Aspermatogenic, Agglutinating and Immobilizing Antigens: Extraction, Separation, Purification and Molecular Weight Determinations by Equilibrium Ultracentrifugation

2015 ◽  
pp. 270-277
Author(s):  
Seymour Katsh ◽  
Henry Klostergaard ◽  
Grace F. Katsh ◽  
Jimmy Klostergaard
Keyword(s):  
Molecular Weight ◽  
Extraction Separation ◽  
Equilibrium Ultracentrifugation

Jack bean urease (EC 3.5.1.5). IV. The molecular size and the mechanism of inhibition by hydroxamic acids. Spectrophotometric titration of enzymes with reversible inhibitors

1980 ◽  
Vol 58 (12) ◽  
pp. 1323-1334 ◽  
Author(s):  
Nicholas E. Dixon ◽  
John A. Hinds ◽  
Ann K. Fihelly ◽  
Carlo Gazzola ◽  
Donald J. Winzor ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Active Site ◽  
Binding Sites ◽  
Spectrophotometric Titration ◽  
Hydroxamic Acids ◽  
Jack Bean Urease ◽  
Equivalent Weight ◽  
Guanidinium Chloride ◽  
Jack Bean ◽  
Equilibrium Ultracentrifugation

Kinetic, spectral, and other studies establish that hydroxamic acids bind reversibly to active-site nickel ion in jack bean urease. Equilibrium ultracentrifugation studies establish that the molecular weight of native urease is 590 000 ± 30 000 while that of the subunit formed in 6 M guanidinium chloride in the presence of β-mercaptoethanol is ~95 000. Essentially the same subunit molecular weight (~93 000) is found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, subsequent to denaturation in a guanidinium chloride – β-mercaptoethanol system at various temperatures. Coupled with an equivalent weight of 96 600 for binding of the inhibitors acetohydroxamic acid and phosphoramidate, these results establish securely that urease is a hexamer with one active site per 96 600-dalton subunit. Consistent values for the equivalent weight are obtained by a routine spectrophotometric titration of the active site of freshly prepared urease with trans-cinnamoylhydroxamic acid. General equations are derived which describe spectrophotometric titrations of binding sites of any enzyme with a reversible inhibitor. These equations allow the evaluation of the difference spectrum of the protein–inhibitor complex even when the binding sites cannot readily be saturated with the inhibitor or vice versa.

scholarly journals Physical properties and subunit structure of l-asparaginase isolated from Erwinia carotovora

1972 ◽  
Vol 126 (2) ◽  
pp. 361-379 ◽  
Author(s):  
K. A. Cammack ◽  
D. I. Marlborough ◽  
D. S. Miller
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Molecular Conformation ◽  
Sodium Dodecyl ◽  
Erwinia Carotovora ◽  
Optical Rotatory Dispersion ◽  
Spherical Particles ◽  
Sedimentation Equilibrium ◽  
Subunit Structure ◽  
Equilibrium Ultracentrifugation

1. l-Asparaginases from Erwinia carotovora and Escherichia coli (EC2 enzyme) are both capable of inhibiting and eliminating certain types of tumour cells. The Er. carotovora enzyme is a more basic protein, however, and in contrast with the EC2 enzyme it contains neither tryptophan nor cystine, and disulphide bonds are therefore absent. The molecule is very stable in solution from pH3.0 to about pH12.0, and is somewhat more stable at alkaline pH than is the Esch. coli enzyme. Calculations based on a s020,w 7.43S and a sedimentation-equilibrium molecular weight of 135000±10000 give a frictional ratio (f/f0) of 1.08. The molecular conformation is therefore very compact in solution, and the electron microscope shows the negatively stained molecules as almost spherical particles with a diameter of 7.2±0.7nm. 2. Sedimentation-velocity and equilibrium ultracentrifugation, in 5–8m solutions of urea and guanidinium chloride, and also electrophoresis in sodium dodecyl sulphate–polyacrylamide gel, reveal a dissociation of the native protein molecule into four subunits of similar molecular weight in the range 32500–38000. The enzymically inactive subunits can be physically reassembled into an active tetramer when urea is removed by dialysis. Although the subunit structures of the Er. carotovora enzyme and the Esch. coli enzyme molecules are similar, the secondary bonding forces holding the subunits together in the tetramer are somewhat stronger in the Er. carotovora enzyme. 3. The optical-rotatory-dispersion (o.r.d.) parameters that characterize the Cotton effects arising from ordered structure in the molecule are [m′]233=−3522±74° and [m′]200=9096±1700°. These show very marked changes as the secondary structure is disrupted and the molecule dissociates into subunits. A correlation pathway was traced on the basis of o.r.d. parameters and enzyme activity as the polypeptide chains were denatured and renatured (and reconstituted) into active molecules after the dilution of solutions in urea. Subunits resulting from treatment with sodium dodecyl sulphate do not show the typically disordered o.r.d. profile, but nevertheless they are inactive.

APPLICATION OF THE ARCHIBALD ULTRACENTRIFUGAL PROCEDURE TO LYSOZYME AND APURINIC ACID: EVALUATION USING A MECHANICAL INTEGRATOR

1956 ◽  
Vol 34 (3) ◽  
pp. 364-370 ◽  
Author(s):  
David B. Smith ◽  
G. C. Wood ◽  
P. A. Charlwood
Keyword(s):  
Molecular Weight ◽  
Sedimentation Rate ◽  
Computational Work ◽  
Transient Data ◽  
Equilibrium Ultracentrifugation

The method outlined by Archibald for handling transient data in equilibrium ultracentrifugation yields a molecular weight for lysozyme that conforms to those given by other methods. A method for applying the Archibald procedure to apurinic acid which is polydisperse and has a concentration dependent sedimentation rate is outlined. The use of a mechanical integrator for reducing the computational work is described.

Solvent Cutting to Make Superior Coal Tar Pitches

2011 ◽  
Vol 239-242 ◽  
pp. 1296-1299 ◽  
Author(s):  
Jian Shi Zhou ◽  
Xian Yong Wei ◽  
You Quan Dou ◽  
Gui Zhen Gong ◽  
Peng Li ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polycyclic Aromatic Hydrocarbon ◽  
Carbon Materials ◽  
Coal Tar ◽  
Separation System ◽  
Proper Order ◽  
Extraction Separation ◽  
Polycyclic Aromatic ◽  
Heavy Fractions ◽  
Light Components

Using solvents extraction separation system of self-designed make superior coal tar pitches for carbon materials. In proper order choose petroleum ether, methanol and carbon disulfide to cut coal tar into several fractions under different condition. The first solvent can take light components out from coal tar. Then drag out those chemical that contain heteroatom such as nitrogen, sulfide applying the second solvent. The last is to dig out higher molecular weight polycyclic aromatic hydrocarbon from coal tar deeply. Heavy fractions are vacuumed to drive out any solvents. The superior chirpy coal tar pitches for carbon materials acquired.

scholarly journals Physicochemical properties of Planorbis corneus erythrocruorin

1975 ◽  
Vol 149 (2) ◽  
pp. 437-445 ◽  
Author(s):  
E J Wood ◽  
L J Mosby
Keyword(s):  
Molecular Weight ◽  
High Speed ◽  
Sedimentation Coefficient ◽  
Sedimentation Equilibrium ◽  
Alkaline Conditions ◽  
Planorbis Corneus ◽  
A Subunit ◽  
Partial Dissociation ◽  
Equilibrium Ultracentrifugation ◽  
Intermediate Value

The erythrocruorin from the snail Planorbis corneus had a sedimentation coefficient, s020,w, of 33.5 ± 0.31S, and a molecular weight of 1.65 × 10(6) ± 0.04 × 10(6) by high-speed sedimentation-equilibrium ultracentrifugation. The amino acid composition and absorption spectrum of the protein are reported. A very low number of half-cystine residues was found, corresponding to 0.4 residue per haem group. The haem content was 2.76 ± 0.22%, corresponding to a protein molecular weight of about 22300. Under both acid and alkaline conditions partial dissociation took place to yield mixtures of products that could not be identified. A subunit corresponding to that containing one haem group was not obtained under any of the dossociating conditions tried. Electron microscopy revealed a ring-shaped molecule about 12.2 ± 0.5 nm in diameter. The native erythrocruoerin bound O2 co-operatively, the intermediate value of h in Hill plots having values between 1.7 and 3.4 depending on the conditions.

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