PVP-AuNP impedes glycation mediated Hen Egg White Lysozyme aggregation under physiological condition

PVP-AuNP impedes glycation mediated Hen Egg White Lysozyme aggregation under physiological condition | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article PVP-AuNP impedes glycation mediated Hen Egg White Lysozyme aggregation under physiological condition Jennifer Johnson, Tushar Tyagi, Prasenjit Maity, Satish Kumar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3921564/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Protein glycation a non-enzymatic protein modification, alters the structure of biomolecule leading to several neurodegenerative and other disorders. As onset of disorders due to protein glycation is primarily driven by the development of advanced glycation end products (AGEs), therapeutic intervention against related disorders by inhibiting AGEs production is imperative. Nanoparticles have recently gained more prominence as therapeutic agents in biological field such as medicine, drug discovery and diagnosis. In present study, we extensively investigated the effect of chemically synthesized polyvinylpyrrolidone conjugated gold nanoparticles (PVP-AuNP) on D-ribose induced glycation of hen egg white lysozyme (HEWL) under physiological conditions. Our finding shows that AGEs formation was inhibited by PVP-AuNP over the period of 20 days. Interaction of gold nanoparticles prevented glycation induced misfolding and aggregation of lysozyme by stabilizing its native structure, which was evident with static light scattering, ThT, Congo red and ANS fluorescence coupled with CD spectroscopy. Further, by estimating carbonyl content and thiol group, our study suggests that PVP-AuNP possesses antioxidant property thus prevent the HEWL against glycation driven oxidative damage. Present study therefore elucidates that PVP-AuNP a significant antiglycation agent can be used against wide range of disorders induced by AGEs. Aggregation Glycation Gold nanoparticle Lysozyme Neurodegeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Glycation is a prevalent non enzymatic post-translational modification that involves cascade of nucleophilic reaction between the free amino group of the protein and the carbonyl group of reducing sugar. Spontaneous glycation yield formation of a Schiff base intermediates reversibly that translates into an Amadori product, which eventually results into advanced glycation end products (AGE) [ 1 ]. Formation of AGEs alter and damage the overall structure of biological macromolecules especially proteins. The milieu around protein molecule in the cell, being surrounded by sugars, sugar derivatives, xenobiotics including toxins makes it prone to glycation. Accumulation of AGEs is reported to lead several pathophysiological complications such as diabetic nephropathy or retinopathy and atherosclerosis [ 2 ]. Production and accumulation of AGEs also induces protein misfolding and aggregation culminating in several non-neuropathological and neurodegenerative disorders like Alzheimer, Parkinson, and diabetes type II [ 3 , 4 ]. Extracellular or intracellular deposition of protein aggregates called amyloid plaques are the histological hallmark of neurodegenerative disorders. Among several post-translational modifications, glycation plays a crucial role in formation of amyloid both in vitro and in vivo . Effect of glycation on protein folding, aggregation, amyloid formation and cytotoxicity has been reported earlier [ 5 ]. AGE induced amyloid fibrils have been found in the brain tissues of patients suffering from transmissible spongiform encephalopathy, Alzheimer’s disease and patients having islets of Langerhans of diabetes [ 6 , 7 ]. It has been observed that site specific Aβ1–42 glycation affects the formation of amyloid fibrils and generates neurotoxicity to SH-SY5Y cell lines [ 8 ]. Glycation accelerates the aggregation rate of IAPP in the presence of glucose [ 9 ]. Various glycating agents like glucose, D-ribose, methylglyoxal, and glyoxal are found to promote amyloid formation of human albumin under different experimental conditions [ 10 ]. Using drosophila and mice model, Oliveira and co-workers demonstrated that glycation enhance α-synuclein toxicity in vitro and in vivo [ 11 ]. Globally around 55 million people living with dementia have been reported, and this is expected to increase to nearly 78 million by 2030 [ 12 ]. Understanding its socioeconomic burden, several manoeuvres are being looked upon to impede the conversion of native protein to its toxic form which in turn ameliorates aggregation-associated various neurodegenerative and non-neuropathic conditions. Small organic molecules [ 13 ], synthetic peptides [ 14 ], polysaccharides [ 15 ] are reported to inhibit aggregation of proteins under different experimental conditions. Various in vitro and in vivo studies have demonstrated that inhibition of glycation reaction may be used as a therapeutic approach against diabetes related ailments and other metabolic disorders [ 16 , 17 ]. Nanomaterials owing to their smaller size and higher surface to volume ratio are potential agent for the diagnosis and prevention of diseases. Recent studies have reported that gold nanoparticles (AuNPs) can function as an antiglycation agent, thereby inhibiting the formation of AGEs [ 18 , 19 ]. The inhibitory effect of AuNPs on bovine serum albumin glycation in presence of D-ribose advocates that colloidal particles could be a potential therapeutic agent for the treating diabetes and its associated pathophysiological conditions such as hyperglycaemia [ 20 ]. Silver and gold nanoparticles are also observed to demonstrate anti-glycating property upon human serum albumin [ 21 ]. Polyvinylpyrrolidone (PVP) conjugated gold nanoparticles are observed to restrain the hen egg white lysozyme aggregation at relatively lower pH and elevated temperature [ 22 ]. However, the mechanism underlying this study was elusive. Hen Egg White Lysozyme (HEWL) protein serves as an excellent model protein to investigate the structural and conformational changes that a protein undergoes during misfolding and aggregation owing to its globular structure and enzymatic activity [ 23 ]. 60% sequence similarity of HEWL and human lysozyme is observed, that is majorly responsible for hereditary systemic amyloidosis [ 24 ] . In current study, we explored the effect of polyvinylpyrrolidone (PVP) conjugated gold nanoparticle (PVP-AuNP) on structure and function of HEWL in presence of D-ribose under physiological condition, 37°C, pH 7.4. Present study shows that PVP-AuNP reduce the formation of AGEs and inhibit ribose mediated aggregation of HEWL. Our observations suggests that interaction of gold nanoparticle modulates the secondary structure of protein and helps in retaining its catalytic activity in presence of ribose over the period of 20 days. Further, PVP-AuNP was observed to dampen the self-assembly of lysozyme by affecting carbonyl and thiol content. Our results pose PVP-AuNP nanoparticle holds a novel therapeutic potential against AGEs mediated neurodegenerative disorders and other metabolic complications. 2. Materials and methods Hen egg white lysozyme (HEWL: L-6876), D-ribose (analytical grade), Thioflavin T (ThT), Micrococcus lysodeikticus , Chloroauric Acid, Congo Red, 8-anilinonaphthalene-1-sulfonic acid (ANS) dye, Ellman’s reagent, Sodium phosphate monobasic, Polyvinyl pyrrolidone K-30, sodium tetra hydroborate were obtained from Sigma-Aldrich. Sodium azide, 2–4 dinitrophenylhydrazine (DNPH) were purchased from Himedia Laboratories. All other chemicals used belonged to analytical grade. 2.1 Preparation and characterization of Polyvinylpyrrolidone (PVP) conjugated Gold Nanoparticle (AuNP) Polyvinylpyrrolidone conjugated gold nanoparticle (PVP-AuNP) was prepared using standard protocol as reported earlier [ 25 ]. 1 mM HAuCl 4 aqueous solution (30 mL) was mixed with polyvinylpyrrolidone (PVP, K30, 40 kDa) and was subjected to continuous stirring at 273 K for 20 min. Fresh aqueous solutions of PVP/ HAuCl 4 were combined alongwith sodium borohydride (NaBH 4 ) (0.1 M, 1.5 mL, 5 molar equivalent with respect to HAuCl 4 ), a potent reducing agent, and vigorously stirred for 30 minutes at 273 K to produce the conjugated nanoparticle (PVP-AuNP). Next, utilizing a membrane filter for ultra-filtration, the dispersed aqueous nanoparticle was purified. A strong ruby red colour was developed after 15 to 20 minutes, indicating the synthesis of gold nanoparticle (PVP-AuNP). Following that, numerous biophysical approaches were used to characterise this nanoparticle solution. Characterization of PVP- AuNP was done using different biophysical methods such as UV-Visible spectroscopy, X-Ray diffraction patterns, transmission electron microscopy (TEM), dynamic light scattering, and MALDI Mass spectrometry. Fresh HEWL stock solution was prepared in double deionized water. Protein concentration was determined by using a molar extinction coefficient of 37970 M -1 cm -1 and calculating the absorbance (OD) at 280 nm. For the experiment, stock was diluted to 60 µM as final concentration of protein in sodium phosphate buffer (50 mM) with pH 7.4. Stock solution of D-ribose sugar (0.5 M) was prepared in distilled water and kept at 4°C before use. Phosphate buffer was filtered using 0.22 µm filter. 2.3 Protein glycation study To induce glycation, 60 µM HEWL was incubated with 100 µM D-ribose sugar at 37 0 C, pH 7.4. Sodium azide (1 mM) was added to avert any bacterial contamination. To study the effect of nanoparticles on glycated protein, 100 µM and 200 µM PVP-AuNP was incubated individually to HEWL with D-ribose at 37 0 C, pH 7.4 for the time period of 20 days. Protein sample in absence of ribose was considered as a control. Aliquots were withdrawn at specific time point for further analysis. 2.4 AGE Fluorescence spectroscopy Formation of advanced glycated end products (AGE) under different conditions were monitored by measuring the fluorescence spectra as reported earlier [ 26 ]. Glycated HEWL with and without PVP-AuNP along with the control were monitored with excitation at 370 nm with slit width of 2 nm and the emission were monitored in the range of 390–600 nm with slit width of 2 nm. Fluorescence at 440 nm was recorded as a signature of glycated species in present experiment [ 27 ]. 2.5 Light scattering measurement To monitor the presence of glycation induced protein aggregates, scattering intensity of glycated HEWL with and without nanoparticles were measured at 350 nm at a 90 0 angle. Samples were monitored with excitation at 350 nm with 2 nm slit width and the emission spectra was measured at 320–400 nm at 2 nm slit width using Jobin-Yvon Fluoromax 4 spectrofluorometer. Three distinctive spectra were averaged after subtracting with respective blank. 2.6 Thioflavin T (ThT) dye assay Fresh ThT stock was made in double deionized water. Protein samples were diluted in 50 mM phosphate buffer (pH 7.4) such that the molar ratio of protein to ThT dye was 1:2. The protein concentration was 10 µM in the assay. Glycated HEWL with and without PVP-AuNP along with the control at different time points were monitored with excitation at 450 nm with 5 nm slit width, and the emission spectra were measured at 470–550 nm with 10 nm slit width at different time points. Three distinctive spectra were averaged after subtracting with respective blank. 2.7 Congo Red dye assay Fresh Congo Red dye stock solution (4 mM) was formulated and diluted in sodium phosphate buffer (50 mM). Glycated HEWL with and without PVP-AuNP along with the control at different time points were diluted with congo red (in 50 mM phosphate buffer pH 7.4) in a manner that the molar ratio of protein to congo red was ~ 1:2. In the assay, the protein concentration was 10 µM. Absorption spectra were monitored at 400–700 nm wavelength spectrum using cuvette of 1cm path length. 2.8 Dynamic Light Scattering (DLS) Samples were analysed for DLS measurement at 633 nm wavelength through a laser and 90° scattering angle of the detector. All the measurements were done using the Zetasizer Nano S90 DLS (Malvern Instrument Ltd, United Kingdom). 20 measurements were recorded for 2 ml native and glycated protein samples with and without PVP-AuNP at a constant temperature (25 0 C) using quartz cuvette. Data was processed through Malvern Zetasizer Software 7.11. 2.9 Fluorescence microscopic study Fluorescence microscopy was performed by incubating the glycated protein samples with and without PVP-AuNP with ThT dye in a molar ratio of 1:2 for 30 mins in dark, and then mounted on a clean slide and covered using a cover slip. Samples were visualized and the images were obtained by using Nikon DM 5200 Fluorescence microscope. Observations were performed with 10X/0.25 objective lens. Control HEWL (absence of ribose) was also run concurrently. 2.10 Tryptophan fluorescence The intrinsic tryptophan fluorescence was measured by exciting the glycated HEWL with and without PVP-AuNP along with the control for different time at 295 nm with 2 nm slit width and the emission spectra were recorded in the range of 310–400 nm with 10 nm slit width using Jobin-Yvon Fluoromax 4 spectrofluorometer. Three separate spectra were averaged after subtracting with each corresponding blank. 2.11 Circular Dichroism (CD) spectroscopy Circular Dichroism experiment was executed using quartz cuvette with path length (0.1 cm) at 25 0 C with JASCO J815 spectropolarimeter. CD spectra of each sample was generated in the far ultraviolet absorbance region (190–250 nm) by averaging 20 consecutive scans at 50 nm min − 1 scan rate with a suitable baseline. The response time was set to 1 s and the band width was achieved at 1 nm. Data was interpreted using the K2D3 software [ 28 ]. 2.12 8-anilinonaphthalene-1-sulfonic acid dye assay ANS (8-anilinonaphthalene-1-sulfonic acid) stock was freshly prepared and diluted with phosphate buffer (pH 7.4). Glycated HEWL with and without PVP-AuNP along with the control were diluted with ANS dye in a manner that the molar ratio of protein to ANS was ~ 1:4. The protein concentration was 10 µM in the assay. Samples were monitored with excitation wavelength at 380 nm with 2 nm slit width, and the emission wavelength at 400–600 nm with slit width of 10 nm at different time points. Three separate spectra were averaged after subtracting with each corresponding blank. 2.13 HEWL lytic assay Fresh Micrococcus lysodeikticus stock solution was prepared in deionized water. This stock was further diluted to a concentration of 78 µg/mL in assay medium (phosphate buffer). Native and glycated samples with and without the gold nanoparticle (PVP-AuNP) were diluted in a way that the final concentration of protein was 70 nM. The lysozyme activity was monitored using a double beam spectrophotometer by measuring the slope for first 50 seconds and decrease in the initial rate at 450 nm absorbance range. 2.14 Determination of carbonyl content Carbonyl content of the protein under different conditions was measured using 2,4 dinitrophenylhydrazine reagent (2,4 DNPH). Stock solution of 2,4-DNPH (10 mM) was made in 2.5 M Hydrochloric acid. Next 2,4 DNPH reagent (400 µL) was added to 100 µL protein samples, which was then followed by incubation at room temperature for 1 hour. After incubation, 500 µL of chilled 20% Trichloroacetic acid (TCA) solution were added to the samples. These samples were centrifuged for 5 minutes, 10000 rpm at 4 0 C. 1:1 (v/v) ethanol/ethylacetate solution was used to wash the resulting pellets thrice followed by dissolving the pellets in guanidine hydrochloride solution (6M). Protein carbonyl content was calculated using the molar coefficient 22000 M − 1 cm − 1 at 370 nm using UV-Visible spectrophotometer [ 29 ]. 2.15 Determination of thiol groups Thiol group was estimated using a standard Ellman’s method [ 30 ]. The samples were mixed with 1 mM ethylenediaminetetraacetic acid (EDTA) and 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) reagent and then incubated for 30 minutes at 37 0 C. The final volume of all the samples were made up to 1ml using Tris-HCl buffer (pH 8). Final absorbance was calculated at 412 nm using the molar coefficient 14000 M − 1 cm − 1 using spectrophotometer. 3. Results and discussion 3.1 Characterization of Polyvinylpyrrolidone (PVP) conjugated Gold Nanoparticle (AuNP) After synthesis, characterization of PVP-AuNP was done using biophysical techniques like UV-Visible spectroscopy, Dynamic light scattering (DLS), X-ray diffraction (XRD) analysis, MALDI and imaging by Transmission Electron Microscope (TEM). The details characterizations of the synthesized nanoparticles are shown below (Fig. 1). UV-Vis Spectroscopy of PVP-AuNP showed featureless exponential absorption profile in the UV (250–400 nm) range indicating formation of ultra small (non-Plasmonic) gold nanoparticles (Fig. 1a). The TEM image with particle size histogram (Fig. 1b) showed the presence of highly monodisperse gold particles with average diameter of 1.5 ± 0.2 nm. XRD diffraction profile of PVP-AuNP along with the standard diffraction profile of bulk gold are shown in Fig. 1c, which showed a broad diffraction profile at 38º (2Ɵ) indicating the presence of (111) crystal plane. The ultra small crystal size of nanoparticles makes the X-ray diffraction profile broad. The MALDI mass spectrum also confirms the presence of gold particles with average nuclearity of 33–35 atoms per particle (Fig. 1d). The hydrodynamic diameter (D h ) of PVP-AuNP dispersed in water were found to be 8.2 ± 0.5 nm (Fig. 1e). Difference in size of PVP-AuNP as monitored by TEM and DLS was owing to the fact that TEM display the size of nanoparticles in solid state while water shell around PVP-AuNP also contribute to the size of nanoparticles monitored by DLS. 3.2 PVP-AuNP inhibits formation of advanced glycated product (AGE) To investigate the formation of advanced glycated product (AGE) products, in vitro glycation of HEWL protein was carried out in the presence of D-ribose sugar at physiological condition; 37°C, pH 7.4. Earlier work has demonstrated the effect of ribose on various protein, however these studies either used very high concentration of ribose or performed at non-physiological conditions [31]. As the concentration of D-ribose present in the blood is about 100 µM [32] we explored the same concentration of sugar to induce glycation on HEWL and to investigate the effect of PVP-AuNP. After incubating 60 µM HEWL in presence 100 µM D-ribose, formation of AGE product was monitored by characteristic fluorescence emission at 440 nm [33]. Figure 2 shows the time dependent AGE production in different experimental condition. Compared to control (HEWL without D-ribose), gradual increase in emission intensity was observed in HEWL with 100 µM D-ribose over the period of 12 days which was saturated thereafter, indicating the formation of AGE product. In presence of 100 µM and 200 µM PVP-AuNP, fluorescence intensity reduced by ~ 21% and ~ 60% respectively, suggesting that functionalized gold nanoparticle inhibit the formation of AGE product in concentration dependent manner. Reduction in AGE product formation was much in line with earlier finding where AgNPs and AuNPs are observed to inhibit AGE formation in HSA [34]. 3.3 PVP-AuNP inhibits AGE induced HEWL aggregation Advanced glycation end products are known for the onset of various diseases such as neurodegenerative disorders including Parkinson's disease, Alzheimer's disease and aging [35]. Accumulation of AGEs in various tissues induce oxidative stress, and inflammation leading to misfolding and aggregation of several proteins. Next, we investigated if gold nanoparticle affects the AGEs induced aggregation of HEWL at physiological condition. Figure 3a shows the static light scattering of glycated lysozyme in presence and absence of 100 µM and 200 µM PVP-AuNP individually. Presence of higher molecular weight species is often characterized by an increase in the scattering intensity which arises due to aggregation of the biomolecule [36]. Marginal increase in scattering intensity of control (HEWL alone) indicates the prevalence of monomeric protein after twenty days of incubation, while D-ribose induced lysozyme aggregation in presence and absence of gold nanoparticle was observed to be sigmoidal in nature. Aggregation kinetics of HEWL with sugar but without gold nanoparticle shows the lag phase of ~ 1 day. Gradual increase in scattering intensity was observed upto 14 days and was saturated thereafter. Scattering intensity drastically declined in presence of 100 µM and 200 µM PVP-AuNP during 20 days of incubation. Compared to HEWL with ribose, nearly 23% and 55% reduction in scattering intensity in presence of 100 µM and 200 µM gold nanoparticle respectively, shows the dose dependent inhibitory effect on lysozyme aggregation. Addition to reduced scattering intensity, Fig. 3a also indicate the increased lag phase in presence of PVP-AuNP. In absence of gold nanoparticle which shows t 1/2 at ~ 3.5 days, presence of 100 µM and 200 µM gold nanoparticle increases the t 1/2 to ~ 5 days thus signifying that PVP-AuNP impedes the growth of HEWL fibrillation in presence of ribose. Changes in scattering intensity advocates the presence of larger sized protein aggregates and not specify if these structures are ordered or amorphous in nature. Ordered protein structures called amyloid fibrils exhibiting enhanced fluorescence subsequent to binding with thioflavin T (ThT), an amyloid specific probe are hallmark of several neurodegenerative disorders [37]. We further investigated whether PVP-AuNP inhibits the formation of amyloid like structure employing ThT fluorescence. Figure 3b depicting time dependent ThT fluorescence of glycated HEWL in absence and presence of PVP-AuNP demonstrate the similar aggregation kinetics as observed by scattering (Fig. 3a). HEWL with ribose in absence of nanoparticles was showing gradual increase in ThT fluorescence till 14 days and was constant for the time window of 20 days (Fig. 3b) indicate the presence of ThT sensitive amyloid structures. Similar observation was reported earlier where enhanced ThT fluorescence was exhibited by lysozyme in presence of 500 µM D-ribose upto 31 days which decreased further [38]. In presence of 100 µM and 200 µM PVP-AuNP nearly 25% and 40% reduction respectively in ThT fluorescence manifest the inhibitory effect of gold nanoparticle on aggregation and amyloid formation of lysozyme in presence of sugar. Aggregation often leads to higher β-sheet structure in protein, which binds with ThT resulting in enhanced fluorescence [39]. In present result, reduced ThT fluorescence of glycated HEWL in presence of gold nanoparticles indicates lesser proportion of β-sheet which are signature of misfolded and aggregated proteins. We however, probed the secondary structural change of HEWL in present study employing circular dichroism (CD) in section 3.5. Result obtained from ThT fluorescence was further validated through Congo red (CR) assay. CR is another amyloid specific probe which intercalates between the β-strands of aggregated protein and exhibit enhanced absorbance at around 500 nm [40]. Compared to HEWL without sugar, enhanced absorbance was observed in presence of 100 µM D-ribose in absence of nanoparticles after 20 days of incubation (Fig. 3c). This was in agreement with earlier study where D-ribose induced protein aggregates demonstrated enhanced CR absorbance [41]. In presence of 100 µM and 200 µM PVP-AuNP, decrease in CR absorbance after the same time period complement our ThT observation that gold nanoparticles prevent formation of sugar induced amyloid structures. 3.4 Characterization of HEWL aggregates in presence and absence of PVP-AuNP After confirming the inhibitory effect of gold nanoparticle on ribose mediated HEWL aggregation, we characterize these structures by dynamic light scattering (DLS) and fluorescence microscopy. DLS has extensively been used to study the growth of protein aggregates and inhibition under different conditions. Figure 4a showing hydrodynamic radii (R h ) of freshly prepared native HEWL as 2.13 nm was in agreement of globular lysozyme as reported earlier [42]. HEWL in presence of D-ribose without gold nanoparticle after 20 days shows the R h of ~ 446 nm manifesting the population of higher order aggregates (Fig. 4b). After the same time interval, sugar incubated HEWL in presence of 100 µM and 200 µM PVP-AuNP demonstrated the hydrodynamic radii of ~ 404 nm and ~ 361 nm respectively (Fig. 4c and 4d) clearly indicating that gold nanoparticles impede the growth of D-ribose induced lysozyme aggregation. Data obtained from scattering, ThT fluorescence, CR, DLS in unison suggests that PVP-AuNP inhibits D-ribose induced HEWL aggregation in concentration dependent manner. Fluorescence microscopy employing Thioflavin T (ThT) fluorescence has also been used to monitor protein aggregation and inhibition [22]. Formation and deposition of ordered protein structures as fibrils or amorphous aggregates are evident in several neurodegenerative disorders. Although from Fig. 3b using ThT fluorescence it was very apparent that gold nanoparticle inhibits the D-ribose mediated lysozyme aggregation, we further investigated the types of structures formed by fluorescence microscopy using ThT as a probe (Fig. 5). HEWL control after 20 days of incubation did not show any fluorescent structure indicating the presence of globular protein (Fig. 5a). In presence of 100 µM D-ribose, without PVP-AuNP, long fibrillar structure exhibiting ThT fluorescence was observed (Fig. 5b), whereas in presence of 100 µM PVP-AuNP after 20 days, no fibrillar structure displaying ThT fluorescence reveal that gold nanoparticles inhibit the formation of HEWL fibrils in presence of sugar (Fig. 5c). As discussed earlier, ThT a positively charged benzothiazole-based fluorophore is hypothesized to bind with ordered β-sheet structure of aggregated protein thus leading to enhance fluorescence owing to restricted torsional relaxation between benzothiazole ring and benzene ring. Several anti-amyloidogenic compounds are known to inhibit protein aggregation by blocking β-pleated sheet structure [43]. Thus, it is possible that functionalized gold nanoparticle also blocks the formation of β- pleated sheet structure which is apparent from diminished ThT fluorescence. 3.5 PVP-AuNP promote native like structure of HEWL in presence of D-ribose Small anti-amyloidogenic molecules subsequent to binding with protein, resists the structural alteration in biomolecule under aggregation prone condition. In order to probe the conformational changes in native protein in the present study, HEWL with D-ribose in absence and presence of PVP-AuNP was subjected to tryptophan (Trp) fluorescence, far-UV circular dichroism spectroscopy and 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence assay after 20 days of incubation. Intrinsic Trp fluorescence is extensively explored to investigate conformational changes in protein under different condition including glycation [44]. We first probed the Trp fluorescence of glycated HEWL in presence of different concentration of functionalized nanoparticles after 20 days at pH 7.4. Compared to HEWL control where λ max was ~ 347 nm, glycated HEWL without gold nanoparticles demonstrated λ max around 342 nm coupled with marked reduction in the fluorescence intensity indicating structural alteration in HEWL (Fig. 6a). This results was in accordance with earlier findings that showed reduction in tryptophan fluorescence and shift in emission maxima of glycated protein at 37 0 C, pH 7.4 [45]. In presence of 100 µM and 200 µM PVP-AuNP on the other hand, Trp fluorescence increased to ~ 1.6 fold and ~ 1.8 fold respectively, in comparison to glycated protein resonates that conjugated gold nanoparticle promotes native like structure of HEWL in presence of sugar. We further explored Circular dichroism (CD) spectroscopy, an extensively used technique to study the secondary structural aspects of protein. CD can easily distinguish changes in helical structure, parallel and antiparallel β-sheets of protein molecules. Native HEWL in absence of sugar showed α-helical structure with negative band at 208 nm and shoulder around 222 nm. In presence of 100 µM D-ribose, helical signals flattened at 208 nm and 222 nm and significantly changed the molar ellipticity around 216–220 nm indicating the decrement in α-helical content and presence of β sheet structure respectively (Fig. 6b). This observation supported earlier studies where glycation was known to induce β sheet structure in globular protein [46]. However, in presence of 100 µM gold nanoparticle, an increase in α-helical and decrease in β-sheet was observed. The secondary structural aspect of the protein was determined using K2D3 software, where in native lysozyme 32.87% α-helix and 12.82% β-sheet content was observed. In ribosylated HEWL the α-helix content reduced to 11.51% and β-sheet content increased to 19.08%. In presence of 100 µM PVP-AuNP, ribosylated HEWL displayed 14.77% α-helical and 18.42% β-sheet. Reduction in β-sheet coupled with increase in α-helix strongly suggests that gold nanoparticles inhibit the formation of β-sheet structure in glycated HEWL. Taken together, data obtained from ThT fluorescence (Fig. 3b), fluorescence microscopy (Fig. 5), CD (Fig. 6b) clearly reveals that PVP-AuNP inhibit sugar induced HEWL aggregation by blocking formation of β-sheet structures. Previous studies have shown that sugar molecules induce molten globule-like structures in HEWL, characterized by exposure of buried hydrophobic patches which eventually act as nucleation factor for inducing protein aggregation. Exposure of hydrophobic regions of protein is characterised by enhanced ANS fluorescence alongwith blue shift in emission maximum [47]. Figure 6c showing emission maximum of native HEWL after 20 days of incubation was ~ 512 nm. In presence of 100 µM D-ribose, ANS fluorescence increased at emission maximum ~ 486 nm coupled with blue shift which signifies the exposure of hydrophobic patches in lysozyme. Presence of 100 µM and 200 µM gold nanoparticle not only reduced the ANS intensity but also changed the emission maximum to ~ 504 nm and ~ 503 nm respectively of glycated HEWL. Result for ANS fluorescence mirrors that PVP-AuNP not only impedes the D-ribose induced exposure of hydrophobic regions of HEWL but also tries to bring the protein near its native confirmation. Owing to globular structure and well-established enzymatic activity, HEWL has been extensively used to study protein aggregation and in its inhibition under varied conditions [48]. As functional activity is a peculiar characteristic of native protein structure, we probed the activity of ribosylated HEWL alongwith 100 µM and 200 µM gold nanoparticle after 20 days of incubation. Figure 7a represents the lytic activity of Micrococcus lysodeikticus , a lysozyme substrate under different experimental conditions. Linear decline in turbidity in initial 50s was used for calculating the HEWL activity by comparing with freshly prepared native HEWL (pH 7.4) whose activity was assumed to be 100%. Figure 7b shows ~ 70% decrease in ribosylated HEWL activity indicating the loss of native protein structure. In presence of 100 µM and 200 µM PVP-AuNP, activity of HEWL was observed to be around 65% and 90% respectively. It has been hypothesized that changes in positive charges on HEWL and motions of the low ordered regions due to ribosylation decrease the HEWL activity [49]. It is thus possible that interaction between negatively charged PVP-AuNP and positively charged HEWL stabilize the protein to retain its activity in presence of sugar. We have earlier demonstrated that chitotriose and brahmi recovers the HEWL activity at pH 12.2 and pH 2 respectively by stabilizing the structure [50, 51]. Data obtained from Trp fluorescence, circular dichroism and ANS fluorescence in association with enzymatic activity strongly advocates that gold nanoparticles stabilize the native structure of HEWL. 3.6 PVP-AuNP modulates the protein-bound carbonyl groups and thiol group in HEWL Non-enzymatic protein glycation involves excessive chemical attachment of sugar to the protein leading to the formation of early glycation products (EGPs). Further oxidation of EGPs generates protein-bound dicarbonyl intermediates, a key mediator whose accumulation in tissue contributes to the formation of AGEs. Increase in protein carbonyl content is reported to develop several neurodegenerative diseases, cancer, atherosclerosis, diabetes, and aging [52] by inducing oxidative stress, we probed the level of carbonyl content (CC) of HEWL with and without gold nanoparticles in presence of D-ribose. Figure 8a depicts CC of samples under varied experimental conditions at different time period. In absence of gold nanoparticles, gradual increase in CC over the time window of 20 days was observed in presence of sugar. A significant increase of CC by 90.65%, compared to unmodified HEWL after 20 days manifest the presence of reactive intermediate which induces oxidative stress and alters the structure of biomolecule. In presence of 100 µM and 200 µM PVP-AuNP dose dependent reduction in CC content was observed. After 20 days, 100 µM and 200 µM PVP-AuNP substantially reduced protein bound CC by 32.65% and 40.77% respectively, compared to glycosylated HEWL without nanoparticles. Positively charged amino acids like lysine and arginine are potential target for glycation thus responsible for elevated carbonyl intermediates [52, 53]. Interaction between lysine residue and gold nanoparticle are suggested to form ionic bridge generating carboxylate-ammonium type of salt. It is thus possible that electrostatic interaction between negatively charged functionalized gold nanoparticles and positively charged lysine/arginine residues hinders the interaction between sugar and protein leading to the reduced production of CC. Since carbonylation of amino acids induces protein aggregation by promoting formation of covalent and non-covalent bonds and protein unfolding [54], reduced CC along with our CD and ANS results suggest that binding of PVP-AuNP resists D-ribose induced conformational alteration in lysozyme in concentration dependent manner. Thiol groups plays a pivotal role in providing stability and solubility of proteins. Glycation and subsequent production of intermediate dicarbonyl compounds react promptly with thiol (-SH) groups of specific amino acids to generate thiol-aldehyde adducts. Further depletion in thiol group augments overall oxidative damage to the biomolecule [55]. As reduction in thiol groups is observed in fructose mediated glycation in BSA culminating in structural changes [56], we studied the same in glycated HEWL in absence and presence of 200 µM and 100 µM PVP-AuNP. Nearly 46% decrease in free thiol content of glycated HEWL was observed compared to native lysozyme after 5 days while after 20 days of incubation ~ 94% reduction in thiol was monitored (Fig. 8b). This finding was in agreement with earlier study where reduction in free thiols was observed in glycated lysozyme [57]. Treatment with gold nanoparticles to glycated HEWL on the other hand attenuated the decrease in thiol group. After 20 days, percentage prevention of depleting thiol group in presence of 100 µM and 200 µM PVP-AuNP was 38.10% and 47.14% respectively. Inhibitory effect of gold nanoparticle in present study supports the previous finding where reduction in thiol group was less in presence of bio-enzymatically synthesized gold and silver nano particles compared to glycated human serum albumin [34]. It has been argued that degradation of Amadori products induces free radical formation through oxidation of proteins which can be estimated through thiol group. Data obtained from above study signify that PVP-AuNP suppress the oxidation of HEWL in presence of sugar in concentration dependent manner and thus inhibits production of AGEs. Here for the first time, we proposing the mechanism explaining how functionalized gold nanoparticle (PVP-AuNP) inhibits D-ribose induced glycation but also retain its activity at physiological pH. D-ribose induces the formation of carbonyl content which eventually induces formation of AGEs. Parallelly, increased carbonyl content also induces oxidative stress leading to reduction in thiol group of protein. Increased carbonyl content, AGE products and reduced thiol collectively destabilizes the tertiary structure of HEWL leading to the exposure of hydrophobic regions coupled with quenching of exposed Trp molecules which ultimately manifested in HEWL aggregation and reduction in its catalytic activity. Addition of PVP-AuNP, on the other hand, inhibited the formation of carbonyl content, AGE production, and also reduced the oxidative stress. Additionally, PVP-AuNP also modulated the structure of HEWL in order to retain its native-like structure as it was reflected by ANS and Trp fluorescence, along with retention of catalytic activity after 20 days of incubation period. Conclusion Inhibiting protein glycation and reduction in AGEs production through small molecules is paramount against several neurodegenerative and other disorders. Although chemically synthesized drugs do inhibit process of protein aggregation, in most cases this inhibition comes at the cost of structural and functional activity of protein. Present study demonstrates that polyvinylpyrrolidone conjugated gold nanoparticles inhibit D-ribose induced HEWL glycation, eventually reducing AGEs formation in concentration dependent manner. Our results reveal that subsequent to interaction, PVP-AuNP prevent D-ribose induced structural alteration in HEWL which in turn modulates the hydrophobic regions and secondary structure of protein. These modifications further stabilize lysozyme’s tertiary structure which was evident from its activity. Preventing the increase in carbonyl content and resisting the decrease in thiol group in presence of gold nanoparticles signifies the antioxidant property of PVP-AuNP which protect the protein against oxidative stress causing misfolding and aggregation, culminating in several disorders. Findings of present study shows that PVP-AuNP possesses significant anti-glycation property and offers as an effective alternate against the treatment of diabetes, Alzheimer disease, Parkinson disease and atherosclerosis. However, more in-vivo and clinical studies are required to reveal the therapeutic effects of PVP-AuNP. Abbreviations AGEs, advanced glycation end products; ANS, 8-anilino-1-naphthalene sulfonate; CD, circular dichroism; DLS, dynamic light scattering; HEWL, hen egg white lysozyme; PVP-AuNP, polyvinylpyrrolidone conjugated gold nanoparticles; ThT, thioflavin T; Trp, tryptophan Declarations Acknowledgements We thank National Forensic Sciences University, Gandhinagar for providing the infrastructure and experimental facilities that made this work possible. We gratefully acknowledge the Central Instrumentation Facility (CIF) at IIT Gandhinagar for providing CD facility. Authorship contribution Jennifer Johnson: Methodology, Validation, Formal analysis, Investigation, Data curation, Writing - original draft, Software. Tushar Tyagi: Methodology, Data curation. Prasenjit Maity: Formal analysis, Writing - review & editing. Satish Kumar: Conceptualization, Project administration, Visualization, Supervision, Writing - review & editing, Methodology, Formal analysis. 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Fructose and methylglyoxal-induced glycation alters structural and functional properties of salivary proteins, albumin and lysozyme. Nagaraj R, editor. PLOS ONE. 2022;17:e0262369. Additional Declarations No competing interests reported. Supplementary Files GA.jpg Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 19 Feb, 2024 Reviews received at journal 16 Feb, 2024 Reviewers agreed at journal 09 Feb, 2024 Reviewers invited by journal 09 Feb, 2024 Editor assigned by journal 09 Feb, 2024 Submission checks completed at journal 08 Feb, 2024 First submitted to journal 02 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3921564","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":272070909,"identity":"1d19e14c-09f4-4f73-9a9e-9e9872f0a7ce","order_by":0,"name":"Jennifer Johnson","email":"","orcid":"","institution":"National Forensic Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"Johnson","suffix":""},{"id":272070910,"identity":"ad8568c9-5a3d-428c-88b0-b24a4408adcc","order_by":1,"name":"Tushar Tyagi","email":"","orcid":"","institution":"National Forensic Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Tushar","middleName":"","lastName":"Tyagi","suffix":""},{"id":272070911,"identity":"67709b73-eab9-456a-92e3-daf2386ee9bf","order_by":2,"name":"Prasenjit Maity","email":"","orcid":"","institution":"National Forensic Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Prasenjit","middleName":"","lastName":"Maity","suffix":""},{"id":272070912,"identity":"1abbe980-7951-4432-b385-5ec999c4fb5b","order_by":3,"name":"Satish Kumar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIie2RsWoCQRCG/2VBmzvrszGvMGFBEBRf5Q5BG4WAL2C1VtE2wZdQbCz32CLNQToRLo0cWF8QAgcWnppomltTptivGgY+5oMBLJZ/imIj0HlKUfvekUngN4W9QPxNwY/Cnati4GH8HobZCoLWerdvrahNim9TPH0UKhR1oN0IdYq7YjaIKJirkvBAu2IFucIkmhT7gg/kwSeFer7XxWHTBGF2Vnp73pCnsPKXUcGmA+XKU1hfcCaJzZVjvkKbhLQrPVGN+0P2LCl41c7Q841hQfKZyebjJO4tkeVhlbfxIk0PhrAL3q85fxT8e4LFYrFYjBwB5KZSbHXIPgYAAAAASUVORK5CYII=","orcid":"","institution":"National Forensic Sciences University","correspondingAuthor":true,"prefix":"","firstName":"Satish","middleName":"","lastName":"Kumar","suffix":""}],"badges":[],"createdAt":"2024-02-02 16:29:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3921564/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3921564/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51011431,"identity":"d24a8ab2-e338-402c-80d8-1bec65464ce7","added_by":"auto","created_at":"2024-02-12 16:59:07","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":74164,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent characterization data of PVP-AuNP synthesized for the present work \u003cstrong\u003e(a)\u003c/strong\u003e UV-Visible spectrum \u003cstrong\u003e(b)\u003c/strong\u003e TEM image with particle size histogram \u003cstrong\u003e(c)\u003c/strong\u003e Powder X-Ray diffraction profile \u003cstrong\u003e(d)\u003c/strong\u003e MALDI Mass spectrum and \u003cstrong\u003e(e)\u003c/strong\u003e DLS spectrum showing hydrodynamic diameter (D\u003csub\u003eh\u003c/sub\u003e) of PVP-AuNP in aqueous dispersion\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3921564/v1/7834c83d95b465d984da4c8a.jpg"},{"id":51011637,"identity":"1915131d-104b-4208-9c23-d3eb8eff8897","added_by":"auto","created_at":"2024-02-12 17:07:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":40094,"visible":true,"origin":"","legend":"\u003cp\u003eTime dependent\u003cstrong\u003e \u003c/strong\u003eAGE Fluorescence of HEWL in different experimental condition. The symbols shown here are as follows: circle, HEWL control; square, glycated HEWL; triangle up, glycated HEWL with 100 μM PVP-AuNP; triangle down, glycated HEWL with 200 μM PVP-AuNP\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3921564/v1/ac491f2fabdb9fee3c78f1b3.jpg"},{"id":51011436,"identity":"7054a2ed-4a24-484c-8d6e-9e6102355210","added_by":"auto","created_at":"2024-02-12 16:59:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":91958,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea)\u003c/strong\u003e Scattering light intensity and \u003cstrong\u003eb) \u003c/strong\u003eThioflavin - T fluorescence of HEWL control, glycated HEWL, and glycated HEWL with 100 µM and 200 µM PVP-AuNP at pH 7.4, 37\u003csup\u003e0\u003c/sup\u003eC for 20 days. The symbols shown are as follows: circle, HEWL control; square, glycated HEWL; triangle up, glycated HEWL with 100 μM PVP-AuNP; triangle down, glycated HEWL with 200 μM PVP-AuNP\u003cstrong\u003e c) \u003c/strong\u003eCongo Red binding of HEWL control, glycated HEWL, and glycated sample with 100 µM and 200 µM PVP-AuNP at pH 7.4, 37\u003csup\u003e0\u003c/sup\u003eC on the 20\u003csup\u003eth\u003c/sup\u003e day. Absorbance recorded between 400-600 nm\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3921564/v1/8d0d2a76d2840bf7a5d9d764.jpg"},{"id":51011432,"identity":"757c496f-039f-4c9a-879b-de87a55ad4cc","added_by":"auto","created_at":"2024-02-12 16:59:07","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":60775,"visible":true,"origin":"","legend":"\u003cp\u003eHydrodynamic radii of \u003cstrong\u003ea)\u003c/strong\u003e native HEWL \u003cstrong\u003eb)\u003c/strong\u003e Glycated HEWL \u003cstrong\u003ec)\u003c/strong\u003e Glycated HEWL with100 µM PVP-AuNP and \u003cstrong\u003ed)\u003c/strong\u003e Glycated HEWL with 200 µM PVP-AuNP after 20 days at pH 7.4, 37\u003csup\u003e0\u003c/sup\u003eC is shown here\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3921564/v1/1311659bdc7b0d2154a59d34.jpg"},{"id":51011638,"identity":"fa5aea78-7381-434b-ac13-b6aeb83c60a3","added_by":"auto","created_at":"2024-02-12 17:07:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29814,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence microscopy images (10x) using Thioflavin T Dye of \u003cstrong\u003ea) \u003c/strong\u003eHEWL control \u003cstrong\u003eb)\u003c/strong\u003e Glycated HEWL \u003cstrong\u003ec)\u003c/strong\u003e Glycated HEWL in presence of 100 µM PVP-AuNP after 20\u003csup\u003e \u003c/sup\u003edays of incubation\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3921564/v1/c9f6619a2a72b259e76c7a1d.jpg"},{"id":51011437,"identity":"2baed166-f924-425f-96bc-183e404f0e82","added_by":"auto","created_at":"2024-02-12 16:59:07","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":84924,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea) \u003c/strong\u003eIntrinsic tryptophan fluorescence \u003cstrong\u003eb)\u003c/strong\u003e CD (Far-UV) spectra \u003cstrong\u003ec) \u003c/strong\u003eANS Fluorescence intensity of HEWL control, glycated HEWL and Glycated HEWL with PVP-AuNP after 20 days of incubation at pH 7.4, 37\u003csup\u003e0\u003c/sup\u003eC. For Trp and ANS fluorescence 100 μM and 200 μM PVP-AuNP was used while CD\u0026nbsp; spectra was performed with 100 μM PVP-AuNP\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3921564/v1/ce4243332818a1eee73218c9.jpg"},{"id":51011438,"identity":"858e815c-3910-4159-947a-66236ec90d71","added_by":"auto","created_at":"2024-02-12 16:59:07","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":65357,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea)\u003c/strong\u003e Lytic activity of glycated HEWL in presence and absence of PVP-AuNP alongwith native HEWL at initial slope of 50s. The lines depicted are as follows: continuous line, native HEWL; dashed line, glycated HEWL; dashed dotted line, glycated HEWL with 100 μM PVP-AuNP; dotted line, glycated HEWL with 200 μM PVP-AuNP \u003cstrong\u003eb)\u003c/strong\u003e Recovered catalytic activity (percentage) of glycated HEWL in presence and absence of PVP-AuNP after 20 days of incubation\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3921564/v1/981def7e594c0dcca13af333.jpg"},{"id":51011440,"identity":"79414e25-04dc-4357-81e0-6a07fc7ae6a2","added_by":"auto","created_at":"2024-02-12 16:59:07","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":117765,"visible":true,"origin":"","legend":"\u003cp\u003eTime dependent\u003cstrong\u003e a)\u003c/strong\u003e Carbonyl content and \u003cstrong\u003eb)\u003c/strong\u003e Thiol estimation of HEWL control, glycated HEWL with 100 μM and 200 μM PVP-AuNP after 20 days of incubation at pH 7.4, 37\u003csup\u003e0\u003c/sup\u003eC\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3921564/v1/65d88c5c81ff2889ea1f6daf.jpg"},{"id":51011435,"identity":"8ed67ad5-84e5-4e63-a22a-c7b42a507c84","added_by":"auto","created_at":"2024-02-12 16:59:07","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":72573,"visible":true,"origin":"","legend":"\u003cp\u003eProposed mechanism of PVP-AuNP mediated inhibition of D-ribose induced glycation and aggregation of HEWL at physiological pH\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3921564/v1/bde105f8f2f8cb1925d2438f.jpg"},{"id":51011900,"identity":"8be5e00d-1d62-481b-8101-bad45bd4f83a","added_by":"auto","created_at":"2024-02-12 17:15:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":975169,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3921564/v1/8467be47-738a-44c7-b590-d0e0ce13baab.pdf"},{"id":51011434,"identity":"a7c7b2ca-cbee-4cfd-a895-1f173b56de57","added_by":"auto","created_at":"2024-02-12 16:59:07","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":75619,"visible":true,"origin":"","legend":"","description":"","filename":"GA.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3921564/v1/fe027f27fc8a781e7a9f89c4.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"PVP-AuNP impedes glycation mediated Hen Egg White Lysozyme aggregation under physiological condition","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGlycation is a prevalent non enzymatic post-translational modification that involves cascade of nucleophilic reaction between the free amino group of the protein and the carbonyl group of reducing sugar. Spontaneous glycation yield formation of a Schiff base intermediates reversibly that translates into an Amadori product, which eventually results into advanced glycation end products (AGE) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Formation of AGEs alter and damage the overall structure of biological macromolecules especially proteins. The milieu around protein molecule in the cell, being surrounded by sugars, sugar derivatives, xenobiotics including toxins makes it prone to glycation. Accumulation of AGEs is reported to lead several pathophysiological complications such as diabetic nephropathy or retinopathy and atherosclerosis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Production and accumulation of AGEs also induces protein misfolding and aggregation culminating in several non-neuropathological and neurodegenerative disorders like Alzheimer, Parkinson, and diabetes type II [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Extracellular or intracellular deposition of protein aggregates called amyloid plaques are the histological hallmark of neurodegenerative disorders.\u003c/p\u003e \u003cp\u003eAmong several post-translational modifications, glycation plays a crucial role in formation of amyloid both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Effect of glycation on protein folding, aggregation, amyloid formation and cytotoxicity has been reported earlier [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. AGE induced amyloid fibrils have been found in the brain tissues of patients suffering from transmissible spongiform encephalopathy, Alzheimer\u0026rsquo;s disease and patients having islets of Langerhans of diabetes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It has been observed that site specific Aβ1\u0026ndash;42 glycation affects the formation of amyloid fibrils and generates neurotoxicity to SH-SY5Y cell lines [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Glycation accelerates the aggregation rate of IAPP in the presence of glucose [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Various glycating agents like glucose, D-ribose, methylglyoxal, and glyoxal are found to promote amyloid formation of human albumin under different experimental conditions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Using drosophila and mice model, Oliveira and co-workers demonstrated that glycation enhance α-synuclein toxicity \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eGlobally around 55\u0026nbsp;million people living with dementia have been reported, and this is expected to increase to nearly 78\u0026nbsp;million by 2030 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Understanding its socioeconomic burden, several manoeuvres are being looked upon to impede the conversion of native protein to its toxic form which in turn ameliorates aggregation-associated various neurodegenerative and non-neuropathic conditions. Small organic molecules [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], synthetic peptides [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], polysaccharides [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] are reported to inhibit aggregation of proteins under different experimental conditions. Various \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies have demonstrated that inhibition of glycation reaction may be used as a therapeutic approach against diabetes related ailments and other metabolic disorders [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eNanomaterials owing to their smaller size and higher surface to volume ratio are potential agent for the diagnosis and prevention of diseases. Recent studies have reported that gold nanoparticles (AuNPs) can function as an antiglycation agent, thereby inhibiting the formation of AGEs [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The inhibitory effect of AuNPs on bovine serum albumin glycation in presence of D-ribose advocates that colloidal particles could be a potential therapeutic agent for the treating diabetes and its associated pathophysiological conditions such as hyperglycaemia [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Silver and gold nanoparticles are also observed to demonstrate anti-glycating property upon human serum albumin [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Polyvinylpyrrolidone (PVP) conjugated gold nanoparticles are observed to restrain the hen egg white lysozyme aggregation at relatively lower pH and elevated temperature [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, the mechanism underlying this study was elusive. Hen Egg White Lysozyme (HEWL) protein serves as an excellent model protein to investigate the structural and conformational changes that a protein undergoes during misfolding and aggregation owing to its globular structure and enzymatic activity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. 60% sequence similarity of HEWL and human lysozyme is observed, that is majorly responsible for hereditary systemic amyloidosis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] .\u003c/p\u003e \u003cp\u003eIn current study, we explored the effect of polyvinylpyrrolidone (PVP) conjugated gold nanoparticle (PVP-AuNP) on structure and function of HEWL in presence of D-ribose under physiological condition, 37\u0026deg;C, pH 7.4. Present study shows that PVP-AuNP reduce the formation of AGEs and inhibit ribose mediated aggregation of HEWL. Our observations suggests that interaction of gold nanoparticle modulates the secondary structure of protein and helps in retaining its catalytic activity in presence of ribose over the period of 20 days. Further, PVP-AuNP was observed to dampen the self-assembly of lysozyme by affecting carbonyl and thiol content. Our results pose PVP-AuNP nanoparticle holds a novel therapeutic potential against AGEs mediated neurodegenerative disorders and other metabolic complications.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003eHen egg white lysozyme (HEWL: L-6876), D-ribose (analytical grade), Thioflavin T (ThT), \u003cem\u003eMicrococcus lysodeikticus\u003c/em\u003e, Chloroauric Acid, Congo Red, 8-anilinonaphthalene-1-sulfonic acid (ANS) dye, Ellman\u0026rsquo;s reagent, Sodium phosphate monobasic, Polyvinyl pyrrolidone K-30, sodium tetra hydroborate were obtained from Sigma-Aldrich. Sodium azide, 2\u0026ndash;4 dinitrophenylhydrazine (DNPH) were purchased from Himedia Laboratories. All other chemicals used belonged to analytical grade.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Preparation and characterization of Polyvinylpyrrolidone (PVP) conjugated Gold Nanoparticle (AuNP)\u003c/h2\u003e \u003cp\u003ePolyvinylpyrrolidone conjugated gold nanoparticle (PVP-AuNP) was prepared using standard protocol as reported earlier [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. 1 mM HAuCl\u003csub\u003e4\u003c/sub\u003e aqueous solution (30 mL) was mixed with polyvinylpyrrolidone (PVP, K30, 40 kDa) and was subjected to continuous stirring at 273 K for 20 min. Fresh aqueous solutions of PVP/ HAuCl\u003csub\u003e4\u003c/sub\u003e were combined alongwith sodium borohydride (NaBH\u003csub\u003e4\u003c/sub\u003e) (0.1 M, 1.5 mL, 5 molar equivalent with respect to HAuCl\u003csub\u003e4\u003c/sub\u003e), a potent reducing agent, and vigorously stirred for 30 minutes at 273 K to produce the conjugated nanoparticle (PVP-AuNP). Next, utilizing a membrane filter for ultra-filtration, the dispersed aqueous nanoparticle was purified. A strong ruby red colour was developed after 15 to 20 minutes, indicating the synthesis of gold nanoparticle (PVP-AuNP). Following that, numerous biophysical approaches were used to characterise this nanoparticle solution. Characterization of PVP- AuNP was done using different biophysical methods such as UV-Visible spectroscopy, X-Ray diffraction patterns, transmission electron microscopy (TEM), dynamic light scattering, and MALDI Mass spectrometry.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFresh HEWL stock solution was prepared in double deionized water. Protein concentration was determined by using a molar extinction coefficient of 37970 M\u003csup\u003e-1\u003c/sup\u003e cm\u003csup\u003e-1\u003c/sup\u003e and calculating the absorbance (OD) at 280 nm. For the experiment, stock was diluted to 60 \u0026micro;M as final concentration of protein in sodium phosphate buffer (50 mM) with pH 7.4. Stock solution of D-ribose sugar (0.5 M) was prepared in distilled water and kept at 4\u0026deg;C before use. Phosphate buffer was filtered using 0.22 \u0026micro;m filter.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Protein glycation study\u003c/h2\u003e \u003cp\u003eTo induce glycation, 60 \u0026micro;M HEWL was incubated with 100 \u0026micro;M D-ribose sugar at 37\u003csup\u003e0\u003c/sup\u003eC, pH 7.4. Sodium azide (1 mM) was added to avert any bacterial contamination. To study the effect of nanoparticles on glycated protein, 100 \u0026micro;M and 200 \u0026micro;M PVP-AuNP was incubated individually to HEWL with D-ribose at 37\u003csup\u003e0\u003c/sup\u003eC, pH 7.4 for the time period of 20 days. Protein sample in absence of ribose was considered as a control. Aliquots were withdrawn at specific time point for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4 AGE Fluorescence spectroscopy\u003c/h2\u003e \u003cp\u003eFormation of advanced glycated end products (AGE) under different conditions were monitored by measuring the fluorescence spectra as reported earlier [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Glycated HEWL with and without PVP-AuNP along with the control were monitored with excitation at 370 nm with slit width of 2 nm and the emission were monitored in the range of 390\u0026ndash;600 nm with slit width of 2 nm. Fluorescence at 440 nm was recorded as a signature of glycated species in present experiment [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Light scattering measurement\u003c/h2\u003e \u003cp\u003eTo monitor the presence of glycation induced protein aggregates, scattering intensity of glycated HEWL with and without nanoparticles were measured at 350 nm at a 90\u003csup\u003e0\u003c/sup\u003e angle. Samples were monitored with excitation at 350 nm with 2 nm slit width and the emission spectra was measured at 320\u0026ndash;400 nm at 2 nm slit width using Jobin-Yvon Fluoromax 4 spectrofluorometer. Three distinctive spectra were averaged after subtracting with respective blank.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Thioflavin T (ThT) dye assay\u003c/h2\u003e \u003cp\u003eFresh ThT stock was made in double deionized water. Protein samples were diluted in 50 mM phosphate buffer (pH 7.4) such that the molar ratio of protein to ThT dye was 1:2. The protein concentration was 10 \u0026micro;M in the assay. Glycated HEWL with and without PVP-AuNP along with the control at different time points were monitored with excitation at 450 nm with 5 nm slit width, and the emission spectra were measured at 470\u0026ndash;550 nm with 10 nm slit width at different time points. Three distinctive spectra were averaged after subtracting with respective blank.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Congo Red dye assay\u003c/h2\u003e \u003cp\u003eFresh Congo Red dye stock solution (4 mM) was formulated and diluted in sodium phosphate buffer (50 mM). Glycated HEWL with and without PVP-AuNP along with the control at different time points were diluted with congo red (in 50 mM phosphate buffer pH 7.4) in a manner that the molar ratio of protein to congo red was ~\u0026thinsp;1:2. In the assay, the protein concentration was 10 \u0026micro;M. Absorption spectra were monitored at 400\u0026ndash;700 nm wavelength spectrum using cuvette of 1cm path length.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Dynamic Light Scattering (DLS)\u003c/h2\u003e \u003cp\u003eSamples were analysed for DLS measurement at 633 nm wavelength through a laser and 90\u0026deg; scattering angle of the detector. All the measurements were done using the Zetasizer Nano S90 DLS (Malvern Instrument Ltd, United Kingdom). 20 measurements were recorded for 2 ml native and glycated protein samples with and without PVP-AuNP at a constant temperature (25\u003csup\u003e0\u003c/sup\u003eC) using quartz cuvette. Data was processed through Malvern Zetasizer Software 7.11.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Fluorescence microscopic study\u003c/h2\u003e \u003cp\u003eFluorescence microscopy was performed by incubating the glycated protein samples with and without PVP-AuNP with ThT dye in a molar ratio of 1:2 for 30 mins in dark, and then mounted on a clean slide and covered using a cover slip. Samples were visualized and the images were obtained by using Nikon DM 5200 Fluorescence microscope. Observations were performed with 10X/0.25 objective lens. Control HEWL (absence of ribose) was also run concurrently.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Tryptophan fluorescence\u003c/h2\u003e \u003cp\u003eThe intrinsic tryptophan fluorescence was measured by exciting the glycated HEWL with and without PVP-AuNP along with the control for different time at 295 nm with 2 nm slit width and the emission spectra were recorded in the range of 310\u0026ndash;400 nm with 10 nm slit width using Jobin-Yvon Fluoromax 4 spectrofluorometer. Three separate spectra were averaged after subtracting with each corresponding blank.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Circular Dichroism (CD) spectroscopy\u003c/h2\u003e \u003cp\u003eCircular Dichroism experiment was executed using quartz cuvette with path length (0.1 cm) at 25\u003csup\u003e0\u003c/sup\u003eC with JASCO J815 spectropolarimeter. CD spectra of each sample was generated in the far ultraviolet absorbance region (190\u0026ndash;250 nm) by averaging 20 consecutive scans at 50 nm min \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e scan rate with a suitable baseline. The response time was set to 1 s and the band width was achieved at 1 nm. Data was interpreted using the K2D3 software [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.12 8-anilinonaphthalene-1-sulfonic acid dye assay\u003c/h2\u003e \u003cp\u003eANS (8-anilinonaphthalene-1-sulfonic acid) stock was freshly prepared and diluted with phosphate buffer (pH 7.4). Glycated HEWL with and without PVP-AuNP along with the control were diluted with ANS dye in a manner that the molar ratio of protein to ANS was ~\u0026thinsp;1:4. The protein concentration was 10 \u0026micro;M in the assay. Samples were monitored with excitation wavelength at 380 nm with 2 nm slit width, and the emission wavelength at 400\u0026ndash;600 nm with slit width of 10 nm at different time points. Three separate spectra were averaged after subtracting with each corresponding blank.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.13 HEWL lytic assay\u003c/h2\u003e \u003cp\u003eFresh \u003cem\u003eMicrococcus lysodeikticus\u003c/em\u003e stock solution was prepared in deionized water. This stock was further diluted to a concentration of 78 \u0026micro;g/mL in assay medium (phosphate buffer). Native and glycated samples with and without the gold nanoparticle (PVP-AuNP) were diluted in a way that the final concentration of protein was 70 nM. The lysozyme activity was monitored using a double beam spectrophotometer by measuring the slope for first 50 seconds and decrease in the initial rate at 450 nm absorbance range.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Determination of carbonyl content\u003c/h2\u003e \u003cp\u003eCarbonyl content of the protein under different conditions was measured using 2,4 dinitrophenylhydrazine reagent (2,4 DNPH). Stock solution of 2,4-DNPH (10 mM) was made in 2.5 M Hydrochloric acid. Next 2,4 DNPH reagent (400 \u0026micro;L) was added to 100 \u0026micro;L protein samples, which was then followed by incubation at room temperature for 1 hour. After incubation, 500 \u0026micro;L of chilled 20% Trichloroacetic acid (TCA) solution were added to the samples. These samples were centrifuged for 5 minutes, 10000 rpm at 4\u003csup\u003e0\u003c/sup\u003eC. 1:1 (v/v) ethanol/ethylacetate solution was used to wash the resulting pellets thrice followed by dissolving the pellets in guanidine hydrochloride solution (6M). Protein carbonyl content was calculated using the molar coefficient 22000 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 370 nm using UV-Visible spectrophotometer [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Determination of thiol groups\u003c/h2\u003e \u003cp\u003eThiol group was estimated using a standard Ellman\u0026rsquo;s method [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The samples were mixed with 1 mM ethylenediaminetetraacetic acid (EDTA) and 5,5\u0026rsquo;-dithiobis-2-nitrobenzoic acid (DTNB) reagent and then incubated for 30 minutes at 37\u003csup\u003e0\u003c/sup\u003eC. The final volume of all the samples were made up to 1ml using Tris-HCl buffer (pH 8). Final absorbance was calculated at 412 nm using the molar coefficient 14000 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using spectrophotometer.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003e3.1 Characterization of Polyvinylpyrrolidone (PVP) conjugated Gold Nanoparticle (AuNP)\u003c/h2\u003e\n \u003cp\u003eAfter synthesis, characterization of PVP-AuNP was done using biophysical techniques like UV-Visible spectroscopy, Dynamic light scattering (DLS), X-ray diffraction (XRD) analysis, MALDI and imaging by Transmission Electron Microscope (TEM). The details characterizations of the synthesized nanoparticles are shown below (Fig.\u0026nbsp;1). UV-Vis Spectroscopy of PVP-AuNP showed featureless exponential absorption profile in the UV (250–400 nm) range indicating formation of ultra small (non-Plasmonic) gold nanoparticles (Fig.\u0026nbsp;1a). The TEM image with particle size histogram (Fig.\u0026nbsp;1b) showed the presence of highly monodisperse gold particles with average diameter of 1.5 ± 0.2 nm. XRD diffraction profile of PVP-AuNP along with the standard diffraction profile of bulk gold are shown in Fig.\u0026nbsp;1c, which showed a broad diffraction profile at 38º (2Ɵ) indicating the presence of (111) crystal plane. The ultra small crystal size of nanoparticles makes the X-ray diffraction profile broad. The MALDI mass spectrum also confirms the presence of gold particles with average nuclearity of 33–35 atoms per particle (Fig.\u0026nbsp;1d). The hydrodynamic diameter (D\u003csub\u003eh\u003c/sub\u003e) of PVP-AuNP dispersed in water were found to be 8.2 ± 0.5 nm (Fig.\u0026nbsp;1e). Difference in size of PVP-AuNP as monitored by TEM and DLS was owing to the fact that TEM display the size of nanoparticles in solid state while water shell around PVP-AuNP also contribute to the size of nanoparticles monitored by DLS.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003e3.2 PVP-AuNP inhibits formation of advanced glycated product (AGE)\u003c/h2\u003e\n \u003cp\u003eTo investigate the formation of advanced glycated product (AGE) products, \u003cem\u003ein vitro\u003c/em\u003e glycation of HEWL protein was carried out in the presence of D-ribose sugar at physiological condition; 37°C, pH 7.4. Earlier work has demonstrated the effect of ribose on various protein, however these studies either used very high concentration of ribose or performed at non-physiological conditions [31]. As the concentration of D-ribose present in the blood is about 100 µM [32] we explored the same concentration of sugar to induce glycation on HEWL and to investigate the effect of PVP-AuNP.\u003c/p\u003e\n \u003cp\u003eAfter incubating 60 µM HEWL in presence 100 µM D-ribose, formation of AGE product was monitored by characteristic fluorescence emission at 440 nm [33]. Figure\u0026nbsp;2 shows the time dependent AGE production in different experimental condition. Compared to control (HEWL without D-ribose), gradual increase in emission intensity was observed in HEWL with 100 µM D-ribose over the period of 12 days which was saturated thereafter, indicating the formation of AGE product. In presence of 100 µM and 200 µM PVP-AuNP, fluorescence intensity reduced by ~ 21% and ~ 60% respectively, suggesting that functionalized gold nanoparticle inhibit the formation of AGE product in concentration dependent manner. Reduction in AGE product formation was much in line with earlier finding where AgNPs and AuNPs are observed to inhibit AGE formation in HSA [34].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003e3.3 PVP-AuNP inhibits AGE induced HEWL aggregation\u003c/h2\u003e\n \u003cp\u003eAdvanced glycation end products are known for the onset of various diseases such as neurodegenerative disorders including Parkinson's disease, Alzheimer's disease and aging [35]. Accumulation of AGEs in various tissues induce oxidative stress, and inflammation leading to misfolding and aggregation of several proteins. Next, we investigated if gold nanoparticle affects the AGEs induced aggregation of HEWL at physiological condition. Figure 3a shows the static light scattering of glycated lysozyme in presence and absence of 100 µM and 200 µM PVP-AuNP individually.\u003c/p\u003e\n \u003cp\u003ePresence of higher molecular weight species is often characterized by an increase in the scattering intensity which arises due to aggregation of the biomolecule [36]. Marginal increase in scattering intensity of control (HEWL alone) indicates the prevalence of monomeric protein after twenty days of incubation, while D-ribose induced lysozyme aggregation in presence and absence of gold nanoparticle was observed to be sigmoidal in nature. Aggregation kinetics of HEWL with sugar but without gold nanoparticle shows the lag phase of ~ 1 day. Gradual increase in scattering intensity was observed upto 14 days and was saturated thereafter. Scattering intensity drastically declined in presence of 100 µM and 200 µM PVP-AuNP during 20 days of incubation. Compared to HEWL with ribose, nearly 23% and 55% reduction in scattering intensity in presence of 100 µM and 200 µM gold nanoparticle respectively, shows the dose dependent inhibitory effect on lysozyme aggregation. Addition to reduced scattering intensity, Fig.\u0026nbsp;3a also indicate the increased lag phase in presence of PVP-AuNP. In absence of gold nanoparticle which shows t\u003csub\u003e1/2\u003c/sub\u003e at ~ 3.5 days, presence of 100 µM and 200 µM gold nanoparticle increases the t\u003csub\u003e1/2\u003c/sub\u003e to ~ 5 days thus signifying that PVP-AuNP impedes the growth of HEWL fibrillation in presence of ribose.\u003c/p\u003e\n \u003cp\u003eChanges in scattering intensity advocates the presence of larger sized protein aggregates and not specify if these structures are ordered or amorphous in nature. Ordered protein structures called amyloid fibrils exhibiting enhanced fluorescence subsequent to binding with thioflavin T (ThT), an amyloid specific probe are hallmark of several neurodegenerative disorders [37]. We further investigated whether PVP-AuNP inhibits the formation of amyloid like structure employing ThT fluorescence. Figure\u0026nbsp;3b depicting time dependent ThT fluorescence of glycated HEWL in absence and presence of PVP-AuNP demonstrate the similar aggregation kinetics as observed by scattering (Fig.\u0026nbsp;3a).\u003c/p\u003e\n \u003cp\u003eHEWL with ribose in absence of nanoparticles was showing gradual increase in ThT fluorescence till 14 days and was constant for the time window of 20 days (Fig.\u0026nbsp;3b) indicate the presence of ThT sensitive amyloid structures. Similar observation was reported earlier where enhanced ThT fluorescence was exhibited by lysozyme in presence of 500 µM D-ribose upto 31 days which decreased further [38]. In presence of 100 µM and 200 µM PVP-AuNP nearly 25% and 40% reduction respectively in ThT fluorescence manifest the inhibitory effect of gold nanoparticle on aggregation and amyloid formation of lysozyme in presence of sugar. Aggregation often leads to higher β-sheet structure in protein, which binds with ThT resulting in enhanced fluorescence [39]. In present result, reduced ThT fluorescence of glycated HEWL in presence of gold nanoparticles indicates lesser proportion of β-sheet which are signature of misfolded and aggregated proteins. We however, probed the secondary structural change of HEWL in present study employing circular dichroism (CD) in section 3.5.\u003c/p\u003e\n \u003cdiv\u003e\n \u003cp\u003eResult obtained from ThT fluorescence was further validated through Congo red (CR) assay. CR is another amyloid specific probe which intercalates between the β-strands of aggregated protein and exhibit enhanced absorbance at around 500 nm [40]. Compared to HEWL without sugar, enhanced absorbance was observed in presence of 100 µM D-ribose in absence of nanoparticles after 20 days of incubation (Fig.\u0026nbsp;3c). This was in agreement with earlier study where D-ribose induced protein aggregates demonstrated enhanced CR absorbance [41]. In presence of 100 µM and 200 µM PVP-AuNP, decrease in CR absorbance after the same time period complement our ThT observation that gold nanoparticles prevent formation of sugar induced amyloid structures.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003e3.4 Characterization of HEWL aggregates in presence and absence of PVP-AuNP\u003c/h2\u003e\n \u003cdiv\u003e\n \u003cp\u003eAfter confirming the inhibitory effect of gold nanoparticle on ribose mediated HEWL aggregation, we characterize these structures by dynamic light scattering (DLS) and fluorescence microscopy. DLS has extensively been used to study the growth of protein aggregates and inhibition under different conditions. Figure\u0026nbsp;4a showing hydrodynamic radii (R\u003csub\u003eh\u003c/sub\u003e) of freshly prepared native HEWL as 2.13 nm was in agreement of globular lysozyme as reported earlier [42]. HEWL in presence of D-ribose without gold nanoparticle after 20 days shows the R\u003csub\u003eh\u003c/sub\u003e of ~ 446 nm manifesting the population of higher order aggregates (Fig. 4b). After the same time interval, sugar incubated HEWL in presence of 100 µM and 200 µM PVP-AuNP demonstrated the hydrodynamic radii of ~ 404 nm and ~ 361 nm respectively (Fig. 4c and 4d) clearly indicating that gold nanoparticles impede the growth of D-ribose induced lysozyme aggregation. Data obtained from scattering, ThT fluorescence, CR, DLS in unison suggests that PVP-AuNP inhibits D-ribose induced HEWL aggregation in concentration dependent manner.\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eFluorescence microscopy employing Thioflavin T (ThT) fluorescence has also been used to monitor protein aggregation and inhibition [22]. Formation and deposition of ordered protein structures as fibrils or amorphous aggregates are evident in several neurodegenerative disorders. Although from Fig.\u0026nbsp;3b using ThT fluorescence it was very apparent that gold nanoparticle inhibits the D-ribose mediated lysozyme aggregation, we further investigated the types of structures formed by fluorescence microscopy using ThT as a probe (Fig.\u0026nbsp;5).\u003c/p\u003e\n \u003cp\u003eHEWL control after 20 days of incubation did not show any fluorescent structure indicating the presence of globular protein (Fig.\u0026nbsp;5a). In presence of 100 µM D-ribose, without PVP-AuNP, long fibrillar structure exhibiting ThT fluorescence was observed (Fig.\u0026nbsp;5b), whereas in presence of 100 µM PVP-AuNP after 20 days, no fibrillar structure displaying ThT fluorescence reveal that gold nanoparticles inhibit the formation of HEWL fibrils in presence of sugar (Fig.\u0026nbsp;5c). As discussed earlier, ThT a positively charged benzothiazole-based fluorophore is hypothesized to bind with ordered β-sheet structure of aggregated protein thus leading to enhance fluorescence owing to restricted torsional relaxation between benzothiazole ring and benzene ring. Several anti-amyloidogenic compounds are known to inhibit protein aggregation by blocking β-pleated sheet structure [43]. Thus, it is possible that functionalized gold nanoparticle also blocks the formation of β- pleated sheet structure which is apparent from diminished ThT fluorescence.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\"\u003e\n \u003ch2\u003e3.5 PVP-AuNP promote native like structure of HEWL in presence of D-ribose\u003c/h2\u003e\n \u003cp\u003eSmall anti-amyloidogenic molecules subsequent to binding with protein, resists the structural alteration in biomolecule under aggregation prone condition. In order to probe the conformational changes in native protein in the present study, HEWL with D-ribose in absence and presence of PVP-AuNP was subjected to tryptophan (Trp) fluorescence, far-UV circular dichroism spectroscopy and 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence assay after 20 days of incubation. Intrinsic Trp fluorescence is extensively explored to investigate conformational changes in protein under different condition including glycation [44]. We first probed the Trp fluorescence of glycated HEWL in presence of different concentration of functionalized nanoparticles after 20 days at pH 7.4. Compared to HEWL control where λ\u003csub\u003emax\u003c/sub\u003e was ~ 347 nm, glycated HEWL without gold nanoparticles demonstrated λ\u003csub\u003emax\u003c/sub\u003e around 342 nm coupled with marked reduction in the fluorescence intensity indicating structural alteration in HEWL (Fig. 6a). This results was in accordance with earlier findings that showed reduction in tryptophan fluorescence and shift in emission maxima of glycated protein at 37\u003csup\u003e0\u003c/sup\u003eC, pH 7.4 [45]. In presence of 100 µM and 200 µM PVP-AuNP on the other hand, Trp fluorescence increased to ~ 1.6 fold and ~ 1.8 fold respectively, in comparison to glycated protein resonates that conjugated gold nanoparticle promotes native like structure of HEWL in presence of sugar.\u003c/p\u003e\n \u003cp\u003eWe further explored Circular dichroism (CD) spectroscopy, an extensively used technique to study the secondary structural aspects of protein. CD can easily distinguish changes in helical structure, parallel and antiparallel β-sheets of protein molecules. Native HEWL in absence of sugar showed α-helical structure with negative band at 208 nm and shoulder around 222 nm. In presence of 100 µM D-ribose, helical signals flattened at 208 nm and 222 nm and significantly changed the molar ellipticity around 216–220 nm indicating the decrement in α-helical content and presence of β sheet structure respectively (Fig.\u0026nbsp;6b). This observation supported earlier studies where glycation was known to induce β sheet structure in globular protein [46]. However, in presence of 100 µM gold nanoparticle, an increase in α-helical and decrease in β-sheet was observed. The secondary structural aspect of the protein was determined using K2D3 software, where in native lysozyme 32.87% α-helix and 12.82% β-sheet content was observed. In ribosylated HEWL the α-helix content reduced to 11.51% and β-sheet content increased to 19.08%. In presence of 100 µM PVP-AuNP, ribosylated HEWL displayed 14.77% α-helical and 18.42% β-sheet. Reduction in β-sheet coupled with increase in α-helix strongly suggests that gold nanoparticles inhibit the formation of β-sheet structure in glycated HEWL. Taken together, data obtained from ThT fluorescence (Fig.\u0026nbsp;3b), fluorescence microscopy (Fig.\u0026nbsp;5), CD (Fig.\u0026nbsp;6b) clearly reveals that PVP-AuNP inhibit sugar induced HEWL aggregation by blocking formation of β-sheet structures.\u003c/p\u003e\n \u003cp\u003ePrevious studies have shown that sugar molecules induce molten globule-like structures in HEWL, characterized by exposure of buried hydrophobic patches which eventually act as nucleation factor for inducing protein aggregation. Exposure of hydrophobic regions of protein is characterised by enhanced ANS fluorescence alongwith blue shift in emission maximum [47]. Figure\u0026nbsp;6c showing emission maximum of native HEWL after 20 days of incubation was ~ 512 nm. In presence of 100 µM D-ribose, ANS fluorescence increased at emission maximum ~ 486 nm coupled with blue shift which signifies the exposure of hydrophobic patches in lysozyme. Presence of 100 µM and 200 µM gold nanoparticle not only reduced the ANS intensity but also changed the emission maximum to ~ 504 nm and ~ 503 nm respectively of glycated HEWL. Result for ANS fluorescence mirrors that PVP-AuNP not only impedes the D-ribose induced exposure of hydrophobic regions of HEWL but also tries to bring the protein near its native confirmation.\u003c/p\u003e\n \u003cp\u003eOwing to globular structure and well-established enzymatic activity, HEWL has been extensively used to study protein aggregation and in its inhibition under varied conditions [48]. As functional activity is a peculiar characteristic of native protein structure, we probed the activity of ribosylated HEWL alongwith 100 µM and 200 µM gold nanoparticle after 20 days of incubation. Figure 7a represents the lytic activity of \u003cem\u003eMicrococcus lysodeikticus\u003c/em\u003e, a lysozyme substrate under different experimental conditions.\u003c/p\u003e\n \u003cp\u003eLinear decline in turbidity in initial 50s was used for calculating the HEWL activity by comparing with freshly prepared native HEWL (pH 7.4) whose activity was assumed to be 100%. Figure\u0026nbsp;7b shows ~ 70% decrease in ribosylated HEWL activity indicating the loss of native protein structure. In presence of 100 µM and 200 µM PVP-AuNP, activity of HEWL was observed to be around 65% and 90% respectively. It has been hypothesized that changes in positive charges on HEWL and motions of the low ordered regions due to ribosylation decrease the HEWL activity [49]. It is thus possible that interaction between negatively charged PVP-AuNP and positively charged HEWL stabilize the protein to retain its activity in presence of sugar. We have earlier demonstrated that chitotriose and brahmi recovers the HEWL activity at pH 12.2 and pH 2 respectively by stabilizing the structure [50, 51]. Data obtained from Trp fluorescence, circular dichroism and ANS fluorescence in association with enzymatic activity strongly advocates that gold nanoparticles stabilize the native structure of HEWL.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\"\u003e\n \u003ch2\u003e3.6 PVP-AuNP modulates the protein-bound carbonyl groups and thiol group in HEWL\u003c/h2\u003e\n \u003cp\u003eNon-enzymatic protein glycation involves excessive chemical attachment of sugar to the protein leading to the formation of early glycation products (EGPs). Further oxidation of EGPs generates protein-bound dicarbonyl intermediates, a key mediator whose accumulation in tissue contributes to the formation of AGEs. Increase in protein carbonyl content is reported to develop several neurodegenerative diseases, cancer, atherosclerosis, diabetes, and aging [52] by inducing oxidative stress, we probed the level of carbonyl content (CC) of HEWL with and without gold nanoparticles in presence of D-ribose.\u003c/p\u003e\n \u003cp\u003eFigure\u0026nbsp;8a depicts CC of samples under varied experimental conditions at different time period. In absence of gold nanoparticles, gradual increase in CC over the time window of 20 days was observed in presence of sugar. A significant increase of CC by 90.65%, compared to unmodified HEWL after 20 days manifest the presence of reactive intermediate which induces oxidative stress and alters the structure of biomolecule. In presence of 100 µM and 200 µM PVP-AuNP dose dependent reduction in CC content was observed. After 20 days, 100 µM and 200 µM PVP-AuNP substantially reduced protein bound CC by 32.65% and 40.77% respectively, compared to glycosylated HEWL without nanoparticles. Positively charged amino acids like lysine and arginine are potential target for glycation thus responsible for elevated carbonyl intermediates [52, 53]. Interaction between lysine residue and gold nanoparticle are suggested to form ionic bridge generating carboxylate-ammonium type of salt. It is thus possible that electrostatic interaction between negatively charged functionalized gold nanoparticles and positively charged lysine/arginine residues hinders the interaction between sugar and protein leading to the reduced production of CC. Since carbonylation of amino acids induces protein aggregation by promoting formation of covalent and non-covalent bonds and protein unfolding [54], reduced CC along with our CD and ANS results suggest that binding of PVP-AuNP resists D-ribose induced conformational alteration in lysozyme in concentration dependent manner.\u003c/p\u003e\n \u003cp\u003eThiol groups plays a pivotal role in providing stability and solubility of proteins. Glycation and subsequent production of intermediate dicarbonyl compounds react promptly with thiol (-SH) groups of specific amino acids to generate thiol-aldehyde adducts. Further depletion in thiol group augments overall oxidative damage to the biomolecule [55]. As reduction in thiol groups is observed in fructose mediated glycation in BSA culminating in structural changes [56], we studied the same in glycated HEWL in absence and presence of 200 µM and 100 µM PVP-AuNP. Nearly 46% decrease in free thiol content of glycated HEWL was observed compared to native lysozyme after 5 days while after 20 days of incubation ~ 94% reduction in thiol was monitored (Fig.\u0026nbsp;8b).\u003c/p\u003e\n \u003cp\u003eThis finding was in agreement with earlier study where reduction in free thiols was observed in glycated lysozyme [57]. Treatment with gold nanoparticles to glycated HEWL on the other hand attenuated the decrease in thiol group. After 20 days, percentage prevention of depleting thiol group in presence of 100 µM and 200 µM PVP-AuNP was 38.10% and 47.14% respectively. Inhibitory effect of gold nanoparticle in present study supports the previous finding where reduction in thiol group was less in presence of bio-enzymatically synthesized gold and silver nano particles compared to glycated human serum albumin [34]. It has been argued that degradation of Amadori products induces free radical formation through oxidation of proteins which can be estimated through thiol group. Data obtained from above study signify that PVP-AuNP suppress the oxidation of HEWL in presence of sugar in concentration dependent manner and thus inhibits production of AGEs.\u003c/p\u003e\n \u003cdiv\u003e\n \u003cp\u003eHere for the first time, we proposing the mechanism explaining how functionalized gold nanoparticle (PVP-AuNP) inhibits D-ribose induced glycation but also retain its activity at physiological pH. D-ribose induces the formation of carbonyl content which eventually induces formation of AGEs. Parallelly, increased carbonyl content also induces oxidative stress leading to reduction in thiol group of protein. Increased carbonyl content, AGE products and reduced thiol collectively destabilizes the tertiary structure of HEWL leading to the exposure of hydrophobic regions coupled with quenching of exposed Trp molecules which ultimately manifested in HEWL aggregation and reduction in its catalytic activity.\u003c/p\u003e\n \u003cp\u003eAddition of PVP-AuNP, on the other hand, inhibited the formation of carbonyl content, AGE production, and also reduced the oxidative stress. Additionally, PVP-AuNP also modulated the structure of HEWL in order to retain its native-like structure as it was reflected by ANS and Trp fluorescence, along with retention of catalytic activity after 20 days of incubation period.\u003c/p\u003e\n \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eInhibiting protein glycation and reduction in AGEs production through small molecules is paramount against several neurodegenerative and other disorders. Although chemically synthesized drugs do inhibit process of protein aggregation, in most cases this inhibition comes at the cost of structural and functional activity of protein. Present study demonstrates that polyvinylpyrrolidone conjugated gold nanoparticles inhibit D-ribose induced HEWL glycation, eventually reducing AGEs formation in concentration dependent manner. Our results reveal that subsequent to interaction, PVP-AuNP prevent D-ribose induced structural alteration in HEWL which in turn modulates the hydrophobic regions and secondary structure of protein. These modifications further stabilize lysozyme’s tertiary structure which was evident from its activity. Preventing the increase in carbonyl content and resisting the decrease in thiol group in presence of gold nanoparticles signifies the antioxidant property of PVP-AuNP which protect the protein against oxidative stress causing misfolding and aggregation, culminating in several disorders. Findings of present study shows that PVP-AuNP possesses significant anti-glycation property and offers as an effective alternate against the treatment of diabetes, Alzheimer disease, Parkinson disease and atherosclerosis. However, more \u003cem\u003ein-vivo\u003c/em\u003e and clinical studies are required to reveal the therapeutic effects of PVP-AuNP.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAGEs, advanced glycation end products; ANS, 8-anilino-1-naphthalene sulfonate; CD, circular dichroism; DLS, dynamic light scattering; HEWL, hen egg white lysozyme; PVP-AuNP, polyvinylpyrrolidone conjugated gold nanoparticles; ThT, thioflavin T; Trp, tryptophan\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank National Forensic Sciences University, Gandhinagar for providing the infrastructure and experimental facilities that made this work possible. We gratefully acknowledge the Central Instrumentation Facility (CIF) at IIT Gandhinagar for providing CD facility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJennifer Johnson:\u003c/strong\u003e Methodology, Validation, Formal analysis, Investigation, Data curation, Writing - original draft, Software. \u003cstrong\u003eTushar Tyagi:\u003c/strong\u003e Methodology, Data curation. \u003cstrong\u003ePrasenjit Maity:\u003c/strong\u003e Formal analysis, Writing - review \u0026amp; editing. \u003cstrong\u003eSatish Kumar:\u003c/strong\u003e Conceptualization, Project administration, Visualization, Supervision, Writing - review \u0026amp; editing, Methodology, Formal analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial interests -\u0026nbsp;\u003c/strong\u003eThe authors declare they have no financial interests to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMonnier VM. 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BMC Complement Altern Med. 2015;15:27.\u003c/li\u003e\n\u003cli\u003eMuraoka MY, Justino AB, Caixeta DC, et al. Fructose and methylglyoxal-induced glycation alters structural and functional properties of salivary proteins, albumin and lysozyme. Nagaraj R, editor. PLOS ONE. 2022;17:e0262369.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Aggregation, Glycation, Gold nanoparticle, Lysozyme, Neurodegeneration","lastPublishedDoi":"10.21203/rs.3.rs-3921564/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3921564/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProtein glycation a non-enzymatic protein modification, alters the structure of biomolecule leading to several neurodegenerative and other disorders. As onset of disorders due to protein glycation is primarily driven by the development of advanced glycation end products (AGEs), therapeutic intervention against related disorders by inhibiting AGEs production is imperative. Nanoparticles have recently gained more prominence as therapeutic agents in biological field such as medicine, drug discovery and diagnosis. In present study, we extensively investigated the effect of chemically synthesized polyvinylpyrrolidone conjugated gold nanoparticles (PVP-AuNP) on D-ribose induced glycation of hen egg white lysozyme (HEWL) under physiological conditions. Our finding shows that AGEs formation was inhibited by PVP-AuNP over the period of 20 days. Interaction of gold nanoparticles prevented glycation induced misfolding and aggregation of lysozyme by stabilizing its native structure, which was evident with static light scattering, ThT, Congo red and ANS fluorescence coupled with CD spectroscopy. Further, by estimating carbonyl content and thiol group, our study suggests that PVP-AuNP possesses antioxidant property thus prevent the HEWL against glycation driven oxidative damage. Present study therefore elucidates that PVP-AuNP a significant antiglycation agent can be used against wide range of disorders induced by AGEs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"PVP-AuNP impedes glycation mediated Hen Egg White Lysozyme aggregation under physiological condition","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-12 16:59:02","doi":"10.21203/rs.3.rs-3921564/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-02-19T16:50:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-16T07:47:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"c97f3764-6ea3-4298-bfc5-1fc8281741ad","date":"2024-02-09T10:18:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-09T10:15:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-09T10:10:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-09T04:41:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioNanoScience","date":"2024-02-02T16:23:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3afa82da-5cf7-4441-82ce-8a751c0013ee","owner":[],"postedDate":"February 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-04-06T09:59:54+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-12 16:59:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3921564","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3921564","identity":"rs-3921564","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}