Use of optical spectroscopic methods to study the thermodynamic stability of proteins

2000 ◽  
Author(s):  
Maurice R. Eftink ◽  
Haripada Maity
Keyword(s):  
Mole Fraction ◽  
Protein Unfolding ◽  
Weighted Average ◽  
Optical Methods ◽  
Spectroscopic Methods ◽  
Scanning Calorimetry ◽  
Biophysical Characterization ◽  
Intrinsic Signal ◽  
The Stability ◽  
Mutant Forms

The biophysical characterization of globular proteins will almost always include some type of study of the unfolding of protein to obtain thermodynamic parameters. The basic idea is that a transition between a native and unfolded state, induced by temperature, pH, or denaturant concentration, can serve as a standard reaction for obtaining a thermodynamic measure of the stability of the native state. For example, the free energy change for the unfolding reaction can be used to compare the stability of a set of mutant forms of a protein (1-4). This type of analysis is based both on assumptions of the thermodynamic model for the unfolding process and on assumptions in the way the data are analysed; some of these assumptions and their limitations will be discussed below. There are a variety of methods that can be used to monitor an unfolding process. A common method is differential scanning calorimetry, DSC, which measures the variation in the specific heat of a protein-containing solution as a protein is thermally unfolded (5-7). DSC is a popular method for this purpose, but optical methods can also provide suitable information for tracking the unfolding of a protein The spectroscopic signals for the native and unfolded states of a protein can give some insight regarding the structure of the states, and often can provide advantages of economy, ease of measurement and amenability to a wide range of sample concentration. The optical spectroscopic methods that have been used most often for this purpose are absorption spectroscopy, circular dichroism and fluorescence, which will be discussed in this chapter. A key to each of these methods and their use in protein unfolding studies is that the signal is a mole fraction weighted average of the signals of each thermodynamic state. That is, the observed signal, S, can be expressed as . . . S = ∑XiSi . . . . . . 1 . . . where Xi is the mole fraction of species i and si is the intrinsic signal of species i. In order for a particular spectroscopic signal to be useful for tracking a N ↔ U transition of a protein, the signal must be sufficiently different for the N and U states.

scholarly journals A DSC study of zinc binding to bovine serum albumin (BSA)

2007 ◽  
Vol 72 (4) ◽  
pp. 331-337 ◽  
Author(s):  
Sanja Ostojic ◽  
Vida Dragutinovic ◽  
Miodrag Kicanovic ◽  
Branislav Simonovic
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Thermal Denaturation ◽  
Protein Unfolding ◽  
Protein Denaturation ◽  
Binding Constants ◽  
Zinc Binding ◽  
Scanning Calorimetry ◽  
The Stability

The thermal denaturation of bovine serum albumin (BSA) is a kinetically and thermodynamically controlled process. The effects of zinc binding to bovine serum albumin (BSA), followed by differential scanning calorimetry (DSC), were investigated in this work, with the purpose of obtaining a better understanding of the albumin/zinc interaction. From the DSC curves, the thermodynamic parameters of protein denaturation were obtained, i.e., the temperature of thermal transition maximum (T m), calorimetric enthalpy (?Hcal), van't Hoff enthalpy (?HvH), the number of binding sites (I, II), the binding constants for each binding site (K bI, K bII) and the average number of ligands bound per mole of native protein X N. The thermodynamic data of protein unfolding showed that zinc binding to bovine serum albumin increases the stability of the protein (higher values of ?Hcal) and the different ratio ?Hcal/?HvH indicates the perturbation of the protein during thermal denaturation.

scholarly journals High-Throughput Screening of Formulations to Optimize the Thermal Stability of a Therapeutic Monoclonal Antibody

2014 ◽  
Vol 20 (4) ◽  
pp. 552-559 ◽  
Author(s):  
Anita Niedziela-Majka ◽  
Elaine Kan ◽  
Perry Weissburg ◽  
Upasana Mehra ◽  
Scott Sellers ◽  
...  
Keyword(s):  
Thermal Stability ◽  
High Throughput Screening ◽  
Protein Unfolding ◽  
Scanning Calorimetry ◽  
Differential Scanning Fluorimetry ◽  
Environmentally Sensitive ◽  
The Stability ◽  
The Many ◽  
Thermal Stability Of ◽  
Good Agreement

Monoclonal antibodies (mAbs) are an important class of biotherapeutics. Successful development of a mAb depends not only on its biological activity but also on its physicochemical properties, such as homogeneity and stability. mAb stability is affected by its formulation. Among the many techniques used to study the stability of mAbs, differential scanning fluorimetry (DSF) offers both excellent throughput and minimal material consumption. DSF measures the temperature of the protein unfolding transition (Tm) based on the change in fluorescence intensity of the environmentally sensitive dye SYPRO Orange. With DSF adapted to a 96-well plate format, we have shown that low-pH or high-salt concentrations decrease the thermal stability of mAb1, whereas some excipients, such as sucrose, polysorbate 80, and sodium phosphate, increase its stability. The basal fluorescence of SYPRO Orange was enhanced by the presence of detergents, limiting the use of this approach to diluted detergent solutions. Throughput of DSF can be increased further with the use of a 384-well plate. DSF thermograms are in good agreement with the melting profiles obtained by differential scanning calorimetry (DSC). The Tms determined by DSF and DSC were well correlated, with the former being on average lower by 3 °C.

scholarly journals On the optimal control of coronavirus (2019-nCov) mathematical model; a numerical approach

2020 ◽  
Vol 2020 (1) ◽  
Author(s):  
N. H. Sweilam ◽  
S. M. Al-Mekhlafi ◽  
A. O. Albalawi ◽  
D. Baleanu
Keyword(s):  
Mathematical Model ◽  
Optimal Control ◽  
Weighted Average ◽  
Necessary Optimality Conditions ◽  
Numerical Approach ◽  
Population Number ◽  
Infected People ◽  
The Stability ◽  
Novel Coronavirus ◽  
Theoretical Results

Abstract In this paper, a novel coronavirus (2019-nCov) mathematical model with modified parameters is presented. This model consists of six nonlinear fractional order differential equations. Optimal control of the suggested model is the main objective of this work. Two control variables are presented in this model to minimize the population number of infected and asymptotically infected people. Necessary optimality conditions are derived. The Grünwald–Letnikov nonstandard weighted average finite difference method is constructed for simulating the proposed optimal control system. The stability of the proposed method is proved. In order to validate the theoretical results, numerical simulations and comparative studies are given.

scholarly journals Investigation of the Stability of Yeast rad52 Mutant Proteins Uncovers Post-translational and Transcriptional Regulation of Rad52p

Genetics ◽  
2003 ◽  
Vol 163 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Erin N Asleson ◽  
Dennis M Livingston
Keyword(s):  
Half Life ◽  
Translational Regulation ◽  
Cellular Level ◽  
Missense Mutations ◽  
Wild Type ◽  
Mutant Proteins ◽  
Gal1 Promoter ◽  
Rad52 Protein ◽  
The Stability ◽  
Mutant Forms

Abstract We investigated the stability of the Saccharomyces cerevisiae Rad52 protein to learn how a cell controls its quantity and longevity. We measured the cellular levels of wild-type and mutant forms of Rad52p when expressed from the RAD52 promoter and the half-lives of the various forms of Rad52p when expressed from the GAL1 promoter. The wild-type protein has a half-life of 15 min. rad52 mutations variably affect the cellular levels of the protein products, and these levels correlate with the measured half-lives. While missense mutations in the N terminus of the protein drastically reduce the cellular levels of the mutant proteins, two mutations—one a deletion of amino acids 210-327 and the other a missense mutation of residue 235—increase the cellular level and half-life more than twofold. These results suggest that Rad52p is subject to post-translational regulation. Proteasomal mutations have no effect on Rad52p half-life but increase the amount of RAD52 message. In contrast to Rad52p, the half-life of Rad51p is >2 hr, and RAD51 expression is unaffected by proteasomal mutations. These differences between Rad52p and Rad51p suggest differential regulation of two proteins that interact in recombinational repair.

scholarly journals Integration of Stable Ionic Liquid-Based Nanofluids into Polymer Membranes. Part I: Membrane Synthesis and Characterization

Nanomaterials ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 607
Author(s):  
Carolina Hermida-Merino ◽  
Fernando Pardo ◽  
Gabriel Zarca ◽  
João M. M. Araújo ◽  
Ane Urtiaga ◽  
...  
Keyword(s):  
Polymer Membranes ◽  
Small Addition ◽  
Polymeric Membranes ◽  
Synthesis And Characterization ◽  
Complete Disappearance ◽  
Optical Profilometry ◽  
Scanning Calorimetry ◽  
Exfoliated Graphene ◽  
The Stability ◽  
Scanning Electron

In this work, polymeric membranes functionalized with ionic liquids (ILs) and exfoliated graphene nanoplatelets (xGnP) were developed and characterized. These membranes based on graphene ionanofluids (IoNFs) are promising materials for gas separation. The stability of the selected IoNFs in the polymer membranes was determined by thermogravimetric analysis (TGA). The morphology of membranes was characterized using scanning electron microscope (SEM) and interferometric optical profilometry (WLOP). SEM results evidence that upon the small addition of xGnP into the IL-dominated environment, the interaction between IL and xGnP facilitates the migration of xGnP to the surface, while suppressing the interaction between IL and Pebax®1657. Fourier transform infrared spectroscopy (FTIR) was also used to determine the polymer–IoNF interactions and the distribution of the IL in the polymer matrix. Finally, the thermodynamic properties and phase transitions (polymer–IoNF) of these functionalized membranes were studied using differential scanning calorimetry (DSC). This analysis showed a gradual decrease in the melting point of the polyamide (PA6) blocks with a decrease in the corresponding melting enthalpy and a complete disappearance of the crystallinity of the polyether (PEO) phase with increasing IL content. This evidences the high compatibility and good mixing of the polymer and the IoNF.

scholarly journals Thermal Properties and Non-Isothermal Crystallization Kinetics of Poly (δ-Valerolactone) and Poly (δ-Valerolactone)/Titanium Dioxide Nanocomposites

Crystals ◽  
2018 ◽  
Vol 8 (12) ◽  
pp. 452 ◽  
Author(s):  
Waseem Saeed ◽  
Abdel-Basit Al-Odayni ◽  
Abdulaziz Alghamdi ◽  
Ali Alrahlah ◽  
Taieb Aouak
Keyword(s):  
Titanium Dioxide ◽  
Crystallization Kinetics ◽  
Isothermal Crystallization ◽  
Three Dimensional ◽  
Degree Of Crystallinity ◽  
Simultaneous Occurrence ◽  
Scanning Calorimetry ◽  
Isothermal Crystallization Kinetics ◽  
The Stability ◽  
Kinetics Of

New poly (δ-valerolactone)/titanium dioxide (PDVL/TiO2) nanocomposites with different TiO2 nanoparticle loadings were prepared by the solvent-casting method and characterized by Fourier transform infra-red, differential scanning calorimetry, X-ray diffraction and scanning electron microscopy, and thermogravimetry analyses. The results obtained reveal good dispersion of TiO2 nanoparticles in the polymer matrix and non-formation of new crystalline structures indicating the stability of the crystallinity of TiO2 in the composite. A significant increase in the degree of crystallinity was observed with increasing TiO2 content. The non-isothermal crystallization kinetics of the PDVL/TiO2 system indicate that the crystallization process involves the simultaneous occurrence of two- and three-dimensional spherulitic growths. The thermal degradation analysis of this nanocomposite reveals a significant improvement in the thermal stability with increasing TiO2 loading.

scholarly journals Stability of Wheat Genotypes by AMMI and Yield Evaluated under Peninsular Zone of India

2021 ◽  
Author(s):  
Ajay Verma ◽  
G.P. Singh
Keyword(s):  
Weighted Average ◽  
Principal Component ◽  
Efficient Estimation ◽  
High Yield ◽  
First Year ◽  
Wheat Genotypes ◽  
Stability Measure ◽  
Linear Behaviour ◽  
The Stability ◽  
Selection Of

Background: Efficient estimation of main and interaction effects under multi environment trials had been carried out by AMMI. Studies with low proportion of the variance explained by first interaction principal component IPCA1 under AMMI analysis had been reported biased interpretation for the stability of the genotypes.Methods: Weighted Average of Absolute scores (WAASB), quantitative stability measure had been recommended for selection of productive genotypes with broad adaptation. The selection of promising genotypes had been assisted by use of superiority index (WAASBY) that considered simultaneous use of yield and stability by allowing variable weighting mechanism for yield and stability.Result: Wheat GW509 and HI1633 genotypes had been identified by stability measure WAASB for the first year. While Superiority index considered stability and high yield settled for HI1633 and Raj4083 genotypes. Analytic measures of adaptability PRVG and MHPRVG observed suitability of HI1633 and Raj 4083. SI expressed positive linear behaviour with yield, MHPRVG and PRVG measures. While stability measure WAASB maintained moderate negative correlation with yield, SI, MHPRVG and PRVG. Wheat genotypes HI1633, HI1641 and HI1646 identified by stability measure for the next year of study. Superiority index selected HI1641, HI1633 and MACS6752 genotypes. SI expressed direct linear relation with yield, MHPRVG and PRVG. Stability measure exhibited indirect relationships with SI, MHPRVG, PRVG and yield Wheat genotypes HI1633, Raj 4083 for first year and HI1641, MACS6752 for the second year of study.

Biochemical and Biophysical characterization of the main protease, 3-chymotrypsin-like protease (3CLpro), from the novel coronavirus disease 19 (COVID-19)

2020 ◽  
Author(s):  
Juliana C. Ferreira ◽  
Wael M. Rabeh
Keyword(s):  
Antiviral Drug ◽  
Analytical Techniques ◽  
The Novel ◽  
Buffer Solution ◽  
Biophysical Characterization ◽  
Main Protease ◽  
Wide Ph Range ◽  
The Stability ◽  
Ph Range ◽  
Novel Coronavirus

Abstract Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is responsible for the novel coronavirus disease 2019 (COVID-19). An appealing antiviral drug target is the coronavirus 3C-like protease (3CLpro) that is responsible for the processing of the viral polyproteins and liberation of functional proteins essential for the maturation and infectivity of the virus. In this study, multiple thermal analytical techniques have been implemented to acquire the thermodynamic parameters of 3CLpro at different buffer conditions. 3CLpro exhibited relatively high thermodynamic stabilities over a wide pH range; however, the protease was found to be less stable in the presence of salts. Divalent metal cations reduced the thermodynamic stability of 3CLpro more than monovalent cations; however, altering the ionic strength of the buffer solution did not alter the stability of 3CLpro. Furthermore, the most stable thermal kinetic stability of 3CLpro was recorded at pH 7.5, with the highest enthalpy of activation calculated from the slope of Eyring plot. The biochemical and biophysical properties of 3CLpro explored here will improve the solubility and stability of 3CLpro for optimum conditions for the setup of an enzymatic assay for the screening of inhibitors to be used as lead candidates in the drug discovery and antiviral design for therapeutics against COVID-19.

scholarly journals Study on the Structure and Dielectric Properties of Zeolite/LDPE Nanocomposite under Thermal Aging

Polymers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2108
Author(s):  
Bai Han ◽  
Chuqi Yin ◽  
Jiaxin Chang ◽  
Yu Pang ◽  
Penghao Lv ◽  
...  
Keyword(s):  
Dielectric Properties ◽  
Aging Time ◽  
Thermal Aging ◽  
Aging Treatment ◽  
Dielectric Strength ◽  
Breakdown Field ◽  
Scanning Calorimetry ◽  
Aging Resistance ◽  
The Stability ◽  
Breakdown Field Strength

Nanodoping is an effective way to improve the dielectric properties and the aging resistance of polyethylene. Nano-zeolite has a nano-level porous structure and larger specific surface area than ordinary nano-inorganic oxide, which can be used to improve dielectric properties of low-density polyethylene (LDPE) nanocomposite. The zeolite/LDPE nanocomposites were prepared and subjected to thermal aging treatment to obtain samples with different aging time. Using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and the differential scanning calorimetry (DSC) test to study the microscopic and structure characteristics, it was found that nano-zeolite doping can effectively reduce the thermal aging damage to the internal structure of the nanocomposite; carbonyl and hydroxyl decreased significantly during the thermal aging time, and the crystallinity effectively improved. Nano-zeolite doping significantly improved the morphology and strengthened the aging resistance of the nanocomposite. In the dielectric strength test, it was found that nanodoping can effectively improve the direct current (DC) and alternating current (AC) breakdown field strength and the stability after the thermal aging. The dielectric constant of nanocomposite can be reduced, and the dielectric loss had no obvious change during the aging process. Moreover, the zeolite/LDPE nanocomposite with the doping concentration of 1 wt % had the best performance, for the nano-zeolite was better dispersed.

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