Stability of acetylcholinesterase in guanidine hydrochloride solution

1981 ◽  
Vol 59 (7) ◽  
pp. 551-555 ◽  
Author(s):  
Faizan Ahmad
Keyword(s):  
Molecular Weight ◽  
Guanidine Hydrochloride ◽  
Sedimentation Coefficient ◽  
Conformational State ◽  
Random Coil ◽  
Aromatic Residues ◽  
Activity Measurements ◽  
Irreversible Unfolding ◽  
Hydrochloride Solution ◽  
Circular Dichroic

The irreversible unfolding of acetylcholinesterase (acetylcholine hydrolyase, EC 3.1.1.7) by guanidine hydrochloride was studied by difference spectral, circular dichroic, and enzyme activity measurements. At pH 7.0 and in 1.1 M denaturant solution, a conformational state in which enzyme is completely inactive was detected. It is identical to the native enzyme as far as sedimentation coefficient and molecular weight are concerned, but differs from the native molecule by a slight loss in secondary structure and by a small perturbation of aromatic residues. Acetylcholinesterase in concentrated guanidine hydrochloride solution containing β-mercaptoethanol dissociates and exists as a random coil of molecular weight 68 000.

scholarly journals SOME PROPERTIES OF THE PROTEIN FORMING THE OUTER FIBERS OF CILIA

1968 ◽  
Vol 36 (1) ◽  
pp. 79-90 ◽  
Author(s):  
F. L. Renaud ◽  
A. J. Rowe ◽  
I. R. Gibbons
Keyword(s):  
Aqueous Solution ◽  
Molecular Weight ◽  
Amino Acid ◽  
Guanidine Hydrochloride ◽  
Sedimentation Coefficient ◽  
Tetrahymena Pyriformis ◽  
Acetone Powder ◽  
Fiber Protein ◽  
Hydrochloride Solution ◽  
Outer Fiber

Cilia were isolated from Tetrahymena pyriformis by an ethanol-calcium procedure. Solutions of outer-fiber protein were obtained either by aqueous extraction of an acetone powder of whole cilia, or by dissolving the isolated outer-fibers in 0.6 M KCl. In aqueous solution, the outer-fiber protein has a sedimentation coefficient of 6.0S and a molecular weight of 104,000 ± 14,000. In 5 M guanidine hydrochloride solution the molecular weight falls to 55,000 ± 5,000. After reduction and alkylation in 8 M urea, about 95% of the protein migrates as a single band on electrophoresis in polyacrylamide gel at pH 8.9; the migration velocity is identical with that of reduced and alkylated actin. Freshly prepared outer-fiber protein contains about 7.5 sulfhydryl groups per 55,000 g of protein. The amino acid composition of outer-fiber protein resembles that of actin, with such differences as occur being of the same order as those between actins from different species of animal.

Nuclear magnetic resonance spectroscopy of denatured proteins

1969 ◽  
Vol 22 (5) ◽  
pp. 1083 ◽  
Author(s):  
JH Bradbury ◽  
NLR King
Keyword(s):  
Molecular Weight ◽  
Magnetic Resonance ◽  
Magnetic Resonance Spectroscopy ◽  
Trifluoroacetic Acid ◽  
Guanidine Hydrochloride ◽  
Dichloroacetic Acid ◽  
Random Coil ◽  
Resonance Spectroscopy ◽  
Line Broadening ◽  
Line Widths

The proton magnetic resonance spectroscopy of 11 proteins (molecular weight range 5700-650000) has been investigated in five denaturing solvents, viz., trifluoroacetic acid-d, formic acid, dichloroacetic acid, 6M guanidine hydrochloride in D2O, and 8M urea in D2O. The chemical shifts, line-widths, and intensities of the resonances have been measured of the histidine C2 protons, the methionine SCH3 protons and methyl protons of leucine, isoleucine, and valine, the aromatic protons, and the α-CH protons. ��� It is found that, with some exceptions delineated below, the line- widths of the methyl resonances are constant for a particular solvent, independent of the molecular weight of the protein. This indicates that, in general, the proteins behave as random coil structures in these solvents, which confirms the conclusion reached by Tanford and co-workers1-4 for 6M guanidine hydrochloride. ��� However, methyl line broadening occurs in dichloroacetic acid for catalase and fibrinogen, in guanidine hydrochloride for insulin, and in urea for insulin and lysozyme. Furthermore, the C 2 histidine resonance is absent in dichloroacetic acid solutions of thyroglobulin, catalase, and fibrinogen; the SCH3 resonance is absent in myoglobin in trifluoroacetic acid-d and occurs as a doublet for trypsin in guanidine hydrochloride and in urea. A general line broadening of resonances indicates association and/or incomplete unfolding of molecules, whereas perturbations of only one particular resonance, as in the cases detailed above, are probably due to intramolecular non-covalent interactions which involve the perturbed group and another unspecified group in the protein. ��

scholarly journals The Isolation and Properties of Some Soluble Proteins From Wool VI. The Physicochemical Properties of Sgmkb2

1963 ◽  
Vol 16 (1) ◽  
pp. 252 ◽  
Author(s):  
JM Gillespie ◽  
BS Harrap
Keyword(s):  
Molecular Weight ◽  
No Reduction ◽  
Sedimentation Coefficient ◽  
Chain Structure ◽  
Optical Rotatory Dispersion ◽  
Random Coil ◽  
Merino Wool ◽  
Frictional Properties ◽  
Dispersion Data ◽  
End Group

The molecular weight of a high-sulphur protein (SCMKB2) from Merino wool has been determined by the Archibald technique and by light scattering, values of 22,600 and 22,100, respectively, being obtained. Optical rotatory dispersion data show that in aqueous solution the protein behaves as a random coil. This is consistent with the frictional properties of the molecule as deduced from its sedimentation coefficient and intrinsic viscosity. The protein appears to have one N-terminal arginyl end group; since it also contains two acyl groups per 22,000 molecular weight unit, the possibility of a multi�chain structure for the protein unit has been considered. However, no reduction in molecular weight could be achieved in any of several disaggregating solvents.

PHYSICOCHEMICAL STUDIES OF ALKALI LIGNINS: III. SIZE AND SHAPE OF THE MACROMOLECULE

1960 ◽  
Vol 38 (2) ◽  
pp. 270-279 ◽  
Author(s):  
P. R. Gupta ◽  
D. A. I. Goring
Keyword(s):  
Molecular Weight ◽  
Sedimentation Coefficient ◽  
Random Coil ◽  
Alkali Lignin ◽  
Molecular Weights ◽  
A Value ◽  
Huggins Constant ◽  
Physicochemical Studies ◽  
Lignin Macromolecule ◽  
Very High

Light-scattering measurements were made on the alkali lignin fractions described in a previous paper. The range of molecular weights found was from 50,000 to 48 × 106. The usual logarithmic graph of intrinsic viscosity and molecular weight was linear and gave a value of 0.32 for the exponent. From the logarithmic sedimentation coefficient – molecular weight relationship, the exponent was found as 0.52. Flory's hydrodynamic parameter [Formula: see text] was 2.3 × 106. These results suggested that the configuration of the alkali lignin macromolecule conformed to a structure between that of a random coil and an Einstein's sphere impenetrable to solvent. The branching parameter, g, introduced by Zimm and Stockmayer, decreased with an increase in molecular weight as expected. Most of the values of Huggins' constant, k′,were between 1 and 2 which indicated a compact particle. A marked increase in k′ was noted for fractions of low or very high molecular weight. The significance of the data is discussed and a model tentatively suggested for the macromolecule.

Germin: physicochemical properties of the glycoprotein which signals onset of growth in the germinating wheat embryo

1987 ◽  
Vol 65 (12) ◽  
pp. 1039-1048 ◽  
Author(s):  
William C. McCubbin ◽  
Cyril M. Kay ◽  
Theresa D. Kennedy ◽  
Byron G. Lane
Keyword(s):  
Circular Dichroism ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Helical Structure ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Cross Linking ◽  
Wheat Embryo ◽  
Circular Dichroïsm

The size and structure of germin, the homooligomeric glycoprotein which marks the onset of growth in germinating wheat embryos, has been examined by gel filtration, ultracentrifugation, electron microscopy, chemical cross-linking, and optical techniques (circular dichroism). Germin has a sedimentation coefficient (S20,w) of 7.3S, and a Stokes' radius (RS) of 4.5 nm, the latter value being compatible with the dimensions of the particle observed by negative staining in the electron microscope. By three methods (sedimentation equilibrium, sodium dodecyl sulphate (SDS) – polyacrylamide electrophoresis, S20,w/RS), the mean particle mass of the two closely related forms of germin (G and G′) is ca. 130 kilodaltons (kDa). Cross-linking with dimethyl suberimidate indicates that the oligomer is homopentameric, compatible with the molecular mass of the protomer (ca. 26 kDa) as determined by SDS–polyacrylamide gel electrophoresis. Using the Provencher and Glockner analysis to interpret circular dichroism measurements (in the far ultraviolet), both forms of germin contain about 10–20% α-helical structure, 50–60% β-sheet/turn structure, and 20–30% random coil. In a structure-inducing environment (45% trifluoroethanol), the α-helical structure increases to a value (35–40%) similar to that predicted by Chou–Fasman analysis of the protein sequence deduced by cDNA sequencing.

Bestimmung des Molekulargewichtes der 20-β-Hydroxy-steroid-dehydrogenase

1962 ◽  
Vol 17 (7) ◽  
pp. 432-436 ◽  
Author(s):  
Matatiahu Gehatia
Keyword(s):  
Molecular Weight ◽  
Diffusion Coefficient ◽  
Diffusion Coefficients ◽  
Specific Volume ◽  
Sedimentation Coefficient ◽  
Partial Specific Volume ◽  
Hydroxy Steroid ◽  
Steroid Dehydrogenase

The enzyme 20-β-Hydroxy-steroid-dehydrogenase obtained from the culture of Streptomyces hydrogenans and dissolved in 0.05 M Tris puffer, pH 7.3, has been investigated by means of a ultracentrifuge at 20 °C. The sedimentation- as well as the diffusion-coefficients obtained from various solutions at different concentrations were extrapolated to the concentration c = 0. The resulting zero-value for the sedimentation coefficient is s0 = 6.64 s and for the diffusion coefficient is D0 = 5.51 × 10-7 cm2/sec. Supposing the partial specific volume of the enzyme under consideration analogously to other similar proteins is V+=0.749 ml/g, the molecular weight has been estimated as M = 118 400.

STUDIES ON SMALL MOLECULAR WEIGHT ADHESION PROTEINS (SAPs) FROM CONNECTIVE TISSUES

1987 ◽  
Author(s):  
S Wasi ◽  
P Alles ◽  
D Gauthier ◽  
U Bhargava ◽  
J Farsi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyclonal Antibody ◽  
Type I Collagen ◽  
Cell Attachment ◽  
Binding Capacity ◽  
Guanidine Hydrochloride ◽  
Cell Types ◽  
Type I ◽  
Cell Binding ◽  
Connective Tissues

We have identified a family of low molecular weight proteins with cell attachment properties in a variety of soft and mineralised connective tissues (Wong et al., Biochem. J. 232, 119, 1985). For further characterisation of these proteins we extracted porcine bones with 4 M guanidine hydrochloride and purified the proteins on a series of gel filtration columns The purifed SAPs comprise three bands with Mr -14 000 -17 000. All three proteins bound to heparin-sepahrose in both the presence and absence of 4M urea, and when eluted with 2 M NaCl they retained their cell binding capacity. These proteins promoted the adhesion and spreading of a variety of cell types, including normal fibroblasts, osteoblasts, and epithelial cells, and tumour (osteosarcoma) cells. On Western blotting SAPs did not cross-react with antibodies against fibronectin, laminin or type I collagen; however, they were recognised by a monoclonal antibody to human vitronectin, a polyclonal antibody to bovine vitronectin and polyclonal antibody to human somatomedin B. Dose response experiments indicated that maximum attachment of human gingival fibroblasts occurred in the presence or absence of fetal bovine serum on wells precoated with 2.5 μg/cm2 of SAPs. Attachment of cells to these proteins was partially inhibited by the synthetic pentapeptide Gly-Arg-Gly-Asp-Ser. Utilising the nitrocellulose cell binding assay of Hayman et al (J. Cell. Biol. 95, 20, 1982), the cell attachment to these proteins could be completely inhibited by heparin (100 units/mL) whereas up to 1000 units/mL of heparin had no inhibitory effect on cell attachment to fibronectin and vitronectin. The occurrence of these proteins in a variety of connective tissues and their recognition by different cell types may reflect their general biological role in adhesive mechanisms in both hard and soft connective tissues. Currently, we are investigating the relationship between SAPs and vitronectin, since it is possible that SAPs represent a tissue-processed form of vitronectin or may be novel attachment proteins with regions of homology with vitronectin

scholarly journals The composition of peptidochitodextrins from sarcophagid puparial cases

1971 ◽  
Vol 125 (3) ◽  
pp. 703-716 ◽  
Author(s):  
H. Lipke ◽  
T. Geoghegan
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Protein Complex ◽  
Covalent Bonds ◽  
Aromatic Residues ◽  
Similar Molecular Weight ◽  
X Ray ◽  
Molecular Weights ◽  
Physical Interactions ◽  
The Stability

1. N-Bromosuccinimide cleaved proteins and pigments from fly puparia, increasing the chitin:protein ratio from 0.5 to 1.5. The product afforded subfractions (ratio 5:1) of molecular weights of 1200 and 1600 devoid of aromatic residues and N-terminal β-alanine, direct aryl links between polysaccharide chains being discounted. 2. The chitin–protein complex decreased in molecular weight when treated with Pronase, which suggested polypeptide bridges within the native chitin micelle. The limit dextrins generated by chitinase were mixtures of unsubstituted dextrins and peptidylated oligosaccharides, with the former predominating. 3. Peptidochitodextrins of similar molecular weight but markedly different solubility were prepared, which were indistinguishable with respect to amino acid, glucosamine, acetyl, X-ray or infrared characteristics. It is suggested that physical interactions contribute to the stability of the integument in addition to the covalent bonds that form during sclerotization.

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