2. Experimental procedures
2.1. Modification of plasma fibrinogen by MGO
The modification was carried out by incubating 0.3 µM of plasma fibrinogen, dissolved in phosphate buffer saline (50 mM sodium phosphate buffer, pH 7.4 containing 150 mM NaCl) with 7.5 mM concentrations of MGO for varying incubation (0, 7, and 14th day ) at 37?C. The unreacted molecules were removed by extensive dialysis against sodium phosphate buffer.
2.2. Absorbance spectroscopy
The absorption profiles of native and MGO-modified plasma fibrinogen were recorded on Shimadzu Spectrophotometer (UV 1700 model) in the wavelength range of 220–400 nm 32.
Protein samples (20 µl each) were loaded into the wells of 10% reducing SDS polyacrylamide gel 33. Electrophoresis was per-formed at 70 V for 4 h at room temperature. The gels were stained with silver nitrate and then take photograph. All other experiments were carried out with 0.3 µM of native fibrinogen and its counterpart modified by 7.5 mM MGO.
2.4. Fluorescence studies
Fluorescence spectra were recorded on Shimadzu (RF-5301-PC) spectrofluorophotometer at 25 ºC in a 1 cm path length at 10 nm slit width, AGEs fluorescence was measured by exciting the protein samples at 355 nm and 370 nm and emission spectra were recorded in 400-600 and 370-600 nm range respectively34. Gain in the fluorescence intensity (F.I.) ware calculated using the following equation:
2.5. Circular dichroism determination (CD)
CD spectra of native and 7.5 mM MGO-treated samples (7th day, 14th day) were carried on J-815 JASCO spectropolarimeter using 1 mm pathlength cuvette in the far UV region (190–250 nm). Data are expressed as molar residual ellipticity ? (degcm-2dmol-1) at a wavelength ?, based on the following equation 35
Where is the observed ellipticity in degrees, n is the number of amino acid residues in human plasma fibrinogen, Cp is the molar fraction and l is the path length in centimeter. The ? helix content of native and MGO-modified plasma fibrinogen samples were calculated from K2D3 software.
2.6. Fourier transform infrared spectroscopic analysis (FTIR)
FTIR spectroscopy of the protein samples was performed on Perkin Elmer FT-IR spectrophotometer 32, 36. Infrared spectra were recorded between 400 cm-1 and 4000 cm-1. FTIR studies were carried out at 1 mg/ ml of protein concentration.
2.7 Scanning electron microscopy (SEM)
Native and MGO modified plasma fibrinogen were prepared for scanning electron microscope experiments by fixation, dehydration, critical point drying, and sputter coating with gold as described previously (Langer et al., 1988; Weisel, 1992). Sample images were recorded using a JEOL JSM-6510LV microscope working at an acceleration voltage of 15 kV.
2.43 Transmission electron microscopy (TEM)
Transmission electron microscopy was performed on a JEOL electron microscope at 200 kV and magnification of 12,000x 37.
Chromatographic Assay of AGE-CML, Pentosidine and argpyrimidine
CML, pentosidine and argpyrimidine in the MGO modified plasma fibrinogen were estimated by high performance liquid chromatography (HPLC) as described earlier 38. All procedures for enzymatic hydrolysis were carried out under nitrogen.
High Performance Liquid Chromatography Analysis
HPLC analysis was performed to detect the CML, pentosodine and argpyrimidine in native fibrinogen and MGO-modified plasma fibrinogen samples after acid hydrolysis. The retention time of commercially procured CML, pentosidine and argpyrimididine was taken as reference for comparison. Brief explanation of sample preparation is as follows: The native and MGO-glycated fibrinogen samples were first hydrolyzed with 6 N HCl for 24 h at 110 ºC. The hydrolyzed samples were then filtered by 0.42 µM Millex filter for ultrafiltration. The filtered samples were analyzed for CML, pentosidine and argpyrimidine by an ion exchange HPLC column (2622 SC, 4.6 mm 60 mm; Hitachi, Japan) as described previously 39, 40.
Proton nuclear magnetic resonance measurements (1H NMR)
1H NMR spectra were recorded on a Bruker DRX- 400 MHz FT NMR spectrometer. All samples for NMR spectroscopy were first lyophilized and prepared using DMSO as a solvent. The chemical shifts in parts per million (ppm) are expressed with respect to tetramethylsilane (TMS) as a reference.
Detection of Amadori adduct by NBT reagent
The native and MGO-modified plasma fibrinogen samples were subjected to NBT reduction assay for Amadori adduct (ketoamine) as described 41. Samples (300 ?l) were mixed with 3 ml of 100 mM sodium carbonate buffer containing 0.25 mM NBT and incubated at 37ºC for 2 h and absorbance was read at 525 nm against distilled water. The ketosamine content was calculated using molar extinction coefficient of 12,640 M-1cm-1 for monoformazan. Increase in ketosamine content (KC) was calculated from the following equation:
Detection of 5-hydroxymethylfurfural (HMF) in native and modified plasma fibrinogen samples
Formation of 5-hydroxymethylfurfural (HMF) from the Amadori product (ketoamine) of modified plasma fibrinogen was detected by thiobarbituric acid (TBA) reaction according to the method described by Ney et al 42. Briefly, 1 ml each of native and modified plasma fibrinogen samples were mixed with 1 ml of oxalic acid (1M) and incubated at 100 ºC for 2 h. Then, the protein in the assay mixture was removed by precipitation with 40% trichloroacetic acid. 0.25 ml of TBA (0.05 M) was added to 0.75 ml of protein free filtrate and incubated at 40 ºC for 40 min. The colour was read at 443 nm and amount of HMF was calculated using molar extinction coefficient of 40,000 cm-1mol-1.
Lysine modification was determined by using TNBSA 43. To determine lysine modification AGEs modified plasma fibrinogen was mixed with 0.01 % TNBSA for 2 h at room temperature. This was followed by the addition of 10 % SDS and 1 N HCl. The absorbance of the sample was read at 335 nm. Freshly prepared fibrinogen was used for control.
The quantification of modified arginine residues was carried out as described previously 44. Briefly, modified plasma fibrinogen samples were mixed with 9,10-phenanthrenequinone and 2 M NaOH. Incubation was carried out for 3 h at 60 °C followed by the addition of 1 N HCl which was further incubated in the dark at room temperature. Fluorescence measurements were done with ?ex = 312 and ?em = 395 nm. Freshly prepared fibrinogen sample was used as a control.
Determination of free sulfhydryl groups
The free sulfhydryl groups in native and modified plasma fibrinogen samples were determined by Ellman’s reagent 45. Absorbance was recorded at 412 nm. The free sulfhydryl content was determined using molar extinction coefficient of 13,600 M-1cm-1.
Determination of protein bound carbonyl
Carbonyl content of native and modified fibrinogen samples was determined after reaction with
DNPH 46. The final absorbance was read at 360 nm and the carbonyl content was determined by using molar extinction coefficient of 22,000M-1cm-1.
Measurements of thiobarbituric acid reactive substance (TBARS)
The level of TBARS is an important index of oxidative stress and lipid peroxidation 47. Native and modified plasma fibrinogen (100 ?l) was incubated for 1 h at 37 °C. After that, 1.6 ml of TBA reagent was added to all incubated samples. The samples were mixed and then incubated for 1 h at 95 °C and the reaction mixture was cooled with tap water centrifuged at 4000 rpm for 10 min. Supernatant were separated and measured spectrophotometrically at 532 nm. TBARS values were expressed as OH radical (hydroxyl radical) equivalents. Concentration was calculated using extinction coefficient of the TBA reagent =1.56×105 mol-1cm-1. Hydroxyl radical concentration is expressed in terms of n moles/ml of protein.
Determination of superoxide radical
Reduction of Cytochrome c by native and modified plasma fibrinogen (1 mg/ml) was mixed with 32 mM cytochrome c in 20 mM phosphate buffer (pH 7.4). The reaction was monitored at 550 nm at 37 °C. The experiment was carried out in UV-visible spectrophotometer.