scholarly journals Occurrence and characterization of stable intermediate state(s) in the unfolding of ovomucoid by guanidine hydrochloride

1978 ◽  
Vol 171 (1) ◽  
pp. 89-97 ◽  
Author(s):  
Masroor A. Baig ◽  
A. Salahuddin
Keyword(s):  
Intrinsic Viscosity ◽  
Intermediate State ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Random Coil ◽  
Native Structure ◽  
Difference Spectra ◽  
Tyrosine Residues ◽  
Sodium Phosphate Buffer ◽  
Interaction Factor

Reversible unfolding of ovomucoid by guanidine hydrochloride, as followed by viscosity and difference-spectral measurements at 25°C, pH6, occurred in two distinct steps involving at least three major conformational states, namely the native, intermediate and completely denatured states, occurring respectively in 60mm-sodium phosphate buffer, 3.5m-guanidine hydrochloride and 6m-guanidine hydrochloride. The overall native conformation of ovomucoid, as indicated by its intrinsic viscosity (5.24ml/g) and gel-filtration behaviour, differs significantly from that of a typical globular protein. Exposures of tyrosine residues in native ovomucoid measured by difference spectroscopy following perturbation with glycerol, ethylene glycol and dimethyl sulphoxide were, respectively, 0.42, 0.56 and 0.57. Of the exposed phenolic groups only one titrated normally (pKint., 9.91, electrostatic-interaction factor, w, 0.04). Results on difference spectra, solvent perturbation, phenolic titration and intrinsic viscosity (7.4ml/g) taken together showed that, although ovomucoid in 3.5m-guanidine hydrochloride was significantly unfolded, it retained a degree of native structure, removable with 6m-guanidine hydrochloride. In the latter, all the six tyrosine residues were available for titration, and the intrinsic viscosity of ovomucoid increased to 9.4ml/g. Furthermore, the characteristic fine structures in circular-dichrosim spectra of ovomucoid, associated with the elements of native structure, were abolished in 6m-guanidine hydrochloride, suggesting that the completely denatured state is structureless and presumably behaves as a cross-linked random coil. The latter state has been shown by analysis of the results on guanidine hydrochloride-dependence of the transition, intermediate⇌denatured, to be less stable than the intermediate state under native conditions by about 46kJ/mol at 25°C. Attempts have been made to interpret the above results in the light of available information on the amino acid sequence of ovomucoid.

scholarly journals Assessment of Conformational Changes in Low-Sulphur S-Carboxymethylkerateine from Wool

1967 ◽  
Vol 20 (4) ◽  
pp. 827 ◽  
Author(s):  
GM Bhatnagar ◽  
WG Crewther
Keyword(s):  
Absorption Spectrum ◽  
Conformational Changes ◽  
Guanidine Hydrochloride ◽  
Ultraviolet Absorption Spectrum ◽  
Spectral Measurements ◽  
Characteristic Difference ◽  
Concentrated Solutions ◽  
Difference Spectra ◽  
The Difference ◽  
Negative Difference

The effects of urea and guanidine hydrochloride on the ultraviolet absorption spectrum of the low-sulphur S-carboxymethylkerateine fraction of wool have been measured. In concentrated solutions of urea characteristic difference spectra were obtained with maxima of negative absorbance at 288, 280, and 240 miL. The addition of guanidine hydrochloride or an increase in temperature gave similar negative difference maxima at the higher wavelengths. Calculation of the extent of unfolding of the protein chains from the difference in absorbance at all three maxima showed that the unfolding was 50% complete at a urea concentration of about 1� 8M whereas a urea concep.tration of about 4� 3M was required to decrease the helix content by 50%. Similar measurements on components 7 and 8, the two major constituents of SCMKA, showed that a 50% decrease in helix content was obtained with 2�8M and O� 8M urea respectively whereas the corresponding values for 50 % unfolding assessed from difference spectral measurements were 2� 2M and 1� 2M urea respectively. It is suggested that the helical regions of components 7 and 8 aggregate specifically and that spectral measurements relate largely to non-helical portions of the chains.

scholarly journals The Ensemble of Conformations of Antifreeze Glycoproteins (AFGP8): A Study Using Nuclear Magnetic Resonance Spectroscopy

Biomolecules ◽  
2019 ◽  
Vol 9 (6) ◽  
pp. 235
Author(s):  
Cheenou Her ◽  
Yin Yeh ◽  
Viswanathan V. Krishnan
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Three Dimensional ◽  
Random Coil ◽  
The Arctic ◽  
Native Structure ◽  
Antifreeze Glycoproteins ◽  
Structural Motifs ◽  
Ice Surface ◽  
Nuclear Magnetic

The primary sequence of antifreeze glycoproteins (AFGPs) is highly degenerate, consisting of multiple repeats of the same tripeptide, Ala–Ala–Thr*, in which Thr* is a glycosylated threonine with the disaccharide beta-d-galactosyl-(1,3)-alpha-N-acetyl-d-galactosamine. AFGPs seem to function as intrinsically disordered proteins, presenting challenges in determining their native structure. In this work, a different approach was used to elucidate the three-dimensional structure of AFGP8 from the Arctic cod Boreogadus saida and the Antarctic notothenioid Trematomus borchgrevinki. Dimethyl sulfoxide (DMSO), a non-native solvent, was used to make AFGP8 less dynamic in solution. Interestingly, DMSO induced a non-native structure, which could be determined via nuclear magnetic resonance (NMR) spectroscopy. The overall three-dimensional structures of the two AFGP8s from two different natural sources were different from a random coil ensemble, but their “compactness” was very similar, as deduced from NMR measurements. In addition to their similar compactness, the conserved motifs, Ala–Thr*–Pro–Ala and Ala–Thr*–Ala–Ala, present in both AFGP8s, seemed to have very similar three-dimensional structures, leading to a refined definition of local structural motifs. These local structural motifs allowed AFGPs to be considered functioning as effectors, making a transition from disordered to ordered upon binding to the ice surface. In addition, AFGPs could act as dynamic linkers, whereby a short segment folds into a structural motif, while the rest of the AFGPs could still be disordered, thus simultaneously interacting with bulk water molecules and the ice surface, preventing ice crystal growth.

Thrombin Binding To Factor VIII/Von Willebrand Factor Protein

1981 ◽  
Author(s):  
M E P Switzer ◽  
P A McKee
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Substrate Binding ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Void Volume ◽  
Peptidase Activity ◽  
Sds Page ◽  
Von Willebrand ◽  
Willebrand Factor

Thrombin (IIa) both activates and inactivates the procoagulant activity of Factor VIII/von Willebrand Factor (FVIII/vWF). The level of activation increases as the IIa: FVIII/vWF ratio approaches 1:1, suggesting that IIa might bind stoichiometrically to FVIII/vWF either during or after activation. We approached this question by gel filtration and ultracentrifugation studies of FVIII/vWF and l25I-IIa, which activated FVIII/vWF as well as unlabeled IIa. When the mixture of 125I-IIa and FVIII/vWF was chromatographed on 4% agarose a peak of 125I-IIa was eluted with the FVIII/ vWF in the void volume (V0). Similarly, when 125I-IIa was ultracentrifuged with FVIII/vWF, a peak of radioactivity sedimented with the FVIII/vWF protein. 125I-aibumin, used to approximate a control, did not bind to FVIII/vWF. The 125I-IIa-FVIII/vWF complex isolated from the 4% agarose filtration retained ∼50% peptidase activity. The ability to activate additional FVIII/vWF or to clot fibrinogen was <10% of that of free IIa isolated from the same chromatogram. Both the FVIII and vWF moieties appear to be important in binding, since VD protein isolated from the gel filtration of FVIII/vWF on 4% agarose in 0.25 M CaCl2 binds about 24% as much 125I-IIa as native FVIII/vWF. When the isolated 125I-IIa-FVIII/vWF complex was rechromatographed on 4% agarose in 0.15 M NaCl, essentially no dissociation occurred. When these experiments were repeated in 4 M guanidine hydrochloride (GnHCl), ∼30% of the IIa remained bound. When the 125I-IIa-FVIII/vWF complex was isolated from the GnHCl chromatography and analyzed by SDS-PAGE, 58% of the IIa remained bound to the FVIII/vWF before reduction and 43% of the IIa remained bound even after reduction with β-mercaptoethanol for 3 hours at 37°. Thus FVIII/vWF binds at least some of the IIa very tightly. Since FVIII/vWF-bound thrombin is essentially inactive toward macromolecular substrate, binding of thrombin to FVIII/vWF is most likely a mechanism for removing active thrombin from the circulation.

Structure of β-casein

1979 ◽  
Vol 46 (2) ◽  
pp. 281-282 ◽  
Author(s):  
Shunrokuro Arima ◽  
Ryoya Niki ◽  
Kenji Takase
Keyword(s):  
Electron Microscopy ◽  
Phosphate Buffer ◽  
Analytical Ultracentrifugation ◽  
Hydrophobic Interactions ◽  
Gel Filtration ◽  
Random Coil ◽  
Free Energies ◽  
Gel Filtration Chromatography ◽  
Increasing Temperature

SUMMARYThe temperature and concentration dependent association of β-casein was studied by means of viscometry, gel filtration chromatography, electron microscopy, analytical ultracentrifugation and UV difference spectrophotometry. Degrees of polymerization of 12, 22 and 49 and free energies of association of –21, –23 and – 25 kJ/mole monomer were found at temperatures of 10, 15 and 20 °C respectively in 0·2 M Na phosphate buffer pH 6·7.Monomeric β-casein was not a completely random coil but became more compact with increasing temperature, due to hydrophobic interactions.

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. ��

Purification and some properties of the glutamate dehydrogenase of Salmonella typhimurium

1973 ◽  
Vol 19 (4) ◽  
pp. 427-438 ◽  
Author(s):  
J. W. Coulton ◽  
M. Kapoor
Keyword(s):  
Salmonella Typhimurium ◽  
Glutamate Dehydrogenase ◽  
Ammonium Sulfate ◽  
Gradient Centrifugation ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient Centrifugation ◽  
Ammonium Sulfate Fractionation ◽  
Sulfate Fractionation

NADP-specific glutamate dehydrogenase (GDH) from Salmonella typhimurium was purified 190-fold by heat treatment, ammonium sulfate fractionation, DEAE-Sephadex chromatography, reverse ammonium sulfate fractionation, and gel filtration. The enzyme proved to be stable to 55 °C, and displayed a pH optimum at 8.6 in the amination reaction. The sedimentation coefficient of GDH, as determined by sucrose density gradient centrifugation, was about 10.3 S. From gel filtration chromatography, the molecular weight and Stokes' radius for the enzyme were estimated at 280 000 daltons and 54 × 10−8 cm, respectively. Unusual resistance was displayed by the enzyme to high concentrations of the protein denaturants, urea, SDS, and guanidine hydrochloride.

scholarly journals Gene Cloning, Purification, and Characterization of a Novel Peptidoglutaminase-Asparaginase from Aspergillus sojae

2012 ◽  
Vol 78 (15) ◽  
pp. 5182-5188 ◽  
Author(s):  
Kotaro Ito ◽  
Kenichiro Matsushima ◽  
Yasuji Koyama
Keyword(s):  
Gene Cloning ◽  
Expressed Sequence Tag ◽  
Gel Filtration ◽  
Fermented Foods ◽  
Gene Encoding ◽  
Native Form ◽  
Content Type ◽  
Aspergillus Sojae ◽  
Umami Taste ◽  
Sodium Phosphate Buffer

ABSTRACTGlutaminase is an enzyme that catalyzes the hydrolysis ofl-glutamine tol-glutamate, and it plays an important role in the production of fermented foods by enhancing the umami taste. By using the genome sequence and expressed sequence tag data available forAspergillus oryzaeRIB40, we cloned a novel glutaminase gene (AsgahA) fromAspergillus sojae, which was similar to a previously described gene encoding a salt-tolerant, thermostable glutaminase ofCryptococcus nodaensis(CnGahA). The structural gene was 1,929 bp in length without introns and encoded a glutaminase, AsGahA, which shared 36% identity with CnGahA. The introduction of multiple copies of AsgahAintoA. oryzaeRIB40 resulted in the overexpression of glutaminase activity. AsGahA was subsequently purified from the overexpressing transformant and characterized. While AsGahA was located at the cell surface in submerged culture, it was secreted extracellularly in solid-state culture. The molecular mass of AsGahA was estimated to be 67 kDa and 135 kDa by SDS-PAGE and gel filtration chromatography, respectively, indicating that the native form of AsGahA was a dimer. The optimal pH of the enzyme was 9.5, and its optimal temperature was 50°C in sodium phosphate buffer (pH 7.0). Analysis of substrate specificity revealed that AsGahA deamidated not only freel-glutamine andl-asparagine but also C-terminal glutaminyl or asparaginyl residues in peptides. Collectively, our results indicate that AsGahA is a novel peptidoglutaminase-asparaginase. Moreover, this is the first report to describe the gene cloning and purification of a peptidoglutaminase-asparaginase.

Application of the plastein reaction to caseins and to skim-milk powder: II. Chemical and physical properties of the plasteins and the mechanism of plastein formation

1982 ◽  
Vol 49 (2) ◽  
pp. 279-293 ◽  
Author(s):  
Gonca Sukan ◽  
Anthony T. Andrews
Keyword(s):  
Physical Properties ◽  
Peptide Mapping ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Milk Powder ◽  
Skim Milk ◽  
Peptide Bond ◽  
Skim Milk Powder ◽  
Peptide Bond Formation ◽  
Hydrophobic Peptides

SUMMARYChemical and physical properties of plasteins derived from caseins and skim-milk powder have been investigated. Plasteins were very similar in properties regardless of starting material or of the proteinase used in their production. Plastein formed from Na caseinate hydrolysate at pH 5 was stable over the pH range 1·5–9·0 and had the properties of a thixotropic gel. More extreme pH values caused dissolution as did various chaotropic reagents. 8 M-urea disrupted plastein structure almost entirely, as did various organic acids at concentrations of 50% (v/v) and strongly ionic detergents. Non-ionic or weakly cationic detergents and 6 M-guanidine hydrochloride were less effective. Treatment with strong salt solutions, Ca2+ and EDTA had no effect. Gel filtration experiments demonstrated that plasteins were completely disrupted under dissociating conditions and peptide mapping confirmed that all the peptides originally present in the hydrolysate were also present in plastein. Amino group contents were unchanged on plastein formation. These experiments together with gel electrophoresis studies clearly showed that only hydrophobic and ionic bonding mechanisms were involved in plastein formation and no detectable peptide bond formation or transpeptidation occurred. In accordance with this, amino acid analysis suggested that hydrophobic peptides were preferentially incorporated. Implications for use of plasteins in food products are discussed.

Sign in / Sign up

Export Citation Format

Share Document