scholarly journals Effect of Temperature on Oxymyoglobin and Metmyoglobin Denaturation Properties

2019 ◽  
Vol 3 (2) ◽  
Author(s):  
T. S. Belem ◽  
B. Chaudhary ◽  
S. Mohanty ◽  
G. Mafi ◽  
D. VanOverbeke ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Circular Dichroism ◽  
Foodborne Pathogens ◽  
Protein Unfolding ◽  
Circular Dichroism Spectroscopy ◽  
Color Appearance ◽  
Effect Of Temperature ◽  
Significance Level ◽  
Circular Dichroïsm ◽  
Cooked Color

ObjectivesPremature browning is a condition where the interior of patty/steak will appears fully cooked before the temperature necessary to kill foodborne pathogens is reached. Previous research reported that approximately 50% of ground beef retailed in the US is susceptible to premature browning. Myoglobin form present in the interior of steak or patties determines the cooked color appearance. Although previous studies noted that myoglobin denaturation is primarily responsible for the cooked color appearance, limited knowledge is currently available about the effect of temperature on oxymyoglobin and metmyoglobin denaturation properties. The objective of the current study was to determine the effects of myoglobin forms on thermal stability using circular dichroism spectroscopy.Materials and MethodsOxymyoglobin and metmyoglobin solutions at pH 5.6 in 50 mM sodium phosphate buffer were incubated in a continuous heat increment water bath for 10 min. At specific temperature points (65, 71, 73, and 76°C), myoglobin denaturation was determined by changes in myoglobin concentration and by protein unfolding (fluorescence and absorbance) methods. The myoglobin thermal stability was also determined by circular dichroism spectroscopy. Changes in secondary protein structure were determined every 2°C from 52 to 92°C. The data were analyzed as completely randomized using the Mixed Procedure of SAS. A significance level of 0.05 was used to determine differences between means.ResultsOxymyoglobin had greater (p < 0.05) unfolding (as indicated by absorbance changes) than metmyoglobin at all temperatures. However, at 65, 71, and 73°C there were no differences (p > 0.05) in fluorescence intensities between myoglobin forms. Circular dichroism spectroscopy indicates that oxymyoglobin is more heat labile than metmyoglobin.ConclusionThe results indicate that oxymyoglobin had greater denaturation and unfolding than metmyoglobin. Use of appropriate myoglobin denaturation quantification technique will help characterize premature browning.

scholarly journals Thermal Stability of Heterotrimeric pMHC Proteins as Determined by Circular Dichroism Spectroscopy

BIO-PROTOCOL ◽  
2017 ◽  
Vol 7 (13) ◽  
Author(s):  
Anna Fuller ◽  
Aaron Wall ◽  
Michael Crowther ◽  
Angharad Lloyd ◽  
Alexei Zhurov ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Circular Dichroism ◽  
Circular Dichroism Spectroscopy ◽  
Circular Dichroïsm ◽  
Thermal Stability Of

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.

scholarly journals Conformation Transition Kinetics of Silk Fibroin in Aqueous Solution Explored Using Circular Dichroism Spectroscopy

2021 ◽  
Vol 6 (8) ◽  
pp. 1735-1740
Author(s):  
Sora Lee ◽  
Soo Hyun Kim ◽  
You‐Young Jo ◽  
Wan‐Taek Ju ◽  
Hyun‐Bok Kim ◽  
...  
Keyword(s):  
Aqueous Solution ◽  
Circular Dichroism ◽  
Silk Fibroin ◽  
Circular Dichroism Spectroscopy ◽  
Kinetics Of ◽  
Circular Dichroïsm ◽  
Conformation Transition

Enantiopure methoxetamine stereoisomers: chiral resolution, conformational analysis, UV-circular dichroism spectroscopy and electronic circular dichroism

2021 ◽  
Author(s):  
Kun Won Lee ◽  
Ahmed H. E. Hassan ◽  
Youngdo Jeong ◽  
Seolmin Yoon ◽  
Seung-Hwan Kim ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Conformational Analysis ◽  
Absolute Configuration ◽  
Circular Dichroism Spectroscopy ◽  
Chiral Resolution ◽  
Treatment Resistant Depression ◽  
Treatment Resistant ◽  
Electronic Circular Dichroism ◽  
Resistant Depression ◽  
Circular Dichroïsm

Enantioseparation and assignment of absolute configuration of methoxetamine (MXE) enantiopure stereoisomers; a promising novel antidepressant for management of treatment-resistant depression.

scholarly journals Circular dichroism spectroscopy of membrane proteins

2016 ◽  
Vol 45 (18) ◽  
pp. 4859-4872 ◽  
Author(s):  
A. J. Miles ◽  
B. A. Wallace
Keyword(s):  
Circular Dichroism ◽  
Membrane Proteins ◽  
Circular Dichroism Spectroscopy ◽  
Circular Dichroism Spectra ◽  
Helical Bundle ◽  
Beta Barrel ◽  
Circular Dichroïsm

Circular dichroism spectra of helical bundle (red), beta barrel (blue), and mixed helical/sheet/unordered (green) membrane proteins.

scholarly journals Detection of Protein-Protein Interactions in the Alkanesulfonate Monooxygenase System from Escherichia coli

2006 ◽  
Vol 188 (23) ◽  
pp. 8153-8159 ◽  
Author(s):  
Kholis Abdurachim ◽  
Holly R. Ellis
Keyword(s):  
Circular Dichroism ◽  
Protein Interactions ◽  
Visible Region ◽  
Circular Dichroism Spectroscopy ◽  
Flavin Mononucleotide ◽  
Stable Complex ◽  
Monooxygenase System ◽  
Protein Protein Interactions ◽  
Alkanesulfonate Monooxygenase ◽  
Circular Dichroïsm

ABSTRACT The two-component alkanesulfonate monooxygenase system utilizes reduced flavin as a substrate to catalyze a unique desulfonation reaction during times of sulfur starvation. The importance of protein-protein interactions in the mechanism of flavin transfer was analyzed in these studies. The results from affinity chromatography and cross-linking experiments support the formation of a stable complex between the flavin mononucleotide (FMN) reductase (SsuE) and monooxygenase (SsuD). Interactions between the two proteins do not lead to overall conformational changes in protein structure, as indicated by the results from circular dichroism spectroscopy in the far-UV region. However, subtle changes in the flavin environment of FMN-bound SsuE that occur in the presence of SsuD were identified by circular dichroism spectroscopy in the visible region. These data are supported by the results from fluorescent spectroscopy experiments, where a dissociation constant of 0.0022 ± 0.0010 μM was obtained for the binding of SsuE to SsuD. Based on these studies, the stoichiometry for protein-protein interactions is proposed to involve a 1:1 monomeric association of SsuE with SsuD.

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