Investigating the binding interaction of quinoline yellow with bovine serum albumin and anti-amyloidogenic behavior of ferulic acid on QY-induced BSA fibrils.

Protein aggregation induces profound changes in the structure along with the conformation of the protein, and is responsible for the pathogenesis of a number of neurodegenerative conditions such as Huntington's, Creutzfeldt-Jacob, Type II diabetes mellitus, Alzheimer's, etc. Numerous multi-spectroscopic approaches and in-silico experiments were utilized to investigate BSA's biomolecular interaction and aggregation in the presence of quinoline yellow. The present research investigation evaluated the interaction of BSA with the food colorant (QY) at two different pH (7.4 and 2.0). The development of the BSA-QY complex was established with UV visible and fluorescence spectroscopy. The quenching of fluorescence upon the interaction of BSA with QY revealed the static nature of quenching mechanism. The Kb value obtained from our result is 4. 54 × 10-4 M-1. The results from the competitive site marker study infer that quinoline yellow is binding with the sub-domain IB of bovine serum albumin, specifically on site III. Three-dimensional fluorescence and synchronous fluorescence spectroscopy were applied for monitoring the alterations in the microenvironment of BSA upon the addition of quinoline yellow. The results from turbidity and RLS studies showed that higher concentrations of QY (80-400 µM) triggered bovine serum albumin (BSA) aggregation at pH 2.0. At pH 7.4, QY couldn't manage to trigger bovine serum albumin aggregation, perhaps because of the repulsion between negatively charged dye (QY) and anionic bovine serum albumin. The results from far-UV CD, Congo Red, and scanning electron microscopy implicate that the QY-induced aggregates exhibit amyloid fibril-like structures. Molecular docking results revealed that hydrophobic interactions, hydrogen bonding, and Pi-Sulfur interactions contribute to QY-induced aggregation of BSA. Further, the amyloid inhibitory potential of ferulic acid (FA), a phenolic acid on QY-induced aggregation of BSA, has also been assessed. The QY-induced amyloid fibrils are FA-soluble, as confirmed by turbidity, RLS, and far-UV CD studies. Far-UV CD results showed that FA retains α helix and inhibits cross ß sheet formation when the BSA samples were pre-incubated with increasing concentrations of FA (0-500 µM). Our findings conclude that QY dye successfully stimulates BSA aggregation, but ferulic acid inhibits QY-induced aggregation of BSA. Thus, FA can serve as a therapeutic agent and can help in the treatment of various amyloid-related conditions.

Methotrexate for Drug Repurposing as an Anti-Aggregatory Agent to Mercuric Treated α-Chymotrypsinogen-A.

Protein aggregation is related to numerous pathological conditions like Alzheimer's and Parkinson's disease. In our study, we have shown that an already existing FDA-approved drug; methotrexate (MTX) can be reprofiled on preformed α-chymotrypsinogen A (α-Cgn A) aggregates. The zymogen showed formation of aggregates upon interaction with mercuric ions, with increasing concentration of Hg2Cl2 (0-150 µM). The hike in ThT and ANS fluorescence concomitant with blue shift, bathochromic shift and the hyperchromic effect in the CR absorbance, RLS and turbidity measurements, substantiate the zymogen ß-rich aggregate formation. The secondary structural alterations of α- Cgn A as analyzed by CD measurements, FTIR and Raman spectra showed the transformation of native ß-barrel conformation to ß-inter-molecular rich aggregates. The native α- Cgn A have about 30% α-helical content which was found to be about 3% in presence of mercuric ions suggesting the formation of aggregates. The amorphous aggregates were visualized by SEM. On incubation of Hg2Cl2 treated α- Cgn A with increasing concentration of the MTX resulted in reversing aggregates to the native-like structure. These results were supported by remarkable decrease in ThT and ANS fluorescence intensities and CR absorbance and also consistent with CD, FTIR, and Raman spectroscopy data. MTX was found to increase the α-helical content of the zymogen from 3 to 15% proposing that drug is efficient in disrupting the ß-inter-molecular rich aggregates and reverting it to native like structure. The SEM images are in accordance with CD data showing the disintegration of aggregates. The most effective concentration of the drug was found to be 120 µM. Molecular docking analysis showed that MTX molecule was surrounded by the hydrophobic residues including Phe39, His40, Arg145, Tyr146, Thr151, Gly193, Ser195, and Gly216 and conventional hydrogen bonds, including Gln73 (bond length: 2.67Å), Gly142 (2.59Å), Thr144 (2.81Å), Asn150 (2.73Å), Asp153 (2.71Å), and Cys191 (2.53Å). This investigation will help to find the use of already existing drugs to cure protein misfolding-related abnormalities.

Protective role of chlorogenic acid in preserving cytochrome-c stability against HFIP-induced molten globule state at physiological pH.

Numerous neurodegenerative disorders are characterized by protein misfolding and aggregation. The mechanism of protein aggregation is intricate, and it is very challenging to study at cellular level. Inhibition of protein aggregation by interfering with its pathway is one of the ways to prevent neurodegenerative diseases. In the present work, we have evaluated the protective effect of a polyphenol compound chlorogenic acid (CGA) on the native and molten globule state of horse heart cytochrome c (cyt c). A molten globule state of this heme protein was achieved in the presence of fluorinated alcohol 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) at physiological pH, as studied by UV-Vis absorption, circular dichroism, intrinsic and ANS fluorescence. We found that at 50 % (v/v) HFIP, the native cyt c transformed into a molten globule state. The same techniques were also used to analyze the protective effect of CGA on the molten globule state of cyt c, and the results show that the CGA prevented the molten globular state and retained the protein close to the native state at 1:1 protein:CGA sub molar ratio. Molecular dynamics study also revealed that CGA retains the stability of cyt c in HFIP medium by preserving it in an intermediate state close to native conformation.

Bovine serum albumin prevents human hemoglobin aggregation and retains its chaperone-like activity.

This study investigates the ability of bovine serum albumin (BSA) to act as an extracellular chaperone (EC) on human hemoglobin (Hb) at a pH of 7.4. The best temperature for studying this behavior was determined by analyzing Hb's aggregation kinetics at multiple temperatures. 55 °C was chosen as the optimal temperature for forming Hb amyloids. BSA was then tested at various concentrations (20-100 µM) to assess its chaperone-like activity on Hb at 55 °C. At a concentration of 100 µM, BSA exhibits chaperone-like activity with a client protein:BSA ratio of 1:10. The high ratio implies that the chaperone activity of BSA is favored by the effects of macromolecular crowding. The results showed that BSA has the potential to inhibit Hb's dissociation into alpha and beta subunits and protein aggregation by inhibiting secondary nucleation. BSA also causes the depolymerization of fibrils over time. The results were validated using molecular docking and all-atom molecular dynamics simulations. MD analysis such as RMSD, RMSF, Rg, SASA, Hydrogen bond, PCA, Free energy landscape (FEL) revealed that the stability of hemoglobin is greater when it is bound to BSA compared to unbound state. The study suggests that BSA can potentially bind to Hb dimers and reduce excitonic interactions, which reduces Hb aggregation. These results are consistent with the aggregation kinetics experiments.Communicated by Ramaswamy H. Sarma.

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.

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.

The modulation of structural stability of horseradish peroxidase as a consequence of macromolecular crowding.

Macromolecular crowding plays an inevitable role in all biological processes influencing association, conformation, and other characteristics of proteins. Present study is based on the effect of macromolecular crowding on structure of horseradish peroxidase (HRP) enzyme. Concentration-dependent conformational changes induced by crowding agents, dextran 70 and polyethylene glycol (PEG)-4000, were monitored employing a range of biophysical techniques. The intrinsic fluorescence spectra showed transition of protein from native to unfolded state. Marked increase in 8-Anilino-1-naphthalene-sulphonoic acid and Thioflavin T fluorescence indicated presence of non-native moieties with 80 mg/mL dextran. Enhanced absorbance in turbidity, Soret, and Congo red in corroboration with scattering intensity at 350nm results revealed incidence of HRP aggregates. A new peak around 218 nm in CD spectra pointed towards change in secondary structure towards ß-sheets. Significant loss of enzyme activity upon structural disruption was seen. Comet assay demonstrated DNA damage and genotoxic nature of HRP aggregates, supporting spectroscopic, and fluorescence results. The normalized results were obtained with 120 mg/mL PEG-4000 close to that of native HRP implying no disruptive effect on structure. It can be hypothesized that macromolecular crowding is a vital element, which can have diverse effects. In this study, dextran 70 was observed to have pro-aggregatory effect while enhanced stability of native enzyme was witnessed with PEG. Hence, it can be stated that PEG has potentially better crowder as it helps retain the native enzyme structure. Routine addition of crowding agents is recommended if biological molecules are to be studied under more physiologically appropriate environments.

Macromolecular crowding stabilises native structure of α-chymotrypsinogen-A against hexafluoropropanol-induced aggregates.

Cell interior is extremely congested with tightly packed biological macromolecules that exerts macromolecular crowding effect, influencing biophysical properties of proteins. To have a deeper insight into it we studied consequences of crowding on aggregation susceptibility and structural stability of α-chymotrypsinogen-A, pro-enzyme of serine protease family, upon addition of co-solvent reported to exert stress on polypeptides crafting favourable conditions for aggregation. Hexafluoropropan-2-ol (HFIP), a fluorinated alcohol caused structural disruption at 5% v/v unveiled by reduced intrinsic intensity and blue shifted ANS spectra. Significantly enhanced, red-shifted ThT and Congo red spectra sustained conformational changes concomitant with aggregation. FTIR and CD results confirmed transition of native structure to non-native extended, cross-linked beta-sheets. Transmission electron micrographs visibly exhibited incidence of amorphous aggregates. Macromolecular crowding, typically mimicked by concentrated solutions of dextran 70, was noticeably witnessed to defend conformational stability under denaturing condition. The native structure was retained maximally in presence of 100 mg/ml followed by 200 and 300 mg/ml dextran indicating concentration dependent deceleration of aggregate formation. It can be established that explicit consideration of crowding effects using relevant range of inert crowding agents must be a requisite for presumptions on intracellular conformational behaviour of proteins deduced from in vitro experiments.

Consequence of macromolecular crowding on aggregation propensity and structural stability of haemoglobin under glycating conditions.

Cell interiors are extremely congested with biological macromolecules exerting crowding effect, influencing various physiognomies of protein life. Present work deals with effect of crowding on folding behaviour of haemoglobin (Hb) under glycating conditions. Macromolecular crowding was mimicked by concentrated solutions of dextran 70. Hb with 0.2 M fructose and ribose was incubated separately for 96 h in dilute and crowded solution to analyse conformational changes. Reduced intrinsic and ANS fluorescence, decreased Soret absorbance, enhanced turbidity, browning of protein, red shift in ThT and Congo red spectra significantly unveiled protein aggregation. FTIR and CD results revealed transition from α-helix to ß-sheets confirming aggregation. Transmission electron microscopy exhibited incidence of aggregates. Macromolecular crowding was witnessed to defend conformational stability of native Hb under stress condition at 100 mg/ml dextran, noticeably indicating deceleration of aggregation. Stabilising effect of crowding was marginally better in fructosylated Hb than with ribose due to difference in their glycation potential. Contrarily, in over-crowded solution where dextran concentration was 500 mg/ml, heightened aggregation was perceived implying concentration dependant, dual nature of macromolecular crowding. The novelty of this study lies in idea of considering macromolecular crowding as a key player in regulation of protein stability which was safely ignored previously.

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.

Carboxylic acids of different nature induces aggregation of hemoglobin.

Misfolded proteins that escape cellular quality control check lay the foundation for several progressively widespread neurodegenerative diseases, diabetes and others. Here, crotonic and citric acid are employed to study aggregation behaviour of hemoglobin (Hb). A systematic investigation on varying concentrations of acids from 0 to 60 mM on Hb gives an idea that transition is taking place in the vicinity of 10-30 mM. Hb showed increased intrinsic Trp fluorescence in the presence of both acids. A red shift of 10 nm in presence of citric acid contrary to a blue shift of 5 nm in presence of crotonic acid is observed. ANS and ThT fluorescence marked aggregation at 50 mM, supported by Congo red and Soret absorbance spectroscopy. CD, RLS and DLS studies also validate the findings. Molecular docking analysis exhibited the binding mode of Hb with acids. Aggregates were dense, beaded structure as visualised under TEM. Crotonic and citric acid at 20 and 30 mM, respectively, induced structural changes in Hb which transmutes to aggregate at higher concentration. These alterations remained almost constant and no significant changes were observed on increasing concentration further. Also, crotonic acid is more noxious, as it instigates conformational alterations at lower concentration than citric acid.

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.

Thermal unfolding of human lysozyme induces aggregation: Recognition of the aggregates by antisera against the native protein.

Protein aggregates are formed due to the inappropriate folding of polypeptides. Human lysozyme (HLZ) plays an important role in the innate immune response of the body and has been used extensively as a model protein to study aggregation. In this study, we showed that HLZ undergoes unfolding induced aggregation when heated by using spectroscopic and microscopic techniques. We further showed that the aggregates were recognized by polyclonal antibodies against the native HLZ. The consequences of these observations are further co-related with mammalian physiology.

A Biophysical and Computational Study of Concanavalin A Immobilized Zinc Oxide Nanoparticles.

BACKGROUND: Con A, a lectin extract from jackbean Canavalia ensiformis is known for its agglutination activity. ZnO nanoparticles promote the faster electron transfer between the lectin immobilized and the target cells. Hence, Con A immobilized on ZnO nanoparticles will agglutinate cells more effectively than the native protein. OBJECTIVES: Concanavalin A (Con A), a lectin was immobilized on the hexagonal zinc oxide (ZnO) nanoparticles to monitor its activity on RBCs and lymphocytes. METHODS: The immobilization of Con A and zinc oxide nanoparticles has been studied by molecular docking, microscopic and genotoxicity assessment techniques. RESULTS: Qualitative assessment using various techniques like atomic force microscopy, scanning electron microscopy and X-ray diffraction showed minor changes in morphology of Con A and ZnO nanoparticles. FT-IR spectroscopy confirmed the linking of Con A amino groups with ZnO nanoparticles. Con A immobilized nanoparticles in contrast to native lectin showed minor changes in hemagglutination activity as confirmed by pH dependence studies using fluorimetry. Con Aimmobilized nanoparticles retained the agglutination activity, this can be indicative of their potential application in detection of virus transformed and neoplastic cells. The Con A immobilized ZnO nanoparticles did not induce any significant but minor damage to whole cell DNA as revealed from comet assay or plasmid DNA. CONCLUSION: Con A immobilized on ZnO nanoparticles showed minor changes in the structure of ZnO nanoparticles and in the conformational of native Con A. However, Con A immobilized ZnO nanoparticles interestingly, showed pH resistance and better hemagglutination activity as well as minor DNA damage to whole cell lymphocytes. Thus, this novel bioaffinity support has prospective clinical implications.

Peroxidase improves the activity of catalase by preventing aggregation during TFE-induced denaturation.

Catalase, a ubiquitous enzyme of the free radical scavenging machinery unfolds and aggregates in the presence of 2,2,2-triflouroethanol (TFE). Catalase molecule aggregates at 50% TFE as evident by high thioflavin T fluorescence, shifted congo red absorbance, change in circular dichroism and soret spectra. TEM images confirmed the nature of catalase aggregates to be oligomers. Organic solvent-induced aggregation of catalase is prevented by the presence of peroxidase (another enzyme of the free radical scavenging machinery). To alter the progress of aggregation in presence of increasing concentration of TFE, we determined the effect of peroxidase on catalase oligomerization by several different techniques, including turbidity measurement, activity assay, thioflavin T fluorescence, circular dichroism, shift in congo red absorbance, transmission electron microscopy (TEM), Rayleigh scattering, soret absorption spectra, and ANS fluorescence. The presence of peroxidase in the vicinity of folded catalase helps it to remain functionally active and inhibited aggregation in the presence of TFE, suggesting that proteins are stable in crowded environments. Moreover, this catalase-peroxidase interaction is biologically significant as it provides insights into how the aggregation process may be altered.

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.

Serotonin abrogates dopamine induced aggregation of cytochrome c.

Importance of cytochrome c arises from its involvement in apoptosis, sequence homology and conserveness during molecular evolution. Dopamine at 25µM concentration for 30h resulted in cytochrome c aggregation as evident by increase in ANS and ThT binding, structural transition from α helix to ß sheet and shift in congo red assay. Interestingly, serotonin at 25µM concentration incubated for 60h abrogates the aggregatory effect on cytochrome c. Fibrillation of human cytochrome c in the presence of dopamine gives the clue about conformational changes taking place in protein and example of natural aggregation and inhibitory effect of serotonin gives the clue about natural defence mechanism occurring in human body.

Aloe emodin, an anthroquinone from Aloe vera acts as an anti aggregatory agent to the thermally aggregated hemoglobin.

Aggregation of proteins is a physiological process which contributes to the pathophysiology of several maladies including diabetes mellitus, Huntington's and Alzheimer's disease. In this study we have reported that aloe emodin (AE), an anthroquinone, which is one of the active components of the Aloe vera plant, acts as an inhibitor of hemoglobin (Hb) aggregation. Hb was thermally aggregated at 60°C for four days as evident by increased thioflavin T and ANS fluorescence, shifted congo red absorbance, appearance of ß sheet structure, increase in turbidity and presence of oligomeric aggregates. Increasing concentration of AE partially reverses the aggregation of the model heme protein (hemoglobin). The maximum effect of AE was observed at 100µM followed by saturation at 125µM. The results were confirmed by UV-visible spectrometry, intrinsic fluorescence, ThT, ANS, congo red assay as well as transmission electron microscopy (TEM). These results were also supported by fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) which shows the disappearance of ß sheet structure and appearance of α helices. This study will serve as baseline for translatory research and the development of AE based therapeutics for diseases attributed to protein aggregation.