39 research outputs found
Thermal inactivation and conformational lock studies on glucose oxidase
In this study, the dissociative thermal inactivation
and conformational lock theories are applied for the
homodimeric enzyme glucose oxidase (GOD) in order to
analyze its structure. For this purpose, the rate of activity
reduction of glucose oxidase is studied at various temperatures
using b-D-glucose as the substrate by incubation of
enzyme at various temperatures in the wide range between
40 and 70 �C using UV–Vis spectrophotometry. It was
observed that in the two ranges of temperatures, the
enzyme has two different forms. In relatively low temperatures,
the enzyme is in its dimeric state and has normal
activity. In high temperatures, the activity almost disappears
and it aggregates. The above achievements are confirmed
by dynamic light scattering. The experimental
parameter ‘‘n’’ as the obvious number of conformational
locks at the dimer interface of glucose oxidase is obtained
by kinetic data, and the value is near to two. To confirm the
above results, the X-ray crystallography structure of the
enzyme, GOD (pdb, 1gal), was also studied. The secondary
and tertiary structures of the enzyme to track the thermal
inactivation were studied by circular dichroism and
fluorescence spectroscopy, respectively. We proposed a
mechanism model for thermal inactivation of GOD based
on the absence of the monomeric form of the enzyme by
circular dichroism and fluorescence spectroscopy
A biophysical study on the mechanism of interactions of DOX or PTX with α-lactalbumin as a delivery carrier
© 2018, The Author(s). Doxorubicin and paclitaxel, two hydrophobic chemotherapeutic agents, are used in cancer therapies. Presence of hydrophobic patches and a flexible fold could probably make α-Lactalbumin a suitable carrier for hydrophobic drugs. In the present study, a variety of thermodynamic, spectroscopic, computational, and cellular techniques were applied to assess α-lactalbumin potential as a carrier for doxorubicin and paclitaxel. According to isothermal titration calorimetry data, the interaction between α-lactalbumin and doxorubicin or paclitaxel is spontaneous and the K (M−1) value for the interaction of α-lactalbumin and paclitaxel is higher than that for doxorubicin. Differential scanning calorimetry and anisotropy results indicated formation of α-lactalbumin complexes with doxorubicin or paclitaxel. Furthermore, molecular docking and dynamic studies revealed that TRPs are not involved in α-Lac’s interaction with Doxorubicin while TRP 60 interacts with paclitaxel. Based on Pace analysis to determine protein thermal stability, doxorubicin and paclitaxel induced higher and lower thermal stability in α-lactalbumin, respectively. Besides, fluorescence lifetime measurements reflected that the interaction between α-lactalbumin with doxorubicin or paclitaxel was of static nature. Therefore, the authors hypothesized that α-lactalbumin could serve as a carrier for doxorubicin and paclitaxel by reducing cytotoxicity and apoptosis which was demonstrated during our in vitro cell studies
Conformational lock and dissociative thermal inactivation of lentil seedling amine oxidase
Effect of covalent attachment of neomycin on conformational and aggregation properties of catalase
189-195<span style="font-size:9.0pt;mso-bidi-font-size:
12.0pt" lang="EN-US">The carboxylic groups of glutamic acid and aspartic acid residues of
catalase (CAT) were chemically modified using the treatment of the enzyme with
1-ethyl-3-(3'-dimethylamino) carbodiimide hydrochloride (EDC) and neomycin. The
effect of covalent attachment of neomycin on the enzymatic activity,
conformational and aggregation properties of CAT was investigated. The
modification of CAT with different concentrations of neomycin showed two
different types of behavior, depending up on the concentration range of
neomycin. In the concentration range from 0.0 to 5.2 mM, neomycin-modified CAT,
compared to the native enzyme exhibited higher α-helix content, reduced surface
hydrophobicity, little enhancement in CAT activity and a better protection
against thermal aggregation, whereas at concentrations greater than 5.2 mM, the
modified enzyme exhibited a significant decrease in CAT activity and an
increase in random coil content which may result in disorder in the protein
structure and increase in thermal aggregation. This modification is a rapid and
simple approach to investigate the role of aspartate and glutamate residues in
the structure, function and folding of CAT.
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Amine oxidase from lentil seedlings: Energetic domains and effect of temperature on activity
Copper/TPQ amine oxidases from mammalian and plant sources have shown many differences in substrate specificity and molecular properties. In this work the activity of lentil seedling amine oxidase was followed at various temperatures in 100 mM potassium phosphate buffer, pH 7, using benzylamine as substrate. The discontinuous Arrhenius plot of lentil amine oxidase showed two distinct phases with a jump between them. Thermal denaturation of the enzyme, using differential scanning calorimetry under the same experimental conditions, showed a transition at the same temperature ranges in the absence of substrate, indicating the occurrence of conformational changes, with an enthalpy change of about 175.9 kJ/mole. The temperature-induced changes of the activity of lentil amine oxidase are compared with those of bovine serum amine oxidase (taken from the literature)
Domain analysis of human apotransferrin upon interaction with sodium n-dodecyl sulphate: Differential scanning calorimetry and circular dichroism approaches
The domain analysis of human apotransferrin (apo hTF) upon interaction with sodium n-dodecyl sulphate (SDS) was studied by differential scanning calorimetry (DSC) and circular dichroism (CD) using Hepes buffer, 100 mM, at pH = 7. The DSC profile for apo hTF depicts two distinct peaks, while when interacted with SDS, the DSC peaks are shifted to the left as well as the area under the peaks are reduced. The DSC profile without the presence of SDS has two dissimilar peaks including two melting points (Tm = 60 °C, Tm = 70 °C). This profile in the presence of low concentrations of SDS shows two nearly similar peaks and decrement of Tm values about 5 °C relative to profile in the absence of SDS. The DSC excess heat capacity was deconvoluted for apo hTF in the presence of SDS. The interaction of SDS with apo hTF induced two dissimilar subdomains for each domain, but each domain having two subdomains similar to each other. The CD experiment of apo hTF-SDS complexes shows the decrement of α-helix content and the increment of β-sheet structure relative to protein in the absence of SDS. The α-helix tendency calculation shows more α-helix content for N domain relative to C domain. Here SDS at low concentration plays a role as a good probe to define the electrostatic moiety for N domain relative to C domain that is initiated from dissimilarity of α-helicity of two domains. © 2003 Elsevier B.V. All rights reserved
Investigation of thermal reversibility and stability of glycated human serum albumin
Protein glycation, the process by which carbohydrates attach to proteins upon covalent binding, can alter protein thermal reversibility and stability. Protein stability and reversibility have important role in protein behavior and function. Also they are benefit properties for drug produce and protein industrial applications. In this research the thermal reversibility and stability changes in human serum albumin (HSA) were studied upon incubation with glucose (GHSA) under physiological conditions for 21 and 35 days. The thermal reversibility and stability changes in GHSA were evaluated using circular dichroism (CD), UV-vis spectroscopy, fluorescence spectroscopy and differential scanning calorimetry (DSC). Our results showed that the glycation of HSA increased its thermal reversibility and stability, but decreased its conformational entropy compared to fresh native HSA and untreated HSA. Free lysine content assay (TNBSA test) indicated glucose can bind to protein covalently. These alterations were mainly attributed to the formation of crosslink between the lysine residues of HSA upon incubation with glucose. © 2013 Elsevier B.V
Thermal reversibility and disaggregation of human serum albumin upon incubation with 3-β hydroxybutyrate: A proposed mechanism for thiol reaction
Thermal inactivation of proteins is a main challenge in food technology and medicine. The 3-β-hydroxybutyrate (3BHB) is the most abundant ketone body with a carboxyl group. Its concentration increases during fasting, prolonged exercise, and in diabetic patients. In this work, human serum albumin (HSA) was incubated with 3BHB for 7, 14, 21, and 35 days under physiological conditions. The thermal reversibility and thermal aggregation of HSA upon incubation with 3BHB were determined by differential scanning calorimetry and free amine content assay. These results indicated that 3BHB binds the lysine residues of HSA through nucleophilic attack leading to formation of covalent bonds. The calorimetric results showed that the modification of lysine residues by 3BHB resulted in partial unfolding of HSA compared to the modification of thiol group of Cys34, which is surrounded by other amino acid side chains. Since thermal aggregation of HSA is related to Cys34, its modification caused a decrease in thermal aggregation and an increase in thermal reversibility of modified HSA. © 2014 Akadémiai Kiadó