scholarly journals Heme Dissociation from Myoglobin in the Presence of the Zwitterionic Detergent N,N-Dimethyl-N-Dodecylglycine Betaine: Effects of Ionic Liquids

Biomolecules ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 126 ◽  
Author(s):  
Eric Kohn ◽  
Joshua Lee ◽  
Anthony Calabro ◽  
Timothy Vaden ◽  
Gregory Caputo
Keyword(s):  
Ionic Liquids ◽  
Circular Dichroism ◽  
Protein Denaturation ◽  
Binding Free Energy ◽  
Protein Stabilization ◽  
Important Data ◽  
Future Studies ◽  
Low Concentrations ◽  
Circular Dichroïsm ◽  
Zwitterionic Detergent

We have investigated myoglobin protein denaturation using the zwitterionic detergent Empigen BB (EBB, N,N-Dimethyl-N-dodecylglycine betaine). A combination of absorbance, fluorescence, and circular dichroism spectroscopic measurements elucidated the protein denaturation and heme dissociation from myoglobin. The results indicated that Empigen BB was not able to fully denature the myoglobin structure, but apparently can induce the dissociation of the heme group from the protein. This provides a way to estimate the heme binding free energy, ΔGdissociation. As ionic liquids (ILs) have been shown to perturb the myoglobin protein, we have investigated the effects of the ILs 1-butyl-3-methylimidazolium chloride (BMICl), 1-ethyl-3-methylimidazolium acetate (EMIAc), and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIBF4) in aqueous solution on the ΔGdissociation values. Absorbance experiments show the ILs had minimal effect on ΔGdissociation values when compared to controls. Fluorescence and circular dichroism data confirm the ILs have no effect on heme dissociation, demonstrating that low concentrations ILs do not impact the heme dissociation from the protein and do not significantly denature myoglobin on their own or in combination with EBB. These results provide important data for future studies of the mechanism of IL-mediated protein stabilization/destabilization and biocompatibility studies.

Effect of Temperature and pH on the Secondary Structure and Denaturation Process of Jumbo Squid Hepatopancreas Cathepsin D.

2019 ◽  
Vol 26 (7) ◽  
pp. 532-541 ◽  
Author(s):  
Cadena-Cadena Francisco ◽  
Cárdenas-López José Luis ◽  
Ezquerra-Brauer Josafat Marina ◽  
Cinco-Moroyoqui Francisco Javier ◽  
López-Zavala Alonso Alexis ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Secondary Structure ◽  
Cathepsin D ◽  
Thermal Denaturation ◽  
Protein Denaturation ◽  
Marine Organisms ◽  
Effect Of Temperature ◽  
Jumbo Squid ◽  
Α Helix ◽  
Circular Dichroïsm

Background: Cathepsin D is a lysosomal enzyme that is found in all organisms acting in protein turnover, in humans it is present in some types of carcinomas, and it has a high activity in Parkinson's disease and a low activity in Alzheimer disease. In marine organisms, most of the research has been limited to corroborate the presence of this enzyme. It is known that cathepsin D of some marine organisms has a low thermostability and that it has the ability to have activity at very acidic pH. Cathepsin D of the Jumbo squid (Dosidicus gigas) hepatopancreas was purified and partially characterized. The secondary structure of these enzymes is highly conserved so the role of temperature and pH in the secondary structure and in protein denaturation is of great importance in the study of enzymes. The secondary structure of cathepsin D from jumbo squid hepatopancreas was determined by means of circular dichroism spectroscopy. Objective: In this article, our purpose was to determine the secondary structure of the enzyme and how it is affected by subjecting it to different temperature and pH conditions. Methods: Circular dichroism technique was used to measure the modifications of the secondary structure of cathepsin D when subjected to different treatments. The methodology consisted in dissecting the hepatopancreas of squid and freeze drying it. Then a crude extract was prepared by mixing 1: 1 hepatopancreas with assay buffer, the purification was in two steps; the first step consisted of using an ultrafiltration membrane with a molecular cut of 50 kDa, and the second step, a pepstatin agarose resin was used to purification the enzyme. Once the enzyme was purified, the purity was corroborated with SDS PAGE electrophoresis, isoelectric point and zymogram. Circular dichroism is carried out by placing the sample with a concentration of 0.125 mg / mL in a 3 mL quartz cell. The results were obtained in mdeg (millidegrees) and transformed to mean ellipticity per residue, using 111 g/mol molecular weight/residue as average. Secondary-structure estimation from the far-UV CD spectra was calculated using K2D Dichroweb software. Results: It was found that α helix decreases at temperatures above 50 °C and above pH 4. Heating the enzyme above 70°C maintains a low percentage of α helix and increases β sheet. Far-UV CD measurements of cathepsin D showed irreversible thermal denaturation. The process was strongly dependent on the heating rate, accompanied by a process of oligomerization of the protein that appears when the sample is heated, and maintained a certain time at this temperature. An amount typically between 3 and 4% α helix of their secondary structure remains unchanged. It is consistent with an unfolding process kinetically controlled due to the presence of an irreversible reaction. The secondary structure depends on pH, and a pH above 4 causes α helix structures to be modified. Conclusion: In conclusion, cathepsin D from jumbo squid hepatopancreas showed retaining up to 4% α helix at 80°C. The thermal denaturation of cathepsin D at pH 3.5 is under kinetic control and follows an irreversible model.

Glucose in dry and moist ionic liquid: vibrational circular dichroism, IR, and possible mechanisms

2020 ◽  
Vol 22 (19) ◽  
pp. 10726-10737 ◽  
Author(s):  
Jan Blasius ◽  
Roman Elfgen ◽  
Oldamur Hollóczki ◽  
Barbara Kirchner
Keyword(s):  
Ionic Liquid ◽  
Ionic Liquids ◽  
Circular Dichroism ◽  
Molecular Level ◽  
Vibrational Circular Dichroism ◽  
Circular Dichroïsm

The molecular level specification of glucose monomers in ionic liquids and their mixtures with water helps understanding cellulose processing in these liquids.

Light flux density threshold at which protein denaturation is induced by synchrotron radiation circular dichroism beamlines

2008 ◽  
Vol 15 (4) ◽  
pp. 420-422 ◽  
Author(s):  
A. J. Miles ◽  
Robert W. Janes ◽  
A. Brown ◽  
D. T. Clarke ◽  
J. C. Sutherland ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Synchrotron Radiation ◽  
Flux Density ◽  
Protein Denaturation ◽  
Light Flux ◽  
Circular Dichroïsm ◽  
Density Threshold

scholarly journals Physico–Chemical Interaction between Clay Minerals and Albumin Protein according to the Type of Clay

Minerals ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 396 ◽  
Author(s):  
Kim ◽  
Oh
Keyword(s):  
Circular Dichroism ◽  
Fluorescence Quenching ◽  
Conformational Change ◽  
Clay Minerals ◽  
Protein Denaturation ◽  
Chemical Interaction ◽  
Harmful Effect ◽  
Chemical Effect ◽  
Physico Chemical ◽  
Circular Dichroïsm

Clay minerals are widely utilized in pharmaceutical and dermatological sciences as a gastrointestinal medicine or skin remediation agent. In order to verify the feasibility of clays as an injection, pill, or topical agent, it is important to study their interactions with biological components, such as proteins. In this study, we utilized a protein fluorescence quenching assay and circular dichroism spectroscopy to evaluate general aspects of protein denaturation and conformational change, respectively. Three different clays; layered double oxide (LDO), montmorilonite (MMT) and halloysite nanotube (HNT), were treated with albumin and the physico-chemical effect on the protein’s conformation was investigated. MMT was shown to influence the conformational change the most, owing to the large accessible adsorption site. HNT showed meaningful circular dichroism (CD) band collapse as well as fluorescence quenching in the protein, suggesting a potential harmful effect of HNT toward the protein. Among the three tested clays, LDO was determined to affect protein structure the least in terms of three-dimensional conformation and helical structure.

Circular dichroism and difference spectra of members of the troponin–tropomyosin system in cetyltrimethylammonium bromide

1976 ◽  
Vol 54 (11) ◽  
pp. 941-945 ◽  
Author(s):  
William D. McCubbin ◽  
Cyril M. Kay
Keyword(s):  
Circular Dichroism ◽  
Cetyltrimethylammonium Bromide ◽  
Troponin I ◽  
Troponin T ◽  
Troponin C ◽  
Difference Spectra ◽  
Low Concentrations ◽  
Tryptophan Residues ◽  
The Stability ◽  
Circular Dichroïsm

The detergent cetyltrimethylammonium bromide (CTAB) was used as a perturbant to study protein structure. Low concentrations of CTAB induced difference spectra for Ac-Trp-OEt and Ac-Tyr-OEt. The ΔεM values at their difference maxima were found to be 1300 at 292 nm for Ac-Tyr-OEt and 400 at 287 for Ac-Tyr-OEt. These values were used to determine the number of tyrosine residues exposed in tropomyosin and troponin C, as well as the tyrosine and tryptophan residues exposed in troponin I and troponin T. In tropomyosin and troponin C all of the tyorosine residues were accessible to detergent. For TN-T, three of four tyrosines were free while the tryptophan residues were only partially exposed. In the case of TN-I both tyrosines were fully exposed but again evidence was obtained for a partially buried tryptophan chromophore. The stability of these proteins to CTAB was studied by measuring the far-uv circular dichroism spectra. Tropomyosin was quite sensitive to detergent and suffered a 60% loss in ellipticity at the concentration of CTAB used. The troponins, on the other hand, were affected to a lesser extent.

scholarly journals Free Radicals and ROS Induce Protein Denaturation by UV Photostability Assay

2021 ◽  
Vol 22 (12) ◽  
pp. 6512
Author(s):  
Paolo Ruzza ◽  
Claudia Honisch ◽  
Rohanah Hussain ◽  
Giuliano Siligardi
Keyword(s):  
Free Radicals ◽  
Circular Dichroism ◽  
Uv Irradiation ◽  
Protein Denaturation ◽  
Local Heating ◽  
Protein Solutions ◽  
Protein Formulations ◽  
Fluorogenic Probe ◽  
Photo Stability ◽  
Circular Dichroïsm

Oxidative stress, photo-oxidation, and photosensitizers are activated by UV irradiation and are affecting the photo-stability of proteins. Understanding the mechanisms that govern protein photo-stability is essential for its control enabling enhancement or reduction. Currently, two major mechanisms for protein denaturation induced by UV irradiation are available: one generated by the local heating of water molecules bound to the proteins and the other by the formation of reactive free radicals. To discriminate which is the likely or dominant mechanism we have studied the effects of thermal and UV denaturation of aqueous protein solutions with and without DHR-123 as fluorogenic probe using circular dichroism (CD), synchrotron radiation circular dichroism (SRCD), and fluorescence spectroscopies. The results indicated that the mechanism of protein denaturation induced by VUV and far-UV irradiation were mediated by the formation of reactive free radicals (FR) and reactive oxygen species (ROS). The development at Diamond B23 beamline for SRCD of a novel protein UV photo-stability assay based on consecutive repeated CD measurements in the far-UV (180–250 nm) region has been successfully used to assess and characterize the photo-stability of protein formulations and ligand binding interactions, in particular for ligand molecules devoid of significant UV absorption.

scholarly journals Circular dichroism and its uses in biomolecular research - A Review

2021 ◽  
Vol 309 ◽  
pp. 01229
Author(s):  
Sangeeta ◽  
Anu Radha Pathania
Keyword(s):  
Circular Dichroism ◽  
Protein Structure ◽  
Structural Changes ◽  
Recombinant Dna ◽  
Protein Denaturation ◽  
Polarized Light ◽  
Order Structure ◽  
Recombinant Dna Technology ◽  
Ligand Interaction ◽  
Circular Dichroïsm

The higher-order structure of proteins as well as their thermal stability can be determined using the circular dichroism (CD). CD is a common approach for swiftly assessing binding, secondary structure, and folding properties of proteins. In a nutshell, circular dichroism is an absorption spectroscopy technique that employs circularly polarized light to explore structural properties of optically active chiral compounds. Biological molecules, as well as their interactions with metals and other compounds, are studied extensively. Circular dichroism is becoming more widely acknowledged as a useful technique for studying the various conformations taken by proteins and nucleic acids in solution. Because CD is a quantitative approach, it can be used to track protein denaturation and protein-ligand interaction. These CD measures will have two key advantages: they can be performed on small amounts of material in a physiological buffer, and they will provide one of the greatest methods for monitoring any structural changes that occur as a result of changes in environmental conditions. It has proven possible to generate proteins on a big scale for therapeutic reasons utilizing recombinant DNA technology. Circular dichroism is also well-known as a useful method which is used for determining the folding characteristics of proteins. CD is used to see if a purified, produced peptide is either bended or if it has a mutation that impacts its strength and confirmation. The basic steps in getting this CD data, as well as the methodologies for interpreting the spectra in order to predict the protein structure, are summarized in this article. However, many researchers’ value is harmed when they use circular dichroism, either because of poor experimental design or because of insufficient data. The essential steps in getting this CD data, as well as the methodologies for interpreting the spectra in order to predict the protein structure, will be summarized in this article. However, the value of many investigations using circular dichroism is harmed due to insufficient attention to critical components of instrument calibration or sample characterization.

scholarly journals Effect of imidazolium room-temperature ionic liquids on aggregation of amphotericin B: a circular dichroism study

RSC Advances ◽  
2015 ◽  
Vol 5 (98) ◽  
pp. 80325-80329 ◽  
Author(s):  
Laramie P. Jameson ◽  
Sergei V. Dzyuba
Keyword(s):  
Ionic Liquid ◽  
Ionic Liquids ◽  
Circular Dichroism ◽  
Amphotericin B ◽  
Room Temperature ◽  
Room Temperature Ionic Liquid ◽  
Room Temperature Ionic Liquids ◽  
Circular Dichroïsm

The aggregate–monomer equilibrium of amphotericin B could be controlled by the structure of the room-temperature ionic liquid.

Aggregation of a symmetrical metalloporphyrin. Concentration and temperature dependence of the absorption and magnetic circular dichroism spectra of dilute zinc octamethyltetrabenzporphyrin solutions

1981 ◽  
Vol 59 (9) ◽  
pp. 1388-1394 ◽  
Author(s):  
Kenneth J. Reimer ◽  
Monica M. Reimer ◽  
Martin J. Stillman
Keyword(s):  
Circular Dichroism ◽  
Temperature Dependence ◽  
Magnetic Circular Dichroism ◽  
Soret Band ◽  
Blue Shift ◽  
Spectral Changes ◽  
Low Concentrations ◽  
Association Species ◽  
Self Association ◽  
Circular Dichroïsm

Absorption and magnetic circular dichroism (mcd) spectra of toluene solutions of zinc octamethyltetrabenzporphyrin, Zn(OMBP), exhibit marked concentration and temperature dependence. Monomeric Zn(OMBP), 1, characterized by a 429 nm Soret band, dominates at low concentrations (10−7 M, 300 K) and higher temperatures (10−5M, 320 K) in toluene solution. Decreasing temperatures (320 to 235 K for a 10−5 M solution) and increasing concentrations (10−7 M to 10−4M, 300 K) result in 8–10 nm red shifts in this band. These spectral changes are attributed to the concurrent formation of a species 2 with a low extinction coefficient and another compound, 3, with λmax at 439 nm. At the highest concentrations (10−3 M) and lowest temperatures (225–190K) used, a dramatic blue shift (439 to 422 nm) due to a fourth species (4) is observed. All the spectral changes observed are completely reversible. In addition, and consistent with self-association, species 2, 3, and 4 are formed at higher temperatures as the concentration is increased. Possible aggregate structures are proposed and evidence is presented for a novel Zn—N (pyrrole) intermolecular bond.

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