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.