2 Materials and methods

2.1 Materials

BSA was purchased from Phygene Life Sciences Company (Fuzhou, China); ammonium persulfate and tetramethyl ethylenediamine (TEMED) were purchased from Amersham Biosciences (Uppsala, Sweden); trans-trans-2,4-heptadienal, trans-trans-2,4-nonadienal, and trans-trans-2,4-decadienal were purchased from Aladdin Industrial Corporation (Shanghai, China); protein ladder for SDS-PAGE was purchased from Beyotime Biotechnology Company Limited (Shanghai, China); all other chemicals were of analytical reagent grade.

2.2 Preparation of aldehyde-BSA adducts

Aldehyde-BSA adducts were prepared as follows: BSA was dissolved in a buffer containing 50 mM potassium phosphate (pH 7.4) to a final concentration of 10 mg/ml. Aldehyde-BSA adducts were prepared by incubating 10 mg/ml BSA with the modifiers trans,trans-2,4-heptadienal, trans,trans-2,4-nonadienal, or trans,trans-2,4-decadienal at various concentrations (1, 5, 10, 20 ,50 mM) for 24 h at 37℃. Pure BSA was incubated under same conditions as control. Following the reaction, unreacted aldehydes were removed via PD-10 column.

2.3 Determination of protein carbonyl

Protein carbonyls of native and modified BSAs were quantified according to the method described by Indurthi et al.(2012). and Suji et al.(2008) with slightly modifications. Proteins (200 μl at 2.5 mg/ml) were mixed with 1 ml of 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2 M HCL or 2 M HCl (blank control), and were incubated at room temperature (25℃) for 2 h (shake every 15 min). Equal volume of 20% TCA (to a final concentration of 10%) was added to precipitate the protein. The precipitate was collected by centrifugation at 8,000 g for 10 min. The precipitate was washed three times with 5 ml ethanol/ethyl acetate solution (1:1, v/v) to eliminate free DNPH. Protein precipitate was centrifuged again (8,000 g, 5 min) before being suspended in 3 ml of 6 M guanidinium chloride. Samples were incubated at 37℃ for 15-30 min for proteins to completely dissolve. Absorbance was measured at 370 nm. The results were expressed as nmol of carbonyl groups per milligram of soluble protein, with a molar extinction coefficient of 22,000 M-1 cm-1.

2.4 Determination of free amino content

The free amino content of modified and native BSA were determined by the o-phthalic dialdehyde (OPA) method, as described by Adams et al.(2008). The OPA reagent was freshly prepared by dissolving 40 mg of OPA in 1 ml of methanol, followed by the addition of 25 ml of 0.1 M sodium borate, 2.5 ml of 10% SDS, and 0.1 ml 2-mercaptoethanol, finally adjusting to a total volume of 50 ml with distilled water. Protein (120 μl at 2.5 mg/ml) was mixed with 3 ml OPA reagent. After 2 min in the dark at room temperature, the absorbance at 340 nm was recorded against the OPA reagent. A calibration curve was obtained by using L-leucine as the standard.

2.5 SDS-PAGE

Protein crosslinking was analyzed by SDS-PAGE under reducing condition; 5% stacking gel and 10% resolving gel was used for protein separation. Samples were dissolved in the reducing buffer solution containing SDS, and were then boiled for 5 min before loading. Migration was carried out at 20 mA constant current. After separation, proteins were stained with 0.1% (w/v) Coomassie brilliant blue R-250 in 45% (v/v) methanol and 10% (v/v) acetic acid, and were de-stained with 10% methanol (v/v) and 10% acetic acid (v/v).

2.6 Determination of surface hydrophobicity

The surface hydrophobicity of control and aldehyde-BSA adducts were determined using the 8-anilino-1-naphthalenesulfonic acid method, as described by Tang et al.(2012). and Lv et al.(2016) . Briefly, 10 μl 1-anilino-8-naphthalene-sulfonate (ANS) was added to 0.5 ml of each protein sample (diluted to 0.005-0.5 mg/ml), and was allowed to react for 3 min at room temperature. Fluorescence intensity was measured with an excitation wavelength of 390 nm and emission wavelength of 490 nm. Surface hydrophobicity was determined as the initial slope of the curve plot of fluorescent intensity versus protein concentration. The result was expressed as follows: % relative value of surface = ANS aldehyde – BSA adducts*100 / ANS control BSA.

2.7 Determination of intrinsic fluorescence

The intrinsic fluorescence of control and aldehyde-modified BSA (0.1 mg/ml) were recorded from 300 to 400 nm with an excitation wavelength of 290 nm (slit 5 nm), as described by Chen et al.(2013).

2.8 ultraviolet–visible (UV-Vis) spectroscopic analysis

UV/Vis Spectra were recorded on a Thermo scientific UV/visible spectrophotometer, using quartz cuvettes with a 10 mm pathlength. Protein concentration was 0.2 mg/ml, and absorbance measurements were done in the 240 to 600 nm range.

2.9 Fluorescent pigments measurement

Fluorescent pigments were determined by methods from Meynier et al.(2004), with slight modifications described as follows. Formation of fluorescent pigments was first investigated by 3D scanning, with excitation and emission wavelengths of 300 – 410 nm and 350 – 550 nm, respectively, adjusted in 2 nm increments. The maximum excitation wavelength obtained from the 3D scanning image was used as a reference. The maximum excitation and maximum emission wavelength were then obtained by combining the wave scan method. Finally, the emission spectra of native BSA and aldehyde-BSA adducts were scanned from 350-550 nm with the excitation wavelength fixed at the maximum excitation wavelengths of various aldehyde-BSA adducts. The spectra were measured at the following conditions: PMT voltage: 700 V; scan speed: 600 nm/min; EM slit: 5 nm; response time 0.1 s.

2.10 Determination of colorimetric values

Colorimetric values of samples were obtained via a colorimeter (model NS810 spectrophotometrically, 3nh); a*(redness/greenness), b*(yellowness/blueness), and L*(lightness) were measured.

2.11 Statistical analysis

Each experiment was performed in triplicates. Data were expressed as means ± standard deviation (n = 3). Analysis of variance (ANOVA) was performed using the SPSS 16.0 software. Statistical significance was set at p < 0.05. Principal component analysis (PCA) was used to standardize and determine the relationship between the variables using the SPSS 16.0 software. This promoted comprehensive evaluation of the effects of different aldehydes on BSA modification.