Binding of low-affinity and high-affinity heparin to antithrombin. Ultraviolet difference spectroscopy and circular dichroism studies

Biochemistry ◽  
1978 ◽  
Vol 17 (16) ◽  
pp. 3339-3344 ◽  
Author(s):  
Birgitta Nordenman ◽  
Ingemar Bjork
Keyword(s):  
Circular Dichroism ◽  
Difference Spectroscopy ◽  
High Affinity ◽  
Circular Dichroïsm

Spectroscopic studies on the binding of divalent cations to porcine intestinal calcium-binding protein

1978 ◽  
Vol 56 (6) ◽  
pp. 492-499 ◽  
Author(s):  
K. J. Dorrington ◽  
D. I. C. Kells ◽  
A. J. W. Hitchman ◽  
J. E. Harrison ◽  
T. Hofmann
Keyword(s):  
Optical Activity ◽  
Calcium Binding ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Cation Binding ◽  
Side Chain ◽  
Difference Spectroscopy ◽  
High Affinity ◽  
Circular Dichroïsm ◽  
Circular Dichroic

Circular dichroism and ultraviolet absorption difference spectroscopy have been used to study the binding of a series of divalent and trivalent cations to porcine intestinal calcium-binding protein (CaBP). When calcium is bound to the single high-affinity site on CaBP, the aromatic optical activity is greatly increased. Analysis of the circular dichroic spectra, obtained in the presence and absence of calcium, suggested that although changes in the optical activity of the single tyrosyl residue accounted for much of the overall change observed upon binding calcium, one or more of the five phenylalanyl residues was also perturbed. All the cations tested, with the exception of lead which gave rise to unique spectral effects, caused the same changes in optical activity between 300 and 250 nm. In the peptide absorption region, CaBP exhibited optical activity typical of an α-helical protein and no significant changes were observed in the presence of any of the cations tested. Cation-binding curves obtained from the circular dichroic data for the cations bound with high affinity (i.e., calcium, strontium, and the trivalent lanthanide ions) showed that the apparent number of binding sites was inversely related to the protein concentration. This phenomenon was accounted for by the concentration-dependent aggregation of CaBP observed in earlier studies. The binding data, obtained using circular dichroism, clearly indicated that the affinity of CaBP for the various cations was related to their ionic radius. Absorption difference spectra were observed when calcium was bound to CaBP. The features of these spectra confirmed that phenylalanyl as well as tyrosyl transitions were perturbed upon calcium binding. The extent to which the tyrosyl side chain was exposed to solvent was determined by solvent perturbation difference spectroscopy using perturbing agents of differing molecular radius. The apparent degree of exposure increased as the perturbant size decreased suggesting that the side chain was located in a cleft. Bound calcium did not change the degree of exposure. These data, together with complementary data obtained with bovine CaBP, were discussed in terms of the geometry of the cation-binding site.

The thermal denaturation of bovine cardiac G-actin

1977 ◽  
Vol 55 (4) ◽  
pp. 325-331 ◽  
Author(s):  
C. C. Contaxis ◽  
C. C. Bigelow ◽  
C. G. Zarkadas
Keyword(s):  
Circular Dichroism ◽  
Conformational Change ◽  
Thermodynamic Analysis ◽  
Thermal Denaturation ◽  
State Transition ◽  
Thermal Unfolding ◽  
Difference Spectroscopy ◽  
Maximum Stability ◽  
Ph Range ◽  
Circular Dichroïsm

The thermal denaturation of bovine cardiac G-actin has been studied by ultraviolet difference spectroscopy and circular dichroism between pH 7.5 and 10.5. As with proteins previously studied, thermal unfolding is incomplete compared with unfolding by urea or GuHCl. However, the same conformational change is observed over the pH range studied, and the available evidence indicates it is a two-state transition. Thermodynamic analysis of the data shows that ΔH0 and ΔS0 are strongly dependent on the temperature, that ΔCp is 1300 cal deg−1 mol−1, and that G-actin has a temperature of maximum stability near −5 °C.

scholarly journals Fluorescence and circular-dichroism studies on the Streptomyces R61 dd-carboxypeptidase–transpeptidase. Penicillin binding by the enzyme

1973 ◽  
Vol 135 (3) ◽  
pp. 493-505 ◽  
Author(s):  
Manuel Nieto ◽  
Harold R. Perkins ◽  
Jean-Marie Frère ◽  
Jean-Marie Ghuysen
Keyword(s):  
Circular Dichroism ◽  
Thermal Denaturation ◽  
Penicillin G ◽  
Affinity Binding ◽  
Guanidinium Chloride ◽  
Positive Maximum ◽  
Peptide Substrates ◽  
High Affinity ◽  
Circular Dichroïsm ◽  
New Hypothesis

The circular dichroism of the dd-carboxypeptidase–transpeptidase from Streptomyces R61 shows in the near u.v. a set of weak extrema at 289nm (positive) and at 282, 275 and 268nm (all negative). In the far u.v. it shows negative extrema at 217–218 and 208nm, crossover at 202nm and a positive maximum at about 194nm. The u.v. absorption of the enzyme shows it to contain tyrosine and tryptophan in approx. 3.4:1 ratio. The enzyme is fluorescent with a maximum emission at 318–320nm. The near-u.v. circular dichroism of the protein is extensively affected by binding of penicillin G, but the far u.v. is unaffected. Binding of the antibiotic also causes quenching of the fluorescence of the enzyme. The latter effect has been used to study the binding of penicillin G to the enzyme and the influence exerted upon it by salts, denaturants and peptide substrates and inhibitors. High-affinity binding of penicillin appears to be comparatively slow and reversible, and can occur under conditions in which the protein is enzymically inactive. The thermal denaturation of the enzyme in guanidinium chloride at pH7 is affected by binding of the antibiotic. The presence of even large concentrations of β-mercaptoethanol neither impaired the activity of the enzyme nor prevented its inhibition by penicillin G or cephalosporin C. A new hypothesis for the molecular mechanism of the interaction of the enzyme with penicillin is proposed.

The quantitative comparison of the effects of denaturants on the stability of proteins

1986 ◽  
Vol 64 (10) ◽  
pp. 993-998 ◽  
Author(s):  
James A. Thomson ◽  
Charles C. Bigelow
Keyword(s):  
Circular Dichroism ◽  
Specific Binding ◽  
Ribonuclease A ◽  
Quantitative Comparison ◽  
Difference Spectroscopy ◽  
Guanidinium Chloride ◽  
Binding Capability ◽  
The Stability ◽  
Circular Dichroïsm

A novel quantitative comparison of denaturants involving the complete reversible unfolding of proteins is presented. Ribonuclease A was denatured with guanidinium chloride in the presence of low fixed concentrations of various partial denaturants, with the unfolding process being monitored by circular dichroism and difference spectroscopy. The major advantage of this method is that it allows a direct quantitative comparison of the effects of denaturants on the stability of proteins. The effect on the stability of ribonuclease A was shown to be linearly dependent upon the concentration of denaturant. An investigation of the constitutive ions of salts revealed that their effects were additive only in the case of salts that have no specific binding capability. This method can also be useful in detecting the specific binding of salts.

scholarly journals Whole-cell circular dichroism difference spectroscopy reveals an in vivo-specific deca-heme conformation in bacterial surface cytochromes

2018 ◽  
Vol 54 (99) ◽  
pp. 13933-13936 ◽  
Author(s):  
Yoshihide Tokunou ◽  
Punthira Chinotaikul ◽  
Shingo Hattori ◽  
Thomas A. Clarke ◽  
Liang Shi ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Analytical Framework ◽  
Difference Spectroscopy ◽  
Whole Cell ◽  
Bacterial Surface ◽  
System P ◽  
Circular Dichroïsm

Our novel analytical framework to identify the inter-heme interaction in deca-heme cytochrome protein MtrC in whole cell revealed that the heme alignment in reduced MtrC is distinct from that in purified system.

Characterization of Tamm-Horsfall Urinary Glycoprotein

1973 ◽  
Vol 31 ◽  
pp. 420-421
Author(s):  
John P. Robinson ◽  
J. David Puett
Keyword(s):  
Electron Microscopy ◽  
Circular Dichroism ◽  
Secondary Structure ◽  
Quaternary Structure ◽  
Normal Adult ◽  
Morphological Properties ◽  
Adult Males ◽  
Circular Dichroïsm ◽  
Urinary Glycoprotein

Much work has been reported on the chemical, physical and morphological properties of urinary Tamm-Horsfall glycoprotein (THG). Although it was once reported that cystic fibrotic (CF) individuals had a defective THG, more recent data indicate that THG and CF-THG are similar if not identical.No studies on the conformational aspects have been reported on this glycoprotein using circular dichroism (CD). We examined the secondary structure of THG and derivatives under various conditions and have correlated these results with quaternary structure using electron microscopy.THG was prepared from normal adult males and CF-THG from a 16-year old CF female by the method of Tamm and Horsfall. CF female by the method of Tamm and Horsfall.

Sign in / Sign up

Export Citation Format

Share Document