Non-enzymatic glycation of human serum albumin modulates its binding efficacy towards bioactive flavonoid chrysin: A detailed study using multi-spectroscopic and computational methods

The non-enzymatic glycation of plasma proteins by reducing sugars have important consequences on the conformational and functional properties of protein. The formation of advanced glycation end products (AGEs) is responsible for cell death and other pathological conditions. We have synthesized the glycated human serum albumin (gHSA) and characterized the same by using differential spectroscopic measurements. The aim of the present study is to determine the effect of glycation on the binding of human serum albumin (HSA) with bioactive flavonoid chrysin, which possesses anti-cancer, anti-inflammatory and anti-oxidant activities. The interaction of chrysin with HSA and gHSA was studied using multi-spectroscopic, molecular docking and molecular dynamics (MD) simulation techniques. Chrysin quenched the intrinsic fluorescence of both HSA and gHSA by static quenching mechanism. The value of the binding constant (Kb) for the interaction of HSA-chrysin complex (4.779 ± 0.623 × 105 M-1 at 300 K) was found to be higher than that of gHSA-chrysin complex (2.206 ± 0.234 × 105 M-1 at 300 K). Hence, non-enzymatic glycation of HSA significantly reduced its binding affinity towards chrysin. The % α-helicity of HSA was found to get enhanced upon binding with chrysin, and minimal changes were observed for the gHSA-chrysin complex. Site marker probe studies indicated that chrysin binds to subdomain IIA and IIIA of both HSA and gHSA. The results from molecular docking and MD simulation studies correlated well with the experimental findings. Electrostatic interactions followed by hydrogen bonding and hydrophobic interactions played major roles in the binding process. These observations may have some useful insights into the field of pharmaceutics.


S1.1. Synthesis of glycated analogue of HSA using D-glucose
The gHSA was prepared by incubating 40 mM HSA with 0.2 M D-Glucose in 0.1 M phosphate buffer of pH 7.2 at 37 °C for 60 days. 1 mM sodium azide (NaN 3 ) was added to the solution to prevent bacterial growth. After 60 days the sample was dialyzed for two times to remove excess amount of glucose. Then the sample was lyophilized and stored at -20 °C for further use (Joseph, Anguizola & Hage 2011).

S1.2.1. UV-vis studies
UV-vis spectra of 10 μM HSA and gHSA were recorded at room temperature. The absorbance of the glycated sample was found out to be more than its native form as shown in the Figure S1. Similar observation was reported by Roy et al. (Singha Roy, Ghosh & Dasgupta 2016). A visible browning of the glycated sample was also noticed. Figure S1. UV-vis spectra of 10 μM HSA (black) and gHSA (red) in phosphate buffer.

S1.2.2. Fluorescence spectroscopic measurements
The Trp fluorescence intensity of the modified HSA was found lower than that of the native HSA. Similar observation was also reported earlier by Roy et al. and Coussons et al.(Coussons et al. 1997;Singha Roy, Ghosh & Dasgupta 2016). The formation of advanced glycation end products was confirmed by fluorescence spectroscopic studies. The fluorescence spectra of the protein samples incubated with glucose were monitored at λ ex = 295, 335, 350 and 370 nm respectively for Trp, pentosidine, other AGEs and malonaldialdehyde. The experimental observations are presented in the Figure S2 (λ ex = 295 and 350 nm). Figure S3 represents the bar diagram of fluorescence intensity of the glycated samples after 60 days.

S1.2.3. Excited state fluorescence lifetime measurements
The excited state lifetime of HSA and gHSA were determined on Pico Master time correlated single photon counting (TCSPC) lifetime instrument (PM-3) provided by Photon Technology International (PTI), USA. A magic angle of 54.7° was used in order to avoid any involvement of the anisotropic decay. The instrument response function (IRF) was measured using a dilute solution of non-dairy coffee whitener. The reliability of the graphical fits were analysed using the following parameters (i) Durbin-Watson (DW) parameter, (ii) χ 2 values and (iii) a visual scrutiny of the fitted function to the data. The average lifetime of the fluorophore present in HSA and gHSA were estimated using the following equation (Eq. 1).

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where, α i is the pre-exponential factor associated with the i th decay time constant τ i . The decay profile of HSA and gHSA are shown in the Figure S4. The average lifetime of HSA was found to be 5.63 ns, which correlates well with the previously reported results (Pastukhov, Levchenko & Sadkov 2007;Wu et al. 2011). The glycated analogue of HSA possess an excited state lifetime of 4.05 ns (Table S1).

S1.2.6. MALDI-ToF analysis
The mass of HSA and its glycated analogue was measured using MALDI-ToF analyser The tryptic digestion analysis was also carried out to determine the amino acid residues that were modified during glycation.

Preparation of the digestion buffer
The digestion buffer was prepared by dissolving 10 mg of ammonium bicarbonate in 2.5 mL of ultrapure water and stored at 4 °C. The concentration of the buffer was 50 mM.

Preparation of the reducing buffer 8
The reducing buffer was prepared by dissolving 8 mg of DTT (dithiothreitol) in 0.5 mL ultrapure water to obtain a 100 mM solution. The solution was kept at -20 °C for further use.

Preparation of the alkylation buffer
The alkylation buffer was prepared by dissolving 9 mg of iodoacetamide in 0.5 mL ultrapure water. The buffer was stored in dark.
The concentration of HSA and gHSA was taken as 1 mg/mL. The tryptic digestion of the samples were carried out by mixing 10 µL of the sample to a solution of 15 µL digestion buffer and 1.5 µL reducing buffer. The solutions were incubated at 90 °C for 5 minutes and allowed to cool down at room temperature. The resulting solution was further incubated in dark for 25 minutes after the addition of 3 µL alkylation buffer. Then 1 µL of trypsin solution (1 µg/mL) was added and incubated at 37 °C for 3 hours. After 3 hours, another 2 µL of trypsin solution was added and incubated further for overnight at 37 °C. The digested samples were mixed with the matrix (50:50 v/v) for MALDI-ToF analyses. The results from the trypic digestion and MALDI-ToF analysis are given in the Table S1.  Figure S6. MALDI-ToF spectrum of HSA.    Figure S10.