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Application of ultra high hydrostatic pressure for investigating the binding of flavor compounds to ß-lactoglobulin via headspace solid phase microextraction-gas chromatography

by 1977- Hoang, Tinyee Arden, PhD

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native and UHP treated proteins, and mixed well by vortexing. After 20 min equilibration
in the dark, the relative fluorescence intensity (RFI) of each solution was determined in
replicate, starting with a buffer blank (buffer plus probe) and then the smallest to the
greatest protein concentration. The fluorometer quartz cell (Starna Cells, Inc, Atascadero,
CA, no.3-Q-10) was rinsed between fluorescence assays with a small volume of the
future solution being assayed. The RFI of each protein dilution blank (no probe) was
subtracted from the RFI of each corresponding protein solution with PRODAN to obtain
net RFI. Line fitting analysis (Microsoft Excel 2002 for Windows XP) was used to
determine the linearity of the plot of net RFI versus protein concentration, and the
resulting slope was observed as an index of BLG surface hydrophobicity according to
Alizadeh-Pazdar and Li-Chan (2000).
Determination of the Apparent Dissociation Constants
PRODAN binding properties were calculated with the Cogan method (Cogan and
others 1976). The number of accessible binding sites and apparent dissociation constants
of PRODAN with BLG were calculated with equation (1)
where PB0B is the total protein concentration, α is defined as the fraction of free binding
sites on the protein molecules, n is the number of independent binding sites for
PRODAN, RB0B is total PRODAN concentration, and K’d is the apparent dissociation
constant of a single site. The value of α was calculated with equation (2)


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where F represents the fluorescence intensity of PRODAN at selected RB0B, FBmaxB represents
the fluorescence intensity of PRODAN upon saturation of protein molecules, and FB0B
the initial fluorescence intensity of PRODAN in the absence of BLG.
Statistical Analysis
Data collected from the binding studies of PRODAN to BLG were analyzed using
an analysis of variance (ANOVA) procedure using the General Linear Model, with
further analysis using Tukey’s pairwise comparison test to determine differences (p <
0.05) between UHP and pH treatment means (SAS for Windows, version 9.1 TS Level
1M2, SAS Institute Inc., Cary, NC, USA.).
Conformational Change - Intrinsic Fluorescence
The tryptophan fluorescence spectra of native BLG treated at selected pH values
are presented in Figure 1. A difference in peak intensity of the emission spectra was
observed between native BLG at pH 3.0 and pH 7.0, and can be accounted for the
stability of native BLG through dimerization at the higher pH as observed by Busti and
others (2002). A high fluorescence peak intensity observed for native BLG at pH 5.0
results from the displacement of water through protein-protein interactions of monomers
near its isoelectric point of 5.2 (Phillips and others 1994). In Figure 1, the emission
wavelengths occurring at peak intensity for native BLG at pH 7.0 and pH 9.0 were
observed at 339 and 345 nm. The change in emission wavelength, or red shift, with an
increase in pH indicates that the microenvironment surrounding Trp19 and Trp 61 is
becoming more polar (Lakowicz 1983).



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The tryptophan fluorescence spectra of BLG treated at 600 MPa for selected times
and pH values are presented in Figures 2-5. At pH 3.0 (Figure 2), BLG exhibited a twostage
increase in tryptophan emission peak maximum with the first increase observed
after pressure was applied (come-up time) and the second increase of fluorescence
emission peak maximum after 16 min of pressurization. The increases in tryptophan
fluorescence maxima of BLG upon unfolding by UHP are consistent with the results of
Yang and others (2001) and Liu and others (2005). The increase in tryptophan
fluorescence maxima of BLG during UHP treatment suggests the dissociation of the BLG
dimer. Small increases in tryptophan fluorescence intensities observed after UHP
treatment indicates that unfolding of the compact structure of BLG at acid pH may or
may not be complete (Phillips and others 1994).
After exposure of BLG solutions of pH 5.0 to UHP, precipitation of BLG from
the solution was observed visually. The tryptophan emission peak maximum of native
BLG increased by 18% after UHP come-up time, and remained at this intensity after
UHP of 32 min (Figure 3). After UHP treatment of 16 min, the peak emission wavelength
increased by 2 nm, indicating that the tryptophan microenvironment became more polar.
Although BLG was near its isoelectric point of 5.3, a precipitate was only noticeable
visually after UHP treatment. Around pH 4.5, BLG is prone to form octameric structures,
and Timasheff and others (1966) proposed that the transition between the native
isoelectric form of BLG and the acid forms facilitates this octamer formation. The
applied pressure to BLG at pH 5.0 may have unfolded the subunits exposing the
hydrophobic interior of BLG. The exposed cysteine groups of each BLG molecule then
can readily participate in sulfhydryl-disulfide interchange reactions with neighboring

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BLG molecules (Swaisgood 1996) to minimize unfavorable interactions of hydrophobic
groups with water, thereby causing the precipitation of BLG from solution (Petsko and
Ringe 2004).
Following UHP come-up time at pH 7.0, the tryptophan fluorescence emission

7 7

peak maximum decreased slightly from 1.37 x 10P


to 1.34 x 10P


with 2 nm red shift in the
tryptophan emission wavelength (Figure 4). By increasing pressure times to 16 min, the


tryptophan fluorescence maximum increased to 1.39 x 10P P. The observed decrease in the
tryptophan emission spectra can be accounted for by the Tanford transition that occurs in
BLG between pH 6.0 and 8.0 (Taulier and Chalikian 2001). Taulier and Chalikian (2001)
believed that the transition induced a change in local microenvironment of aromatic
residues with loosening of the interior packing. This loosening may explain the increase
in the tryptophan emission peak maximum after 16 or 32 min of UHP, and also results
from the transition to the intermediate molten globule structure as reported by Yang and
others (2001). The increase in the fluorescence intensity observed is attributed to the β-
strand EF loop of BLG adopting an “open” conformation that promotes access to large
internal hydrophobic pockets (Adams and others 2006).
The tryptophan emission peak maxima of BLG after UHP treatment at pH 9.0

7 7

exhibited a decrease from 1.36 x 10P


to 1.25 x 10P


after 4 min of UHP and an increase to


1.34 x 10P


after UHP of 16 min (Figure 5). Although lower than the observed
fluorescence emission maxima of BLG at pH 3.0 to 7.0, this value may be possibly due to
the retention of BLG secondary structure (Casal and others 1988). At alkaline pH, BLG is
susceptible to surface denaturation, or base-induced denaturation (Timasheff and others
1966), accompanied by a more open flexible molecular structure (Phillips and others

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1994). The increases in tryptophan intrinsic fluorescence after 16 min of UHP treatment
may indicate further unfolding of the BLG monomer with loosening of secondary
structure as observed for effects of chaotropic agents, such as urea, greater than 1.5 M on
BLG (Phillips and others 1994; Yang and others 2001).
Conformational Change - Extrinsic PRODAN Fluorescence
PRODAN fluorescence emission spectra were observed at 25°C as a function of
UHP (600 MPa) for selected times and pH (Figures 6-9). The PRODAN fluorescence
emission spectrum of untreated BLG exhibited two peaks: free PRODAN showed a green
emission peak at 520 nm, and PRODAN bound to BLG showed a blue emission peak at
450 nm (Hiratsuki 1998). The binding of PRODAN, as observed by increases in extrinsic
fluorescence intensity, did not indicate binding to specific binding sites of BLG, but may
suggest similar binding properties of other nonpolar flavor compounds. It can be seen in
Figure 6 that at pH 3.0 or 5.0, native BLG exhibited an emission wavelength 25 nm less
than observed emission wavelengths of 450 nm for BLG at pH 7.0 or 9.0, suggesting that
the binding areas for PRODAN in BLG is nonpolar at acidic pH. UHP treatment of BLG
at pH 3.0 (Figure 7), after come-up time, resulted in a decrease in the PRODAN extrinsic
fluorescence intensity. The compact structure of BLG at pH less than 4.0 may increase
BLG resistance to pressure denaturation occluding PRODAN molecules from entering
the hydrophobic interior (Phillips and others 1994). However, after 4 min UHP treatment
of BLG, BLG may not resist pressure denaturation hence an observed increase in
extrinsic fluorescence.
At pH 5.0 (Figure 8), the PRODAN fluorescence emission peak of native BLG
observed at 450 nm increased with increasing UHP treatment time. After an 8 min UHP

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treatment of BLG, a 152% increase in PRODAN fluorescence intensity was observed,
indicating large numbers of PRODAN molecules bound to BLG. The binding of
PRODAN molecules suggests that BLG is unfolding under UHP and exposing the
hydrophobic interior, suggesting under these conditions, BLG is favorable for binding of
hydrophobic flavor compounds.
The difference in fluorescence intensity maxima between native BLG at pH 5.0
and 7.0 (Figure 6) and continued increase in intensity after UHP treatment (Figure 9)
indicate that the conformation of a loop containing β-strands, β-E and β-F, changes as a
function of pH, and the EF loop is folded back to reveal the interior of the calyx in an
open conformation (Qin and others 1998).
Unlike the fluorescence intensities as observed of BLG at pH 7.0, the changes in
intensities observed of BLG at pH 9.0 (Figure 10) is the result of base-induced
denaturation disrupting the native dimeric structure into unfolded monomers (Taulier and
Chalikian 2001). The small decreases in PRODAN fluorescence intensities observed after
UHP treatment of BLG may account for hydration of BLG monomers, decreasing access
of PRODAN molecules to the hydrophobic interior.
In summary, BLG may resist pressure-induced denaturation at acid pH, however,
as pH increases towards the isoelectric point (pH 5.3), precipitation of BLG allows
PRODAN molecules to bind BLG. At neutral pH, the molten globule state of BLG occurs
after pressurization, and hydrophobic binding of PRODAN is enhanced at pH 9.0 through
base-induced denaturation.
Surface Hydrophobicity


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Linear plots that were generated from net RFI versus protein concentration gave a
slope as an index of BLG surface hydrophobicity. The quantitation of protein
hydrophobicity is important for predicting protein functionality. Surface or effective
hydrophobicity is termed for a portion of nonpolar amino acid residues, positioned in the
interior of protein molecules in solutions to avoid contact with the aqueous environment,
which participate in polar and nonpolar interactions, and does not correlate with total
hydrophobicity (Nakai 1983).
The pH significantly affected surface hydrophobicity (Figure 11) of untreated
BLG (p < 0.05), with the smallest surface hydrophobicity observed at pH 3.0, which is
consistent with results of Alizadeh-Pasdar and Li-Chan (2000). The findings also support
the suggestion that the E-F loop (residues 86-89) is arranged so as to occlude the open
end of the calyx, giving the closed conformation (Uhrinova and others 2000). UHP
treatment of BLG after 4, 8, or 16 min at pH 3.0 significantly increased surface
hydrophobicity (p < 0.05). UHP treatment come-up time or 4 min significantly decreased
surface hydrophobicity (p < 0.005) at pH 5.0 through formation of protein aggregates.
The formation of disulfide bonds following SH/S-S interchange (Futenberger and others
1995) decreased the accessibility of PRODAN to the surface hydrophobic site, especially
at or near the isoelectric point (pH 5.3) of BLG. UHP treatment had little observed effect
on surface hydrophobicity of BLG at pH 7.0. However, there was a decrease in surface
hydrophobicity observed after UHP of 16 min, probably due to the interaction of partially
denatured proteins resulting in burial of effective hydrophobic regions (Alizadeh-Pazdar
and Li-Chan 2000). Surface hydrophobicity of BLG increased significantly (p < 0.005)
after UHP come-up time at pH 9.0, which are consistent with results of thermal treatment

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of BLG observed using PRODAN from Alizadeh-Pasdar and Li-Chan (2000).
Additionally, the E-F loop is in the open conformation, providing access to the
hydrophobic pocket.
The surface hydrophobic site of BLG at pH 3.0 has little accessibility to
PRODAN molecules. The observed increase in PRODAN extrinsic fluorescence at pH
7.0 may indicate that BLG has other hydrophobic sites to which PRODAN molecules can
bind. At alkaline pH, UHP treatment of BLG after come-up time may partially unfold
BLG at the cleft formed by β strands A, H, and I, thereby increasing BLG surface
hydrophobicity through exposure of other nonpolar amino acid residues.
PRODAN Binding to BLG
Titrations of BLG with PRODAN (Figures 12-35) were plotted according to the
Cogan method (1976). At pH 3.0, native BLG exhibited 2.26 binding sites for PRODAN


per molecule of protein with an apparent dissociation constant of 114.79 x 10P


M (Table
1). At pH 5.0 and 7.0, native BLG exhibited 1 binding site for PRODAN and a K’BdB of

-7 -7

5.57 x 10P


M and 6.14 x 10P


M, respectively. Yang and others (2003) reported that
native BLG at pH 7.0 exhibited 0.41 binding sites for ANS with a dissociation constant

-5 -7

of 4.5 x 10P


M. At pH 9.0, native BLG had 0.80 binding sites with K’BdB of 3.71 x 10P


The number of binding sites of BLG for PRODAN observed in the present research is
higher than the results Yang and others (2003) reported for BLG to ANS, which suggests
that anionic probes, such as ANS, may underestimate the number of binding sites through
ionic interactions between ANS and BLG, or uncharged probes, like PRODAN, may
overestimate the number of binding sites.


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There were significant differences (p < 0.05) between dissociation constants of
BLG for PRODAN as a function of pH and UHP treatment times. A 1.2-fold increase in
the dissociation constant of BLG was observed after 32 min of UHP treatment (Table 1)
at pH 3.0. The number of binding sites at pH 3.0 decreased with UHP treatment from
2.26 to 1.32 after UHP of 8 min. However after 16 min, the number of binding sites
increased to 1.92 probably due to a loosening of the compact structure from an extended
UHP hold time. At pH 5.0, untreated BLG exhibited the largest number of binding sites


and UHP-treated BLG after 16 min exhibited a large dissociation constant of 7.16 x 10P


M, which may indicate a decrease in the binding affinity of BLG for PRODAN. At pH
7.0, there was a slight increase in the number of binding sites after UHP of 4 min with


highest K’d (8.42 x 10P


M) after UHP of 32 min, which may suggest that during UHP
treatment, changes in secondary structure around the loosely structured surface loops may
decrease accessibility to the binding sites for PRODAN (Creamer 1995). At pH 9.0, the
highest n was observed after come-up time and 32 min.
Yang and others (2003) reported that the surface hydrophobic site of UHPinduced
BLG dimers were surrounded by hydrophobic amino acid residues, which
resulted in an increase of hydrophobic affinity of BLG for CPA at the surface
hydrophobic site. This is consistent with the current study in which increases in the
binding affinity of BLG of pH 3.0 for PRODAN were observed after 4 min of UHP
treatment. It can be seen from Table 1 that BLG of pH 3.0 was observed of having the
most number of binding sites, regardless of UHP treatment times, and the greatest
dissociation constants than BLG of other pH values. On the other hand, BLG of pH 7.0
exhibits lower values of dissociation constants compared to BLG of pH 3.0, and may be

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appropriate for further binding experiments due to the number of binding sites of more
than 0.8 under the different UHP treatment times tested. Most foods occur close to
neutral pH, and binding studies of BLG with flavor compounds carried out at pH 7.0 may
show improvement in flavor characteristics of reduced-fat foods.
Tryptophan emission spectra and PRODAN binding provide evidence that UHP
treatment at 600 MPa and pH 7.0 induces BLG to form a molten globule. PRODAN may
be used to determine the surface hydrophobicity of proteins over a wide range of pH
values. In addition to pressure-induced denaturation, the base-induced denaturation of
BLG at alkaline pH may prove useful in increasing the binding properties of BLG.


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