3 Results and discussion
3.1 Effect of aldehydes on side chain modification of
BSA
Formation of protein-bound carbonyls and retention ratio of free amino
group confirmed the covalent binding of the three aldehydes to proteins,
and determined the effect of carbon atom number and concentration on
degree of modification.
3.1.1 The formation of protein bound
carbonyls
The effects of the three kinds of aldehydes on BSA carbonyl contents are
shown in Fig.1. An increase in carbonyl groups content was observed in
the presence of all three aldehydes. This modification was significantly
greater in aldehydes with shorter chains. Incubation of BSA with
heptadienal resulted in the most dramatic increase in protein carbonyl
content. The difference between the BSA carbonyl values caused by the
three aldehydes at 1-10 mM were not significant, however, this was not
the case at high concentrations (50 mM). The carbonyl content of BSA
incubated with 10 mM heptadienal, nonadienal, and decadienal were
approximately 4.83 ± 0.48, 4.08 ± 0.62, and 3.59 ± 0.32 times,
respectively, that of the control. When treated with 50 mM heptadienal,
nonadienal, and decadienal, the content of carbonyls reached 14.65 ±
0.93, 8.66 ± 0.43 and 6.32 ± 0.61 times, respectively, that of the
control. The formation of protein bound carbonyls correlated well with
aldehyde concentrations ( R= 0.984, p < 0.001 for
heptadienal-BSA; R = 0.912, p = 0.011 for nonadienal-BSA; R =
0.868, p = 0.025 for decadienal-BSA).
The addition of α,β-unsaturated aldehydes to nucleophilic amino acids on
proteins occurs in two ways: Michael addition and Schiff base addition.
The strong electron-withdrawing capability of carbonyl groups polarizes
the double bond. This makes the - carbon more electrophilic and
susceptible to Michael addition. The carbonyl group is maintained, and
the conjugated double bond is lost after Michael adducts. When the -
carbon atoms become saturated due to Michael addition, a Schiff’s base
reaction will occur, which will cause the introduced carbonyl to become
undetectable(Yuan et al., 2007).
Wu et al.(2010). found that incubation with acolein induces a
concentration-dependent increase of protein carbonyls. For example, the
soybean protein carbonyl increased 10 times due to incubation with 10 mM
acrolein. Similar result was obtained with BSA exposure to
crotonaldehyde (an unsaturated aldehydes); the increase in protein
carbonyl concentration was highly associated with loss of lysine and
histidine residues (Ichihashi et al., 2001).
3.1.2 Retention ratio of free amino
content
Fig.2 shows the concentration and chain length of modifier-dependent
reduction of amino groups. The three unsaturated aldehydes at
concentration of 1–50 mM had a significant effect on the amino
retention ratio (p < 0.05). The results indicated that
heptadienal has a stronger influence on the amino group when compared to
the other two aldehydes. Free BSA amino group was rapidly decreased with
increasing concentration of heptadienal, especially when it was at the
20-50 mM range. Incubation of BSA with 50 mM heptadienal resulted in an
amino group retention ratio of only 33.99 ±3.61%.
Chopin et al.(2007). found that 2-hexenal and 2,4-hexadienal
significantly reduces the number of amino groups. However, the number of
double bonds exerts little effect on the retention ratio of free amino
groups. In our study, loss of free amino group in BSA was relatively
less after incubation with various concentrations of nonadienal and
decadienal. After 24 h incubation with 50 mM nonadienal and decadienal,
the rentention ratio of BSA amino group was 62.81 ± 7.96% and 77.29 ±
1.25%, respectively. A strong negative correlation was observed between
the retention ratio of free amino content and the concentration of
aldehyde for heptadienal (R= -0.958, p = 0.003), nonadienal (R =
-0.844, p = 0.035), and decadienal (R = -0.765, p =
0.076).
3.2 Effect of aldehydes on BSA aggregation and
cross-linking
Does covalent modification of proteins caused by these three aldehydes
only exist on protein monomolecules or will it also lead to
cross-linking between protein molecules? We used SDS-PAGE to determine
whether protein modification by these three aldehydes will cause
cross-linking, as well as the relationship between degree of aggregation
and modifier concentration. This will also plays an auxiliary role in
determining conformational change of proteins.
SDS-PAGE was performed in order to monitor BSA aggregation that occur
due to the interaction with these three aldehydes (Fig.3). The
electrophoretic pattern of the control BSA was characterized by one
major band with a molecular mass of 66 kD. Aside from the major band,
there were also a small amount of high molecular weight protein stemming
from impurity. We found that all types of aldehydes decreased the BSA
monomer band at 66 kD, and resulted in some form of aggregation; the
degree of aggregation was closely associated with the type and
concentration of the aldehydes.
A faint new band with a size of 95 kD appeared when BSA was treated with
a low concentration aldehydes (1 mM). As the concentration of the three
aldehydes increased to 5 mM, the intensities of the aggregate bands with
molecular weights of 95 kD and >130 kD were increased, and
the bands of the BSA monomer was decreased (Fig.3). Similar to our
results, Liu et al (2007) also found that BSA treated with oxidation DHA
for various length of times led to formation of a single narrow band
with a molecular mass of approximately 93 kD, signifying the presence of
a high-molecular-weight-protein. In addition, binding of acrolein to BSA
also resulted in formation of two broad protein bands with molecular
masses of approximately 80 kD and > 130 kD; binding of
malondialdehyde to BSA resulted in formation of two broad protein bands
with the molecular masses of approximately 97 kD and 200 kD (Liu et al.,
2007).
In heptadienal-BSA reactants, when the heptadienal concentration was 10
– 20 mM, bands of the 95 kD aggregates and BSA monomers became blurred
and diffused, and bands of protein aggregates with molecular weight
above 175 kD was intensified; when the concentration of heptadienal
reached 50 mM, the bands of the aggregates and BSA monomers almost
disappeared. This was accompanied by a band of > 270 kD on
the top of the resolving gel, as well as protein aggregates that were
too large to enter the stacking gel (Fig.3(A)).
Similar results have been previously reported when the soybean protein
was treated with 1-100 mmol/l MDA (Wu et al.,2009). Moreover, this
phenomenon in SDS-PAGE was also observed when unsaturated aldehydes were
allowed to interact with proteins. In the presence of high concentration
of t-2-hexenal, a-lactalbumin disappeared almost completely, and a high
molecular weight protein aggregate band appeared between the stacking
gel and the main gel (Meynier et al., 2004).
When BSA was treated with 10-50 mM nonadienal, the 95 kD aggregate band
and the BSA monomer band were also blurred and diffused; the bands at
> 175 kD were increased. However, when BSA was treated with
50 mM nonadienal, there was no obvious aggregates trapped in the sample
adding port (Fig.3(B)). Similar aggregation pattern was also observed
for proteins in the presence of decadienal, but to a lesser extent. In
addition, the BSA monomer band and 95 kD aggregate band were relatively
clear. No trapping of protein aggregation was observed in the sampling
port at all tested decadienal concentrations (1-50 mM), (Fig.3(C)). Our
study suggested that exposure to heptadienal induces significantly
greater BSA crosslinking than exposure to either nonadienal or
decadienal, especially when the concentration of modifiers are in the
range of 20-50 mM.
3.3 Effect of aldehydes on the structure change of
BSA
The change in protein surface value and intrinsic fluorescence reflects
whether conformation of proteins have changed due to covalent
modification and aggregation, and whether the number of carbon atoms and
the concentration of aldehydes has an effect on protein conformation.
3.3.1 Change of protein surface properties
Hydrophobicity is an indicator of the number of hydrophobic groups on
the surface of the protein that is in contact with the polar aqueous
environment. It is known to be significantly related to the function of
proteins (Tang et al.,2012; Liu et al., 2012). ANS are widely used for
detecting protein surface properties, determining the refolding and
unfolding processes, as well as characterizing aggregations,
fibrillations and molten globule intermediates (Ahmed et al., 2017;
Sattarahmady et al., 2007).
As shown in Fig.4, the extent of decline was closely correlated with the
concentration and type of modifiers (R= -0.827, p = 0.043 for
heptadienal-BSA; R = -0.730, p = 0.099 for nonadienal-BSA; R =
-0.712, p = 0.112 for decadienal-BSA); in general, the higher the
concentration of the modifiers, the lower of the surface hydrophobicity.
Surface hydrophobicity of BSA was found to be greatly reduced by low
concentration of aldehydes; modified with 1 mM heptadienal, nonadienal,
and decadienal, the surface hydrophobic values were 80.14 ± 8.35%,
82.61 ± 4.16%%, and 79.22 ± 4.50% of control BSA, respectively. There
were significant differences between the three groups of
aldehyde-protein adducts and control BSA (p < 0.05),
however, no significant differences were observed among the three groups
of aldehyde-protein adducts (p > 0.05) when the
concentration of modifiers was 1 mM. The surface hydrophobic value of
BSA was steadily decreased steadily with increased concentration of the
three modifiers. All three aldehydes caused sharp decrease in the
surface hydrophobicity of BSA when their concentrations reached 50 mM.
The surface hydrophobicity of heptadienal-BSA, nonadienal-BSA,
decadienal-BSA were only 3.23 ± 0.55%, 20.04 ± 0.82%, and 33.20 ±
2.74% that of BSA control, respectively; the difference between each of
those was significant (p < 0.05).
Based on the change of surface hydrophobicity, it can be inferred that
the native conformation of BSA has also been altered by the three
aldehydes. The decrease in hydrophobic values may be due to modification
of surface hydrophobic groups or extensive protein aggregation, which
resulted in the hydrophobic groups being buried into the aggregates. Wu
et al. (2010). found that adding of 0.01-10 μmol/l acrolein (α,
β-unsaturated aldehyde) to soybean protein also significantly decreased
the surface hydrophobic value. According to the previous report, surface
hydrophobicity was steadily decreased with increased MDA concentration
from 0 to 100 mM , which has been considered to be the result of
formation of hydrophilic groups (e.g protein carbonyls groups), protein
aggregation via hydrophobic interactions, and structural modification of
exposed hydrophobic residues of soy protein (Wu et al., 2009).
Some scholars believed that oxidative modification could also lead to
protein unfolding and exposure of hydrophobic groups. Following that,
the exposed hydrophobic groups can interact with each other, which leads
to protein aggregation (Tang et al., 2012). The observed surface
hydrophobicity value in this paper may be the result of an equilibrium
reached from the exposure and aggregation of hydrophobic groups
(aggregation of proteins shown by electrophoresis).
3.3.2 Change of intrinsic
fluorescence
The maximum emission wavelength of tryptophan is highly dependent on the
polarity of the surrounding microenvironment, which makes it a very
suitable endogenous fluorescence probe to assess changes in protein
conformation via change in its maximum emission wavelength (Girish et
al., 2016; Ahmed et al., 2017 ).
The maximum emission of control BSA was approximately 340 nm. As shown
in Fig.5, a sharp decrease in fluorescent intensity and a significant
blue shift in its maximum emission wavelength were observed when BSA was
incubated with these three kinds of aldehydes.
At a concentration of 1 mM, all the three aldehydes caused an abrupt
decline in intrinsic BSA fluorescence, but did not lead to blue shift in
maximum emission. The intrinsic fluorescence intensity of
heptadienal-BSA, nonadienal-BSA, and decadienal-BSA was reduced to 48.25
± 2.70%, 46.50 ± 0.41%, and 48.80 ± 1.49% that of the control sample,
respectively. Heptadienal-BSA, nonadienal-BSA, and decadienal-BSA
decreased to approximately 7.72 ± 0.49%, 8.77 ± 0.46%, and 19.88 ±
1.38% that of the control, respectively. Moreover, the maximum emission
wavelength showed a significant blue shift to 325-326 nm in the presence
of 5 mM aldehydes. At concentrations of 10-50 mM modifier, there was a
continued reduction in intrinsic fluorescence intensity, but no further
blue shift was observed. The intrinsic fluorescence intensity of
heptadienal-BSA, nonadienal-BSA, and decadienal-BSA was decreased to
2.65 ± 0.43%, 5.99 ± 0.42%, and 9.9 ± 0.65% that of the control,
respectively, when the modifier was at the highest concentration (50
mM). At this concentration, these three dien-aldehydes almost completely
extinguished the intrinsic fluorescence of BSA.
To summarize, all three dien-aldehydes (at 1-50 mM) demonstrated
significant effects on the intrinsic fluorescence of BSA. When the
concentrations of the dien-aldehydes were low (1 mM), there was no
significant change in the BSA intrinsic fluorescence (p> 0.05). The intrinsic fluorescence intensity of BSA
decreased in the following order: heptadienal > nonadienal
> decadienal, when concentrations of modifiers were in the
ranges of 5-50 mM; there were also significant differences among BSAs
treated with different modifiers (p < 0.05).
The maximum intrinsic fluorescence emission wavelength denotes the
relative position of tryptophan residues within proteins (Estévez et
al., 2008).The blue shift of the maximum emission wavelength indicated
that with greater concentration of the modifiers, the conformation of
BSA was gradually destroyed, and the previously exposed tryptophan
residues of the native protein were buried in the interior. The BSA
protein thus became more hydrophobic and less polar, resulting in
aggregation (Wu et al.,2009). However, the decrease in intrinsic
fluorescence intensity is usually explained by protein folding,
aggregation, or/and degradation of tryptophan (the aggregation of
proteins shown by electrophoresis) (Lv et al.,2016).
3.4 UV/VIS, fluorescent spectroscopic and color properties
of modified
BSA
The spectral characteristic of aldehyde-BSA adducts were determined by
ultraviolet visible spectrophotometer and fluorescence; the color
characteristics of the modified protein were given by color parameters.
The effects of length and concentration of aldehydes on these properties
were also determined.
3.4.1 Changes in UV-Vis absorption
characteristics
Fig.6 shows that the UV-Vis absorbance profiles for heptadienal-,
nonadienal-, and decadienal-modified protein were identical; the
absorbances was increased to 270-280 nm, as well as to 300-400 nm
(Fig.6). This suggested formation of structurally similar chromophores
of these aldehyde-BSA adducts (Vetter et al.,2011). Similar absorbance
spectra shapes have been reported to be typical of melanoidins (Adams et
al;2009).
Previous studies have reported that BSA reacts with methylglyoxal lead
to formation of chromophores with absorbances at 300-400 nm and below
290 nm. The authors speculated that absorption at 320-335 nm occurs due
to the newly generated argyrimidine structure, and absorption at 325-335
nm was due to the pentosidine structure (Vetter et al., 2011). The
binding of whey protein to 2-hexenal or hexanal also resulted in the
increase of the absorption at 280 nm and 300-360 nm (Meynier et
al.,2004).
Glyceraldehyde incubated with acetyl-lysine (118 mM) produced new
compounds with maximum absorption at 275.7, 232.2, 268.7, and 349.3 nm;
the maximum absorption wavelength of 297 nm was accompanied by 260 nm
shoulder peaks. Among the new compounds, the maximum absorption of
trihydroxy-triosidine was at 275.7 nm; maximum absorption of Lys-hydroxy
-triosidine was at 232, 269 and 349 nm; maximum absorption of
triosidine-carbaldehyde was at 297 nm. The new compound
Arg-hydroxy-triosidine, with a maximum absorbance at 330 nm, was
produced when acetyl-lysine (55 mM) and acetyl-arginine (50 mM) were
incubated with glyceraldehyde (Tessier et al.,2002).
As shown in Fig.6, light absorption at 270-280 nm by control BSA was
mainly due to amino acid residues with optical properties, such as
tryptophan, tyrosine, phenylalanine. When the concentration of these
three modifiers were low (1 mM), the absorbance of the aldehyde-BSA
adducts at 270-280 nm was slightly decreased, which may be caused by
modification of chromogenic amino acid residues. Lv et al. (2016) showed
that the maximum UV absorption of untreated shrimp protomyosin was at
approximately 275 nm, which was largely due to Tyr and Trp residues.
After shrimp protomyosin was treated with low concentrations of HNE
(0.01-1 mM), the absorption wavelength of Tyr and Trp slightly shifted
and decreased. This was mainly due to modification of Tyr and Trp
residues on the protein surface, which caused a change in the polarity
of the microenvironment.
Fig.6 shows that with greater concentration of the three aldehydes (5-50
mm), absorption of aldehyde-BSA adducts at 270-280 nm was rapidly
increased. These results were similar to those of other studies. Khatoon
et al.(2012). found that HSA modified by HNE led to increased absorbance
at 280 nm, and was also accompanied by a slight increase in absorbance
at 300-390 nm. It was speculated that the increase in absorbance at 280
nm may be due to structural expansion of HSA caused by the addition of
HNE with lysine, histidine, and cysteine.
The absorption of aldehyde-BSA adducts were dependent on modifier
concentration, and the increasing range was negatively correlated to
length of aldehydes. On one hand, carbonylation may have distorted the
native conformation of the BSA, thereby exposing the aromatic amino acid
residues and causing an increase in absorbance at 270-280 nm. On the
other hand, some new compounds may have been formed, which absorb at
270-280 nm (Ahmed et al.,2017).
As shown in Fig.6(A), the absorbance of heptadienal-BSA around 270 nm
increased greatly with greater concentration of the modifiers, which
increased from 0.33 ± 0.01 to 1.91 ± 0.06 and 4.79 ± 0.18 times that of
the control. The maximum absorbance wavelength shifted from
approximately 270 nm at low modifier concentration to approximately 276
nm at high modifier concentration (50 mM).
The absorbance of nonadienal-BSA and decadienal-BSA near 270 nm were
increased from 0.33 ± 0.01 to 1.14 ± 0.08 and 0.69 ± 0.01, which was
2.45 ± 0.24 and 1.20 ± 0.03 times higher than that of the control. The
maximum absorption wavelength of nonadienal-BSA and decadienal-BSA were
shifted to approximately 275 nm and 273 nm, respectively, when the
concentration of the modifiers reached 50 mM (Fig.6(B-C)). A significant
correlation was found between maximum absorption and the concentration
of aldehydes (R = 0.991, p < 0.001 for heptadienal-BSA;
R = 0.984, p < 0.001 for nonadienal-BSA; R= 0.854,p = 0.030 for decadienal-BSA).
Moreover, as presented in Fig.6, these three aldehyde-BSA adducts have
no characteristic absorption peaks in the range of 300-400 nm, and their
optical density increased with decrease in wavelength. This is similar
to the typical ”tail pattern” of melanin, which has a tailing absorption
rage that extends to the visible spectrum. The main reason for this
phenomenon may be due to the many kinds of chromophore structures, which
resulted in a system that has no characteristic absorption peak in this
region (Hrynets et al.,2013).
3.4.2 Formation of fluorescent pigments
Accumulation of fluorescent pigments (also called lipofuscin) is usually
closely related to aging and various chronic degenerative diseases. In
this study, the fluorescent lipofuscin of aldehyde-BSA adducts and
control were detected by fluorescence spectrophotometer. It has been
reported that the maximum excitation and emission wavelengths of
fluorescent lipofuscin fall between 340-375 nm and 420-490 nm,
respectively (Trombly et al.,1975). In the presence of aldehydes, new
fluorescent lipofuscin with the excitation and emission wavelengths at
340-400 nm and 400-500 nm, respectively, were observed (Meynier et
al.,2004). We showed that incubation of unsaturated aldehyde and
oxidized linoleic acids with BSA produced new fluorescent compounds,
which showed maximum excitation and emission wavelengths at 350-380 nm
and 420-450 nm, respectively, similar to previously reported values
(Yamaki et al., 1992).
Based on the previous reports, we first used 3D scanning to determine
the approximate maximum excitation and emission of lipofuscin. However,
data obtained from 3D scanning is affected by the wavelength accuracy
and Rayleigh scattering. Therefore, on the basis of 3D scanning, we
further used two-dimensional of excitation and emission wavelength
scanning to determine the maximum wavelength of fluorescent lipofuscin
from aldehyde-BSA adducts (Considering the results of the
pre-experiment, the aldehyde-BSA adducts lipofuscin fluorescence
intensity was relatively higher when modifier concentrations was at 10
mM ).
The fluorescent lipofuscin emission spectra for each kind of
aldehyde-BSA adducts were obtained with the excitation wavelength set at
their own maximum excitation wavelength. As shown in Fig.7(A-C), the
maximum excitation and emission wavelengths of BSA modified by
heptadienal, nonadienal, and decadienal at 10 mM were 352.3/436.4 nm,
353.3/434.4 nm, and 343.4/430.5 nm, respectively. In addition, Fig.7(A)
also showed that the spectra of heptadienal-BSA exhibited a shoulder
peak at about 465-470 nm; BSA-nonadienal has a defined shoulder peak at
465-470 nm (Fig.7(B)); BSA-decadienal demonstrated two shoulder peaks at
405-410 nm and 465-470 nm ((Fig.7(C)). Moreover, the lipofuscin
excitation and emission spectra were broad, which suggested the presence
of several fluorophores (Hidalgo et al.1993) .
Fig.8 is the emission spectrum of fluorescent lipofuscin from
aldehyde-BSA adducts. We selected the excitation wavelength of 352.3 nm
for detection of heptadienal-BSA and control, as shown in Fig.8(A).
Control BSA exhibited very low fluorescence emission with a broad range.
On the other hand, fluorescence emission intensity of lipofuscin rose
steadily after BSA was modified by heptadienal at concentrations from 0
to 10 mM. From the initial value of 2410.70 ± 103.45 (control),
fluorescence intensity rose to 18716.23 ± 612.66 (concentration of
heptadienal at 10 mM). However, lipofuscin fluorescence intensity was
decreased to 4187.53 ± 452.88 when BSA was incubated with heptadienal at
50 mM. In addition, a red shift in the maximum emission wavelength of
lipofuscin was observed when the concentration of heptadienal reached to
20-50 mM. The maximum emission wavelength of lipofuscin shifted to 468.9
nm when the concentration of heptadienal reach 20 mM, and further
shifted to 477.8 nm when the concentration of heptadienal reached to 50
mM.
Similarly, lipofuscin fluorescence emission intensity increased with
greater nonadienal concentration (0-10 mM ), from 2360.20 ± 64.79 (the
control) to 19989.53 ±603.07 (concentration of nonadienal at 10 mM )
(Fig.8(B)). As the concentration of the modifier continued to increase,
lipofuscin fluorescence emission intensity decreased to 12295.30 ±
738.64 when the concentration of nonadienal reached 50 mM; the maximum
emission wavelength exhibited a weak red shift to 439.3 nm.
As previously reported, in malondialdehyde/glycine reaction systems,
there was a red shift in the maximum emission wavelength of fluorescence
lipofuscin with increased aldehyde concentration (Yin et al.,1994).
Gardner et al.(1979) found that increase in conjugated groups led to the
red shift in maximum fluorescence emission wavelength of lipofuscin.
However, Vetter et al. (2011) pointed out that the change in the maximum
emission wavelength may be due to the existence of multiple fluorophores
with overlapping absorbance and emission spectra. Similar to our
results, the reaction of BSA with oxidized oil also produced fluorescent
lipofuscin, initially with a maximum emission wavelength at 410 nm, but
exhibited an increasingly prominent shoulder at 425 nm with increase in
degree of oxidation (Rampon et al.,2001).
The lipofuscin fluorescence emission intensity increased with greater
concentration of decadienal (0-10 mM) (Fig.8(C)), from the initial
1830.13 ± 122.76 (control) to 22668.23 ± 403.97 (concentration of
decadienal at 10 mM). With the increase in the concentration of
decadienal (20-50 mM), the lipofuscin fluorescence emission intensity
decreased to 17505.17 ±761.58 (50 mM); the maximum emission wavelength
did not show a significant red shift.
Overall, a weak correlation was observed between fluorescent pigments
and concentration of aldehydes (R= -0.284, p = 0.586 for
heptadienal-BSA; R = 0.190, p = 0.719 for nonadienal-BSA; R =
0.435, p = 0.389 for decadienal-BSA).
Only a few of aldehyde-protein adduct products have fluorescence
properties, which are also affected by various factors such as the type
and concentration of aldehydes involved in the reaction, the type of
protein or amino acid, the nature of the medium, the reaction
temperature, and the intermediate products (Chelh et al.,2007).
Fluorescent lipofuscin usually have certain structural characteristics,
such as an electron-donating group in conjugation with an imine, which
is the source of fluorescence (Gardner et al.,1979). Pyridinium and
pyrrole were confirmed to be the main sources of fluorescence during
lipid oxidation and protein reactions by NMR and LC-MS/MS. Notably,
these molecular structures contain the imino-ene conjugation required
for fluorescence, while the other non-major fluorescent structures are
not well-studied (Schaich et al.,2008) .
Model systems are usually used to explain the structure and formation
mechanism of fluorescent lipofuscin. The most widely studied system is
the formation of AGEs (Advanced glycation end products, also one of the
sources of lipofuscin) by reducing sugar (which also has aldehyde groups
that can participate in electrophilic addition) with protein or amino
acid.
The typical fluorescent structures of AGEs are pyrraline, pyrroles,
pentosidines, and crosslines (Méndez et al.,2007;Yin et al.,1996).
Reaction between MDA and protein (or amino acid) results in addition
products formed by lipid oxidation products and proteins (or amino acids
/ biomacromolecules containing free amino acid), which have been
extensively studied. The group of products from MDA/protein reactions
with strong fluorescence properties was proposed to be
1,4-dihydropyridine-3,5-dicarbaldehydes, which has a conjugated electron
donor structure (Itakura et al.,1996) .
In addition, the reaction of 4-hydroxy-2-nonenal (HNE, with hydroxyl
group on the side chain of molecule), 4-oxo-trans-2-nonenal (ONE), and
epoxaldehyde with proteins could also lead to production of fluorescent
lipofuscin products, which usually contain pyridine or pyrrole
structures (Li et al.,2015) . However, there are few reports on the
fluorescence properties of lipofuscins formed by proteins and
unsaturated aldehydes without oxygen-containing groups in the side
chain. Meynier et al.(2004) . pointed out that the maximum excitation
and emission wavelengths of fluorescent compounds produced by 2-octeral
and lysine residues of BSA was 350 nm and 440 nm respectively, and that
the fluorescent compounds were pyridinium adducts. However, fluorescent
compounds produced by dien-aldehyde and protein (or amino acid) have
been less studied. Decadienal was the main research object in previous
reports. Leake et al.(1985). found that reaction between 2,4-decadienal
and lysine or alpha-N-acetyl lysine resulted in strong fluorescence with
excitation at 355 nm and emission at 420 nm, while 2,4-decadienal alone
gave no fluorescence. Zhu et al.(2010). found that the reaction of
2,4-decadienal with L-lysine on β-LG could not only form a Schiff base
structure, but could also form structures such as a pyridinium adduct,
which may be the source of fluorescence.
3.4.3 Changes in color
characteristics
As shown in Fig.9(A-C), our results demonstrated that the various
concentrations of modifiers increased the value of a *and b *, and
decreased the value of L* in BSA (p < 0.05). When the
modifiers concentration was low (1-10 mM), the value of a *and b * of
aldehyde-BSA adducts was rapidly increased, while the value of L * was
sharply decreased. The greater b * value indicated that higher modifier
concentration leads to increased yellowness of aldehyde-BSA adducts; The
increase in a * value was indicative of an increase in the red degree of
the samples, while the decrease in L * indicated that the aldehyde-BSA
adducts complex became darker when the concentration of the modifiers
was increased.
When the modifiers concentration was 20-50 mM, the color of aldehyde-BSA
adducts changed slowly. The results showed that the yellowness and
redness of the protein increased sharply at low aldehyde concentrations.
However, the increasing range of color was weakened at high modifier
concentrations, and the a * and b * values of heptadienal-BSA was
decreased. When the concentration of the modifiers were 10 mM, the a *
values of heptadienal-BSA, nonadienal-BSA, and decadienal- BSA increased
from -0.03 ± 0.03 in the control to 8.39 ± 0.81, 9.17 ± 0.17, and 7.92 ±
0.33, respectively (Fig.8(A)); their b * values increased from 4.24 ±
0.04 in the control to 17.95 ± 0.45, 21.58 ± 0.28, and 20.87 ± 0.16,
respectively (Fig.8(B)).
The value of L * decreased drastically with the concentration of the
modifiers was increased (1-50 mM). The L* value of heptadienal-BSA
decreased sharply with increase in modifiers concentration (20 mM), from
95.13 ± 0.26 to 71.98 ± 1.15, which was 24.33 ± 1.21% lower than that
of the control. However, when modifier concentrations were in the range
of 20-50 mM, the L * value of heptadienal-BSA was reduced as the
concentration of modifiers was increased. L * decreased to 68.63 ± 1.50
when heptadienal concentration was 50 mM, which was 27.86 ± 1.58% lower
than that of the control (Fig.9(C)).
The L * of nonadienal-BSA and decadienal-BSA with 10 mM modifier were
80.48 ± 1.42 and 82.64 ± 0.46, respectively, which were 15.40 ± 1.49%
and 13.13 ± 0.48% lower than those of the control, respectively. When
the concentration of the modifiers was increased to 50 mM, the L * of
nonadienal-BSA and decadienal-BSA were 77.96 ± 1.20 and 81.44 ± 0.64,
respectively, which were 18.05 ± 1.26% and 14.39 ± 0.67% lower than
that of the control, respectively (Fig.9(C)).
The nonenzymatic browning of biomacromolecules is mainly due to the
reaction of carbohydrates or lipid oxidation products (such as
aldehydes) with various nitrogen-containing substances, such as amino
acids, peptides or proteins (Hidalgo et al.,2000). It has been
previously reported that these two kinds of browning reactions are
generally accompanied by an increase in a *, b *, and a decrease in L *
, similar to our results (Bosch et al., 2007) . Moreover, some scholars
proposed that there were no significant chemical differences between
biomacromolecule browning caused by lipid oxidation products such as
aldehydes) and sugars (Hidalgo et al.2000; Hidalgo et al.,1999). The
main melanoidins generated from these two browning reactions are furans,
pyrroles, pyrazines, and pyridines (Wang et al.,2011). Some researchers
pointed out that the Schiff-base structure formed by amino groups and
aldehydes produced from lipid oxidation are source of colored substances
(Gardner et al.,1979) .
4 Principal Component Analysis
(PCA)
PCA was conducted to explore the relationships between the variables,
and to evaluate the effects of these variables on aldehyde-modified BSA.
We obtained two principal components, which accounted for 97.496% of
the total variance; PC1 explained 77.343%, while PC2 explained 20.153%
of the total variance. As can be seen from the factor loading plot
(Fig.10(A)), during formation of protein bound carbonyls, a* and b* were
positively correlated with PC1, the retention ratio of free amino
content, the protein surface value, and the intrinsic fluorescence; L*
was negatively correlated with PC1, while lipofuscin fluorescence was
positively correlated with PC2.
Fig.10(B) shows the total score plots of aldehyde-BSA samples, which
represent the comprehensive adverse effects of these aldehydes on
proteins. As shown by Fig.10(B), the overall adverse effect on BSA are
aldehyde-mediated enlargements. When the concentration of the modifiers
increased to 20-50 mm, heptadienal had the greatest effect on BSA,
followed by nonadienal, and finally decadienal, which was consistent
with results from previous analysis.