The preparation of human hemoglobin α-subunit and a study of its monomer–dimer association

1976 ◽  
Vol 54 (11) ◽  
pp. 1002-1010 ◽  
Author(s):  
Frank A. Terpstra ◽  
David B. Smith
Keyword(s):  
Dissociation Constant ◽  
Gel Filtration ◽  
Sedimentation Velocity ◽  
Sedimentation Equilibrium ◽  
Velocity Sedimentation ◽  
Stable Complex ◽  
Cross Linking ◽  
Human Hemoglobin ◽  
Α Subunit ◽  
Equilibrium Data

An improved procedure for the isolation of the α-subunit of human hemoglobin is described. The monomer–dimer equilibrium in α-subunit solutions has been studied by boundary analysis in gel filtration, sedimentation velocity, sedimentation equilibrium, and cross-linking with dimethyl adipimidate. A dissociation constant has been determined from the sedimentation equilibrium data. The reaction with haptoglobin of cross-linked α-subunit showed that the dimer fraction would form a stable complex.

scholarly journals The structure of L-lactate oxidase from Mycobacterium smegmatis

1977 ◽  
Vol 165 (2) ◽  
pp. 375-383 ◽  
Author(s):  
P A Sullivan ◽  
C Y Soon ◽  
W J Schreurs ◽  
J F Cutfield ◽  
M G Shepherd
Keyword(s):  
Electron Microscopy ◽  
Mycobacterium Smegmatis ◽  
Sedimentation Velocity ◽  
Equilibrium Analysis ◽  
Sedimentation Equilibrium ◽  
Cross Linking ◽  
Velocity Analysis ◽  
Approach To Equilibrium ◽  
Lactate Oxidase ◽  
Dimethyl Suberimidate

1. An improved purification was developed for L-lactate oxidase from Mycobacterium smegmatis. 2. The mol.wt. of the native enzyme by a sedimentation-equilibrium analysis was 345 000, and other ultracentrifuge methods gave values in the range 345 000-350 000. 3. An amino acid analysis, determinations of protein and flavin, a sedimentation-velocity analysis and an approach to equilibrium analysis gave values for the subunit mol.wt. in the range 43 500-47 000. 4. It was concluded that L-lactate oxidase contains eight subunits of mol.wt. 43 500. 5. Cross-linking of the subunits with dimethyl suberimidate and electron-microscopy studies were consistent with an octameric structure.

The reaction between hemoglobin α-subunit and haptoglobin

1976 ◽  
Vol 54 (11) ◽  
pp. 1011-1015 ◽  
Author(s):  
Frank A. Terpstra ◽  
David B. Smith
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Human Hemoglobin ◽  
Gel Filtration Chromatography ◽  
Separation Techniques ◽  
Α Subunit ◽  
Α Subunits

The interaction between human hemoglobin α-subunit and porcine haptoglobin was investigated by polyacrylamide gel electrophoresis, gel filtration chromatography, sedimentation through excess α-subunit and gel filtration in an α-subunit-containing medium. No interaction was detected by the first two methods indicating dissociation of the complex during the application of these separation techniques. The latter two methods, in which the complex is studied in a medium of excess subunits, showed that haptoglobin became saturated with the binding of two α-subunits.

scholarly journals NADP-linked malic enzyme. Purification from maize leaves, Mr and subunit composition

1988 ◽  
Vol 254 (1) ◽  
pp. 229-233 ◽  
Author(s):  
M S Thorniley ◽  
K Dalziel
Keyword(s):  
Malic Enzyme ◽  
Enzyme Purification ◽  
Gel Filtration ◽  
Sedimentation Velocity ◽  
Exchange Chromatography ◽  
Subunit Composition ◽  
Sedimentation Equilibrium ◽  
Apparent Homogeneity ◽  
Maize Leaves ◽  
A Subunit

1. The isolation of NADP-linked malic enzyme (EC 1.1.1.40) from maize leaves is described, together with studies of its Mr and subunit composition. 2. The enzyme was purified to apparent homogeneity by affinity chromatography on N6-aminohexyl-2′,5′-bisphosphoadenosine-agarose, gel filtration with Sephadex G-100 and ion-exchange chromatography on DEAE-Sephadex A-50. A purification of 140-fold with a 30% yield was obtained. 3. A detailed study of the Mr by several methods revealed the existence of different Mr forms in solution. 4. In the presence of dithiothreitol the enzyme appears to be present in triethanolamine buffer, pH 7.5, as a tetramer with a subunit Mr of 60,000 and an S20,w of 10.75 S. 5. In phosphate buffer, pH 7.0, it seems to be a dimer of Mr 120,000 with an S20,w of 7.95 S. 6. In the absence of dithiothreitol, lower-Mr forms were detected by sedimentation-equilibrium and sedimentation-velocity studies in triethanolamine buffer. 7. Results from gel filtration gave Mr values of about 340,000 in both buffers.

scholarly journals The Isolation of Aggregates of Spectrin From Bovine Erythrocyte Membranes

1975 ◽  
Vol 28 (3) ◽  
pp. 259 ◽  
Author(s):  
GB Ralston
Keyword(s):  
Ionic Strength ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Velocity ◽  
Sedimentation Equilibrium ◽  
Erythrocyte Membranes ◽  
Bovine Erythrocyte ◽  
Molecular Weights ◽  
Wide Range

Aggregated states of spectrin from bovine erythrocyte membranes can be detected in sedimentation velocity experiments. These aggregates have been isolated by means of gel filtration on columns of 4 % agarose. They appear to be stable over a wide range of pH and ionic strength, although they are dissociated by sodium dodecyl sulphate. Sedimentation equilibrium measurements yielded values of 960000 and 480000 for the molecular weights of the major aggregates, corresponding to a tetramer and dimer, respectively. The presence of different aggregated states in spectrin preparations may explain the wide variation in the reported physica~ properties of spectrin.

scholarly journals Structural characterization of Myxococcus xanthus MglC, a component of polarity control system, and its interactions with MglB

2020 ◽  
Author(s):  
Srajan Kapoor ◽  
Akriti Kodesia ◽  
Nidhi Kalidas ◽  
Ashish ◽  
Krishan Gopal Thakur
Keyword(s):  
Crystal Structure ◽  
Dissociation Constant ◽  
Myxococcus Xanthus ◽  
Gel Filtration ◽  
Structural Features ◽  
Docking Studies ◽  
Titration Calorimetry ◽  
Stable Complex ◽  
List Type ◽  
Family Protein

AbstractMyxococcus xanthus displays two types of motilities i.e. Social (S) and Adventurous (A). The pole-to-pole reversals of these motility regulator proteins is the key to this process. Here, we determined ~1.85 Å resolution crystal structure of MglC, which revealed that despite sharing <9% sequence identity, both MglB and MglC adopt Regulatory Light Chain 7 (RLC7) family fold. Interestingly, MglC is structurally unique compared to the other known RLC7 family proteins having ~30°-40° shift in the orientation of functionally important α2 helix. Using isothermal titration calorimetry and gel filtration chromatography, we show that MglC binds MglB in 2:4 stoichiometry with submicromolar range dissociation constant. Using combination of small angle X-ray scattering and molecular docking studies, we show that MglBC complex is formed by MglC homodimer sandwiched between two homodimers of MglB.In BriefKapoor et al. report the crystal structure of Myxococcus xanthus MglC, a Roadblock Light Chain 7 (RLC7) family protein, involved in polarity reversal. The structure reveals a distinct orientation of α2 helix compared to other RLC7 proteins. They also demonstrate that MglC binds a GTPase activating protein, MglB, with submicromolar range dissociation constant.HighlightsMglC adopts RLC7 fold and has distinct structural features.MglC interacts MglB to form a stable complex having submicromolar range dissociation constant.MglC homodimer is sandwiched between two MglB homodimers to form a 2:4 stoichiometric complex.

Large-cluster and combined fluorescent and gold immunoprobes

1996 ◽  
Vol 54 ◽  
pp. 892-893
Author(s):  
Richard D. Powell ◽  
James F. Hainfeld ◽  
Carol M. R. Halsey ◽  
David L. Spector ◽  
Shelley Kaurin ◽  
...  
Keyword(s):  
Metal Cluster ◽  
Sulfonyl Chloride ◽  
Gel Filtration ◽  
Cross Linking ◽  
Site Specific ◽  
Fab Fragments ◽  
Cluster Label ◽  
Covalently Linked ◽  
Texas Red ◽  
Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

2018 ◽  
Vol 9 (03) ◽  
pp. 20204-20223
Author(s):  
Maghsoudi, Hossein ◽  
U Pati
Keyword(s):  
Dna Binding ◽  
P53 Protein ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Cross Linking ◽  
Mobility Shift ◽  
Dna Complexes ◽  
P53 Antibodies

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

scholarly journals Covalent alteration of the prosthetic heme of human hemoglobin by BrCCl3. Cross-linking of heme to cysteine residue 93.

1992 ◽  
Vol 267 (13) ◽  
pp. 8739-8743
Author(s):  
J.T. Kindt ◽  
A Woods ◽  
B.M. Martin ◽  
R.J. Cotter ◽  
Y Osawa
Keyword(s):  
Cysteine Residue ◽  
Cross Linking ◽  
Human Hemoglobin

scholarly journals Terpene polyacrylate TPA5 shows favorable molecular hydrodynamic properties as a potential bioinspired archaeological wood consolidant

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Michelle Cutajar ◽  
Fabrizio Andriulo ◽  
Megan R. Thomsett ◽  
Jonathan C. Moore ◽  
Benoit Couturaud ◽  
...  
Keyword(s):  
Molar Mass ◽  
Analytical Ultracentrifugation ◽  
Gel Filtration ◽  
Axial Ratio ◽  
Sedimentation Equilibrium ◽  
Archaeological Wood ◽  
Molecular Hydrodynamic ◽  
Pine Trees ◽  
Minimal Concentration ◽  
Hydrogen Bonding Potential

AbstractThere is currently a pressing need for the development of novel bioinspired consolidants for waterlogged, archaeological wood. Bioinspired materials possess many advantages, such as biocompatibility and sustainability, which makes them ideal to use in this capacity. Based on this, a polyhydroxylated monomer was synthesised from α-pinene, a sustainable terpene feedstock derived from pine trees, and used to prepare a low molar mass polymer TPA5 through free radical polymerisation. This polymer was extensively characterised by NMR spectroscopy (chemical composition) and molecular hydrodynamics, primarily using analytical ultracentrifugation reinforced by gel filtration chromatography and viscometry, in order to investigate whether it would be suitable for wood consolidation purposes. Sedimentation equilibrium indicated a weight average molar mass Mw of (4.3 ± 0.2) kDa, with minimal concentration dependence. Further analysis with MULTISIG revealed a broad distribution of molar masses and this heterogeneity was further confirmed by sedimentation velocity. Conformation analyses with the Perrin P and viscosity increment ν universal hydrodynamic parameters indicated that the polymer had an elongated shape, with both factors giving consistent results and a consensus axial ratio of ~ 4.5. These collective properties—hydrogen bonding potential enhanced by an elongated shape, together with a small injectable molar mass—suggest this polymer is worthy of further consideration as a potential consolidant.

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