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

Comparative thermostability of mesophilic and thermophilic alcohol dehydrogenases: Stability-determining roles of proline residues and loop conformations

The present study demonstrates the comparative thermal, conformational and kinetic stabilities of the three closely related enzymes; the mesophilic yeast alcohol dehydrogenase (YADH), horse liver alcohol dehydrogenase (HLADH), and the extreme-thermophilic Thermoanaerobacter brockii alcohol clehydrogenase (TBADH). The mid-point unfolding temperatures for TBADH and HLADH were at least 10 degrees C and 6 degrees C higher, respectively, than that of YADH. When YADH was completely inactivated by thermal stress, the residual activities of HLADH and TBADH were 70% and 100%, respectively. The optimum temperature (T-opt) activities of HLADH and TBADH were at least 40 degrees C and 55 degrees C higher, respectively, than that of YADH. Due to the higher rigidity of HLADH and TBADH, the enzymatic activation energies of HLADH and TBADH were higher than that of YADH. Geometric X-ray analysis indicated a comparatively higher coil (turn and loop) percentage in TBADH and HLADH than in YADH. Pairwise alignment for TBADH/HLADH exhibited a similarity score approximately 2.5-fold greater than that of the TBADH/YADH pair. Multiple alignments made with ClustalW revealed a higher number of conserved proline residues in the two most stable enzymes (HLADH/TBADH). These extra prolines tend to occur in surface loops and are likely to be responsible for the increased stability of TBADH and HLADH, by loop rigidification

The mechanism of antioxidant activity of IRFI005 as a synthetic hydrophilic analogue of vitamin E.

Developing a rational strategy to control intracellular reactive oxygen species (ROS) requires understanding the mechanism of antioxidant activity. In this investigation the properties of a novel synthetic analog of vitamin E (IRFI005) with potent antioxidant activity are described. A mechanism is proposed for its efficient radical-scavenging effects. Cellular antioxidant and antitoxicity assays showed IRFI005 to freely permeate across cellular membranes, enabling it to be an effective suppressor of intracellular ROS and to protect cells against toxicity induced by free radical generating compounds. The free radical-scavenging activity of IRFI005 examined by UVeVis and electron spin resonance (ESR) techniques clearly confirmed a “two electrons and/or H-atom” donation mechanism for each molecule of IRFI005. Reducing power assay as well as semi-empirical calculations revealed that under physiological conditions (pH 7) almost all IRFI005 molecules are in the anionic state (IRFI005-). Data indicated that the electron donating ability of IRFI005- was dominant at physiological pH because of higher stability of quinine-IRFI005- and less barrier energy of IRFI005- than neutral IRFI005. Consequently, the efficient cellular protection of IRFI005 against toxic free radicals can be explained by a two electron-transfer process, because of reduced inter-frontier molecular orbital energy gap barrier at physiological pH. Our findings suggest that hydrophilic vitamin E-like antioxidants are good candidates in designing novel therapeutic strategies for inhibition of oxidative stress associated with different human diseases

Theoretical and experimental studies on the structure-antioxidant activity relationship of synthetic 4-methylcoumarins

The development of antioxidants as useful drugs for the treatment of neurodegenerative diseases such as Alzheimer's is extremely challenging in medicinal chemistry. Coumarins have attracted great attention as possible therapeutic tools against oxygen radicals in human degenerative diseases. In order to establish the possible structure-antioxidant activity relationship, a series of twenty four 4-methylcoumarin derivatives were examined by employing reducing power measurements, and comparison with bond dissociation enthalpy and ionization potential calculations. Based on the reducing potency of 4-methylcoumarin derivatives with respect to trolox, these compounds were classified into five groups as “most active”, “more active”, “moderately active”, “less active” and “inactive” derivatives. The presence of hydroxyl groups is an essential requirement for the activity, and substitution of hydroxyl groups by methoxy groups leads to non-active derivatives. The results revealed that dihydroxyl groups in the ortho position show a better antioxidant activity with respect to dihydroxyl groups in the meta position. This is ascribed to the ability to construct more stable 4-methylcoumarin radical intermediates by rearrangement of intra-molecular hydrogen bonding. Our findings indicate that other important factors to enhance the antioxidant activity of coumarins are the number of hydroxyl groups, the presence of ester substitutions and a thiono functional group on the pyrone ring. However, bond dissociation enthalpy and ionization potential calculations alone are not sufficient to identify the best antioxidant structures. As a result, chemical and functional properties of molecules such as 4-methylcoumarins should be examined as a whole entity, considering all substitutions versus a single substitution to design functional compounds with good antioxidant activity