Bioflavonoids ameliorate crowding induced hemoglobin aggregation: a spectroscopic and molecular docking approach.

The cellular environment is densely crowded, confining biomacromolecules including proteins to less available space. This macromolecular confinement may affect the physiological conformation of proteins in long-term processes like ageing. Changes in physiological protein structure can lead to protein conformational disorders including neurodegeneration. An intervention approach using food and plant derived bioflavonoids offered a way to find a treatment for these enervating pathological conditions as there is no remedy available. The bioflavonoids NAR (naringenin), 7HD (7 hydroxyflavanone) and CHR (chrysin) were tested for their ability to protect Hb (hemoglobin) against crowding-induced aggregation. Morphological and secondary structural transitions were studied using microscopic and circular dichroism experiments, respectively. The kinetic study was carried out using the relative thioflavin T assay. Molecular docking, AmylPred2, admetSAR and FRET were applied to understand the binding parameters of bioflavonoids with Hb and their drug likeliness. Isolated human lymphocytes were used as a cellular system to study the toxic effects of Hb aggregates. Redox perturbation and cytotoxicity were evaluated by DCFH-DA and MTT assays, respectively. This study suggests that bioflavonoids bind to Hb in the vicinity of aggregation prone amino acid sequences. Binding of the bioflavonoids stabilizes the Hb against crowding-induced structural alterations. Therefore, this study signifies the potential of bioflavonoids for future treatment of many proteopathies including neurodegeneration.Communicated by Ramaswamy H. Sarma.

Probing the interaction of zinc oxide nanorods with human serum albumin: A spectroscopic approach.

Bio-functionalized metal oxide nanoparticles (NPs) have been taken great importance in biomedical fields. The use of nanoparticles as delivery agents of therapeutic molecules led the researchers to emphasize the potential impact of these NPs on bio-macromolecules as protein-nanoparticle complexes, which also extended their importance as vehicles in targeted drug delivery systems due to increased ease of administration, firmness, reduced toxic side effects, and half-life of drugs. Since human serum albumin is the blood protein responsible for transporting materials in the blood system, the interaction of these particles with HSA is essential to be understood before considering the nanoparticles for any individual biomedical application. In the present study, we synthesized zinc-oxide nanorods (ZONRs) using a microwave-assisted synthesis technique, and characterized them by XRD, FTIR, Raman, SEM-EDX, UV-Vis spectroscopy, and photoluminescence (PL) spectroscopy methods. The interaction studies were carried out using fluorescence spectroscopy, and the change in secondary structure was analyzed using CD spectroscopy. The results of MTT cell viability assay demonstrated that the ZONRs has potential cytotoxic properties.

Connecting the Dots: Macromolecular Crowding and Protein Aggregation.

Proteins are one of the dynamic macromolecules that play a significant role in many physiologically important processes to sustain life on the earth. Proteins need to be properly folded into their active conformation to perform their function. Alteration in the protein folding process may lead to the formation of misfolded conformers. Accumulation of these misfolded conformers can result in the formation of protein aggregates which are attributed to many human pathological conditions including neurodegeneration, cataract, neuromuscular disorders, and diabetes. Living cells naturally have heterogeneous crowding environments with different concentrations of various biomolecules. Macromolecular crowding condition has been found to alter the protein conformation. Here in this review, we tried to show the relation between macromolecular crowding, protein aggregation, and its consequences.

Refolding of Hemoglobin Under Macromolecular Confinement: Impersonating In Vivo Volume Exclusion.

Biomacromolecules evolve and function inside the cell under crowded conditions. The effect of macromolecular crowding and confinement on nature and interactions of biomacromolecules cannot be ruled out. This study demonstrates the effect of volume exclusion due to macromolecular crowding on refolding rate of Gn-HCl induced unfolded hemoglobin. The in vivo like crowding milieu was created using dextran 70. Unfolding of Hb was followed by the absorbance at 280 nm and intrinsic fluorescence intensity along with a bathochromic shift that shows the destabilization of Hb in the presence of the denaturing agent. This was supported by a decrease in soret absorbance, increased hydrodynamic radii and loss in secondary structure, evidenced from dynamic light scattering and circular dichroism experiments respectively. Refolding process of Hb was followed by an increase in soret absorbance, decrease in intrinsic fluorescence intensity with a hypsochromic shift, decreased hydrodynamic radii and gain in secondary structural content. The results revealed that the effect of confinement and volume exclusion is insignificant on the process of Hb refolding.

Green synthesis of silver nanoparticles, its characterization, and chaperone-like activity in the aggregation inhibition of α-chymotrypsinogen A.

Consumption of silver nanoparticles (AgNPs) has been increased many folds due to its antimicrobial actions resulting in its widespread incorporation into a wide range of biomedical and consumer products. Still, enough research is needed to clearly understand the effect of these nanoparticles on the conformations of important macromolecules like proteins under different pathophysiological conditions. Pointing towards the situation, we carefully designed an in vitro study to elucidate the effect of green AgNPs on the aggregation pattern of α-chymotrypsinogen A at a human pathological body temperature. We observed that the B-AgNPs inhibited the aggregation in αCgn-A in a concentration-dependent manner showing maximum inhibition at 30 µg/ml above which the effect of aggregation inhibition was reduced as evident at 40 and 50 µg/ml concentrations of B-AgNPs. Further, in our in vitro analysis, we found that the B-AgNPs of lower sizes has potential chaperone-like activity at pathological body temperature, which can be used as a component of the drug to prevent protein aggregation after further verification in animal models.

Inhibition of advanced glycation end products by isoferulic acid and its free radical scavenging capacity: An in vitro and molecular docking study.

Non-enzymatic glycation and Oxidation of some essential biological macromolecules are paramount in the pathogenesis of various diseases including diabetes and atherosclerosis. Hyperglycemia plays a key role in the pathological process of diabetic complications by progressive accumulation of advanced glycation end products (AGEs) in body tissues. Formation of AGEs as a result of protein glycation is followed by an increased free radical activity that additionally contributes towards the bio-macromolecular damage. The present study aimed to evaluate the free radical scavenging and antiglycation capacity of isoferulic acid (IFA). The free radical scavenging activity of IFA was measured using DPPH, FRAP, and metal chelating assays. IFA showed effective reducing power, free radical scavenging activity and metal chelation activity in concentration dependent manner. The antiglycation activity of IFA was studied using various spectroscopic techniques. The obtained results were validated with free amino, sulfhydryl group, carbonyl content and AGEs formation. Secondary structural alterations were monitored using circular dichroism, morphology of aggregates was analyzed using transmission electron microscopy. Molecular docking reveals the possible binding location of IFA with in the sub-domain IIA of human serum albumin (HSA).

Probing the binding of phenolic aldehyde vanillin with bovine serum albumin: Evidence from spectroscopic and docking approach.

The interactions of bovine serum albumin (BSA) with vanillin (VAN) were studied using UV-vis absorption, fluorescence, synchronous fluorescence, three dimensional fluorescence spectroscopy (3D), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and molecular docking techniques. The results revealed that VAN causes the static quenching of BSA by forming BSA-VAN complex. The thermodynamic parameters obtained using isothermal titration calorimetry (ITC) showed that the interaction between BSA and VAN is spontaneous and hydrogen bonding, van der Waals forces are mainly involved in stabilizing the complex. The distance between the donor and the acceptor was analyzed using fluorescence resonance energy transfer (FRET) which showed Forster distance of 2.58 nm. Molecular docking technique was applied to study the modes of interaction between BSA-VAN system and it was found that VAN bound to the sub-domain IIA of BSA. Structural analysis using 3D, synchronous fluorescence FTIR, and CD showed that upon binding of VAN, BSA exhibits small micro-environmental changes around tryptophan amino acid residue.

Aggregation of globular protein as a consequences of macromolecular crowding: A time and concentration dependent study.

The living cells show profoundly crowded condition, called as macromolecular crowding. Crowding essentially impacts on protein structure and lead to its aggregation. Protein aggregates have been involved in a wide range of diseases including Parkinson, Alzheimer's, and Huntington's. Increased in normal physiological macromolecular crowding because of increasing age can be implicated as one of the leading cause of proteopathies. In the present study, we have demonstrated the effect of macromolecular crowding on native structure of hemoglobin using bovine serum albumin as a crowding agent. Conformational changes of Hb at different concentrations of BSA were monitored using intrinsic fluorescence and ATR-FTIR spectroscopy. These results showed the transition of native Hb to a non-native form. Thermodynamic parameters were analyzed by isothermal titration calorimetry. The measurements of turbidity, thioflavin T, congo red and intrinsic fluorescence revealed the formation of significant protein aggregates with time. The kinetics of protein aggregation using relative ThT and 8-anilino-1-naphthalenesulphonic acid spectra clearly showed acceleration of the process with time and in concentration dependent manner. The spectra at 80g/l of BSA incubated for 64h showed formation of maximum Hb aggregates. Transmission electron microscopy results clearly showed the formation of amyloid aggregates structures, thus supporting the spectroscopic data.