are considered and it is concluded that it must arise from heterogeneity of the protein. Optical rotatory dispersion measurements indicate that the proteins probably exist as random coils in dilute aqueous buffer.

Comparison of RNA Terminal Sequences of Phages f2 and Qbeta: Chemical and Sedimentation Equilibrium Studies

Science ◽  
1968 ◽  
Vol 160 (3835) ◽  
pp. 1459-1460 ◽  
Author(s):  
H. L. Weith ◽  
G. T. Asteriadis ◽  
P. T. Gilham
Keyword(s):  
Sedimentation Equilibrium ◽  
Equilibrium Studies

Kinetic and Equilibrium Studies of the Reactions of Cyanide Ion with 1,3,5-Trinitrobenzene, 2,4,6-Trinitroanisole, and 2,4,6-Trinitrotoluene in Isopropanol

1974 ◽  
Vol 52 (1) ◽  
pp. 8-17 ◽  
Author(s):  
Leong Huat Gan ◽  
Albert Richard Norris
Keyword(s):  
Standard Enthalpy ◽  
Equilibrium Constants ◽  
Activation Parameters ◽  
Stopped Flow ◽  
Cyanide Ion ◽  
Equilibrium Studies ◽  
Kinetics Of Formation ◽  
Kinetics Of

Equilibrium constants for the formation of 1:1 cyanide ion σ-complexes with 1,3,5-trinitrobenzene, 2,4,6-trinitroanisole, and 2,4,6-trinitrotoluene have been determined spectrophotometrically over a range of temperatures. Standard enthalpy (ΔH0) and entropy (ΔS0) changes associated with each reaction have been evaluated. The kinetics of formation of the σ-complexes have been investigated by means of a stopped-flow technique and the activation parameters characterizing the formation of each complex have been determined. Evidence is presented which indicates the cyanide ion – 2,4,6-trinitroanisole σ-complex formed in isopropanol contains the cyanide ion bonded exclusively at the C-3 position.

Physicochemical Studies of Conglutin γ, a Storage Globulin From Seeds of Lupinus angustifolius

1980 ◽  
Vol 7 (1) ◽  
pp. 1 ◽  
Author(s):  
RJ Blagrove ◽  
JM Gillespie ◽  
GG Lilley ◽  
EF Woods
Keyword(s):  
Molecular Weight ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Optical Rotatory Dispersion ◽  
Sedimentation Equilibrium ◽  
Native Protein ◽  
Equilibrium Studies ◽  
Physicochemical Studies ◽  
Α Helix

Physicochemical studies are reported for conglutin �, the minor globulin isolated from seeds of L. angustifolius cv. Uniwhite. Isoelectric focusing of the native protein in polyacrylamide gel slabs resolved major and minor broad bands near pH 8.0 and 7.8 respectively. Following reduction of disulfide bonds with β-mercaptoethanol in 8 M urea, the smaller polypeptide chain of known sequence focused near pH 6.9 while the larger chain focused near pH 8.0. Sedimentation equilibrium studies showed that the major component in aqueous buffers at neutral pH is a hexamer of molecular weight 280 000 which dissociates to the monomer of molecular weight 47 000 at pH 4.8. The sequence molecular weight of the small subunit polypeptide is 16 517 [Elleman, T.C. (1977). Aust. J. Biol. Sci. 30, 33-45]. The molecular weights determined for the larger chain by sedimentation equilibrium or column chromatography in 6 M guanidine hydrochloride, and by dodecyl sulfate-polyacrylamide gel electrophoresis, were in the range 28 000-30 000. Optical rotatory dispersion and circular dichroism measurements have been used to establish the approximate proportions of α-helix (15%), β-structure (35%), β-turns (18%) and unordered regions (32%) in the native protein. The denaturation curve for guanidine hydrochloride and the proportions of α-helix (50%), β-turns (18%) and unordered regions (32%) in 80 % trifluoroethanol have been determined.

scholarly journals Sedimentation-equilibrium studies on the heterogeneity of two β-lactamases

1970 ◽  
Vol 118 (3) ◽  
pp. 467-474 ◽  
Author(s):  
P. H. Lloyd ◽  
A. R. Peacocke
Keyword(s):  
Molecular Weight ◽  
Diffusion Coefficients ◽  
Minor Component ◽  
Theoretical Solution ◽  
Sedimentation Equilibrium ◽  
Equilibrium Studies ◽  
Molecular Weights ◽  
A Minor

Solutions of crystalline β-lactamase I and β-lactamase II, prepared by Kuwabara (1970), were examined in the ultracentrifuge and their sedimentation coefficients, diffusion coefficients, molecular weights and heterogeneity determined. Each sample was shown to consist of a major component comprising at least 97% of the material and a minor component of much higher molecular weight. The molecular weights of the major components were 27800 for β-lactamase I and 35600 for β-lactamase II. Emphasis is placed on a straightforward practical way of analysing the sedimentation-equilibrium results on mixtures of two macromolecular components rather than on a strict theoretical solution. Appendices describe the theory of systems at both chemical and sedimentation equilibrium and the procedure for calculating the combined distribution of two components.

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