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www.nature.com/scientificreports/ Experimental mixtures preparation. The glycation procedure involves the use of HSA, MG as a glycating agent and AgNPs as an AGEs inhibitor in the reaction system. Briefly, HSA (100 μM) and MG (1 mM) were mixed with or without varying concentrations (0.09, 0.18 and 0.27 mM) of AgNPs in 0.05 M phosphate buffered saline (PBS, pH 7.4), after which mixtures were incubated for 6 days at 37 °C. Native HSA (without MG and AgNPs) was used as a control. After day 6 of incubation, the solutions were dialyzed against sodium phosphate buffer at 4 °C for 48 h to remove the unbound MG and AgNPs from solution. Following dialysis, samples were appropriately diluted and used immediately for analysis. Determination of free amino groups by fluorescamine. Lysine side chain modifications were mon- itored using fluorescamine as described previously, with slight modification32. This process forms a highly flu- orescent reaction product with amino groups. G-modified HSA with or without AgNPs solution (5μl; approx. 1 mg/ml), 100 μl of 100 mM Na2HPO4, 45 μl aqua dest, and 50 μl fluorescamine reagent (1 mM fluorescamine in acetonitrile) were mixed and incubated for 10 min in the dark in a 96-well plate. The fluorescence of the sample was then measured at excitation/emission wavelengths of 390/490 nm in a FLUORO-STAR plate reader (BMG, Germany). N-α-acetyl-lysine from 0 to 1.5 mM was used to determine the linearity of fluorescence within the expected lysine content of the protein solution. Determination of free arginine side chains by 9,10-phenanthrenequinone. Free arginine was determined as previously described33. Briefly, 50 μl samples were mixed in 150 μl of 9,10-phenanthrenequinone reagent (150 μM in ethanol) and 25 μl of 2 N NaOH. Protein samples without 9,10-phenanthrenequinone were used to correct the results for AGE fluorescence. Samples were incubated at 60 °C for 3 h, after which 40 μl were transferred to a 384 well plate and mixed with 40μl of 1.2N HCl. Fluorescence in the reaction product was allowed to develop for 1 h in the dark at room temperature, then measured with a TECAN Safire spectrometer (USA) at excitation/emission wavelengths of 312/395 nm. To test the linearity of fluorescence within the expected arginine content of the protein solution, N-α-acetyl- arginine from 0 to 0.4 mM was used. Estimation of protein-bound carbonyl contents. Carbonyl contents of native and MG-modified HSA with or without AgNPs samples were determined as previously described, with slight modifications34. Briefly, 15μM native and MG-modified HSA with or without AgNPs samples dissolved in 10mM DNPH (2,4-dinitrophenyl hydrazine) solution was made in 2 N HCl. Samples were then vortexed for 1 h at room temper- ature and precipitated with 0.5 ml of 20% (v/v) TCA, followed by 3 min of centrifugation at 11,000 g at 4 °C. The pellet was washed with 1 ml of ethanol-ethyl acetic acid mixture (1:1; v/v) to remove extra DNPH reagent. Next, samples were incubated at room temperature for 10 min and then centrifuged at 11,000 × g for 5 min at 4 °C. The supernatant was discarded and the pellet was washed twice with ethanol-ethyl acetic acid mixture. The protein pellet was then suspended in 1 ml of 6 M guanidium hydrochloride dissolved in 20 mM potassium phosphate buffer, pH 2.3 (attuned with trifluoroacetic acid), after which samples were incubated at 37 °C for 15–30 min to ensure complete solubility of proteins. All samples were subsequently centrifuged to remove any insoluble material. Carbonyl content was determined in the supernatant based on the absorbance at 370 nm against 6 M guanidium hydrochlorides (as blank) using the molar extinction coefficient of 22,000 M−1 cm−1. Protein carbonyl content was expressed as nmol/mg of protein. Estimation of carboxymethyl lysine (CML) content by ELISA. CML content was measured by ELISA as described earlier, with minor modification35. Briefly, absorbance was measured at 405 nm using a Model 550 BioRad microplate reader. Product formation was then measured with a 405 nm filter in an ELX800 multiwell plate reader (BioTek Instruments, USA). UV-Visible and fluorescence spectroscopy. UV-visible absorption spectra of native and treated HSA were carried out using a Cary Win UV-Vis spectrophotometer (Varian Inc., USA) in the range of 250–500 nm. Fluorescence spectra were obtained on a Jasco FP-6500 (Japan). Samples were positioned in a cuvette with a 1 cm path length. All emission spectra were recorded with 1 nm wavelength intervals. Native HSA and glycated-HSA emission spectra were obtained in the range of 360–600 nm under excitation at 360 nm. To calculate the percent inhibition of AGEs formation by various concentrations of AgNPs, the MG-modified sample was used as a positive control. The percent inhibition was calculated using the following formula: % AGEs = [1 − (fluorescence of the test group/fluorescence of the control group)] × 100%. (2) High performance liquid chromatography (HPLC). HPLC analysis was performed on a Hitachi analyt- ical HPLC system (Japan) composed of L-7100 low-pressure gradient pumps, an L-7200 sequential auto-sampler and a high sensitivity diode array detector (190–800 nm) that was managed by a D-7000 HPLC System Manager software. The products formed in the incubated mixtures of HSA and MG were identified on a Phenomenex Luna 5 u, 100 Å, HPLC column (150 mm × 4.6 mm × 5 μm). Mobile phases A and B were 0.1 M aqueous ammonium acetate and 100% HPLC analytical grade acetonitrile, respectively. Aqueous ammonium acetate solution was prepared in deionized water and both mobile phases were degassed and sonicated for 15 min before use. Prior to HPLC analysis, all samples and solvents were filtered with Millipore 0.22 μm syringe filters. The accuracy and reproducibility of the HPLC method was confirmed by repeated testing. Circular Dichroism (CD). Far-UV CD profiles of samples were recorded on a Jasco spectropolarimeter (J-815, Japan) attached to a Jasco Peltier-type temperature controller (PTC-424S/15). The instrument was cal- ibrated with D-10-camphorsulphonic acid and measurements were carried out at 25 °C using a temperature Scientific RepoRts | 6:20414 | DOI: 10.1038/srep20414 3PDF Image | Green synthesis of silver nanoparticles inhibitory effects on AGEs
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