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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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Figure 1. Ribbon diagram of a single subunit of β-lactoglobulin. The diagram was
reproduced from (Brownlow and others 1997). The triangle and circle dots indicate
positions for the hydrophobic pocket or hydrophobic surface pocket, respectively,
proposed by Sawyer and others (1998).


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Surface Hydrophobicity of Ultra High Pressure Treated β-Lactoglobulin –
PRODAN Fluorescent Probe

1 1 2 1

Tinyee HoangP P, Joseph R. PowersP P, John K. FellmanP P, Stephanie ClarkP


and Barry




(1) Dept. of Food Science & Human Nutrition, (2) Dept. of Horticulture & Landscape
Architecture, Washington State Univ., PO Box 646376, Pullman, WA 99164-6376


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The effects of ultra-high hydrostatic pressure (UHP) treatment at 600 MPa with
treatment times of 0 to 32 min and pH values of 3.0 to 9.0 on intrinsic tryptophan
fluorescence of β-lactoglobulin (BLG) and the binding properties of 6-propionyl-2-
(dimethylamino)-naphthalene (PRODAN) extrinsic probe to BLG were studied. UHP
treatment of BLG at selected pH values resulted in increases in tryptophan fluorescence
peak intensities with unfolding of BLG structure. A red shift in the emission peak
wavelength after UHP come-up time at pH 7.0 indicated an increase in the polarity of the
microenvironment of tryptophan. Significant increases in surface hydrophobicity of
UHP-treated BLG were observed with the greatest value at pH 9.0 after come-up time,
probably resulting from irreversible unfolding and exposure of hydrophobic sites of BLG
to PRODAN. Significant changes in the number of binding sites and increases in
dissociation constants of BLG for PRODAN were observed as a function of pH and UHP
treatment time. A linear relationship was not observed between UHP treatment and
number of binding sites. However, at pH 7.0, BLG had relatively low dissociation
constants, with more binding sites than BLG of other pH values, suggesting that UHPtreated
BLG can be used as an ingredient for improvement of flavor in most reduced fat
foods close to neutral pH.


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Recent developments and advances in ultra high pressure (UHP) processing
technology are now at a stage where certain foodstuffs can be commercially processed
isobarically at pressures on the order of hundreds of mega-pascals (MPa). In contrast to
thermal processing, UHP processing affects the structural stability of food constituents
such as proteins in such a way that can improve their intrinsic functional properties for
new ingredients.
UHP involves subjecting a food material to pressures up to 600 MPa and holding
the food isobarically for up to 30 min before pressure release (Ramaswamy and others).
UHP treatment is sufficient in bringing about necessary molecular change, microbial
deactivation, pasteurization, and extended shelf-life.
High pressures act by altering the balance of intramolecular and solvent-protein
interactions. Pressure-induced denaturation results from the disruption of both
hydrophobic interactions and salt bridges. The extent of the changes in proteins depends
on factors such as temperature, pH, solvent, and ionic strength, as well as on the nature of
the native protein structure and pressure applied to the proteins (Iametti and others 1997).
Pressure-induced protein structure unfolding is accompanied by a reduction in volume
and hydration of nonpolar amino acid residues (Damodaran 1996). The unfolding
mechanism of pressure-induced protein denaturation is not yet fully understood, although
UHP treatment results in changes in the structure of the protein molecules due to the
cleavage of weak hydrogen bonds and van der Waals forces while covalent bonds are
unaffected (Tedford and others 1999).


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Fluorescence spectroscopy is a useful technique to study the structure and
dynamics of protein molecules, providing the primary protein structure harbors intrinsic
chromophores. The sensitivity and noninvasiveness of fluorometry is a promising and
widely used technique in medicine, biology, biochemistry, and molecular biophysics
(Royer 1995). The intrinsic fluorescence of tryptophan residues in proteins is particularly
sensitive to protein microenvironments and provides a sensitive method to study
conformational changes of proteins (Busti and others 2002).
Extrinsic fluorescent probes can bind specifically to proteins, influencing both the
intrinsic fluorescence of the protein and the fluorescence of the probe. PRODAN (6propionyl-2-(dimethylamino)-naphthalene)
exhibits sensitivity to the polarity of the
environment in biological materials (Alizadeh-Pazdar and Li-Chan 2000). Although
PRODAN exhibits low quantum yield in aqueous solutions, PRODAN becomes highly
fluorescent in nonpolar solvents or when bound to hydrophobic sites on proteins or
membranes (Vazquez and others 2005). Without a charge on PRODAN, electrostatic
interactions are eliminated during the determination of surface hydrophobicity. Spectral
shifts of PRODAN fluorescence that reflect changes in polarity of the environment and
absence of ionic interactions due to lack of a permanent charge are supported by studies
on spectral properties of PRODAN (Weber and Farris 1979), and binding with spectrin,
egg and milk proteins (Alizadeh-Pazdar and others 2004), bovine serum albumin,
ovalbumin (Haskard and Li-Chan 1998), thionin (Huang and others 1997), and lipid
bilayers (Kusube and others 2005).
Recently the PRODAN probe was used by Alizadeh-Pasdar and Li-Chan (2000)
for the quantitation of protein surface hydrophobicity at pH 3.0, 5.0, 7.0, and 9.0. In that

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study (Alizadeh-Pazdar and Li-Chan 2000), a method was developed with PRODAN to
quantitate protein surface hydrophobicity comparing the binding of PRODAN versus
aromatic 1-anilinonaphthalene-8-sulfonic acid (ANS) and anionic aliphatic cis-parinaric
acid (CPA) fluorescent probes to whey protein isolate (WPI), β-lactoglobulin (BLG), and
bovine serum albumin (BSA) during thermal treatment at 80°C. Alizadeh-Pazdar and Li-
Chan (2000) reported lower hydrophobicity at pH 3.0 than other pH values for BSA,
BLG, and WPI determined using PRODAN, whereas hydrophobicity determined using
ANS was high at pH 3.0 for the proteins compared to other pH values. They also reported
that heating (80 °C for 30 min) of BSA, BLG and WPI at pH 3.0, 5.0, 7.0, or 9.0 either
had no effect or significantly decreased (p ≤ 0.05) surface hydrophobicity determined
using PRODAN.
Some authors have reported using ANS and CPA to determine hydrophobicity of
UHP treated BLG or whey protein concentrate (Yang and others 2001; Liu and others
2005) and others have reported using PRODAN for characterizing binding sites of human
serum albumin (Moreno and others 1999). It is the aim of this research to use PRODAN
to observe changes in hydrophobicity of BLG as a function of UHP treatment and pH.
β-Lactoglobulin (BLG) and PRODAN were purchased from Sigma Chemical Co.
(St. Louis, MO, no. L-0130) and Molecular Probes (Eugene, OR), respectively.
Ultra-high Hydrostatic Pressure (UHP) Treatment
BLG solutions, at selected protein concentrations of 0.05 to 0.50 mg/mL in 0.05
mg/mL increments were prepared with sodium phosphate buffer (0.01 M) and adjusted to
selected pH values of 3.0, 5.0, 7.0, or 9.0. The concentrations of BLG at pH 7.0 were

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spectrophotometrically determined using a molar extinction coefficient of εB280B = 17600





Pat 280 nm. BLG solutions, at an initial temperature of 4°C, were treated with an
isostatic pressing system (Engineered Pressure Systems, Inc., Andover, MA) composed
of a cylindrical pressure chamber (height = 0.25 m, diameter = 0.10 m) at 600 MPa for 0
(come-up time), 4, 8, 16, or 32 min. The come-up time (5.25 min) is the compression
time required to reach a pressure of 600 MPa. After exposure to high pressure, the BLG
solutions were assayed immediately or stored at 4 °C.
Intrinsic and Extrinsic Fluorescence Spectra
Conformational changes of BLG solutions were monitored by observing intrinsic
tryptophan and extrinsic fluorescence spectra. Intrinsic fluorescence was assayed using
an excitation wavelength of 295 nm to avoid excitation of tyrosine (Bhattacharjee and
Das 2000; Muresan and others 2001) and observing fluorescence intensity at the emission
wavelength of 338 nm. Extrinsic PRODAN fluorescence of BLG solutions was assayed
using an excitation wavelength of 365 nm and observing the fluorescence intensity at the
emission spectra at wavelengths from 375 to 600 nm. Ten microliters of PRODAN
(0.153 mM in methanol) solution was added to 4 mL of untreated or UHP-treated BLG
solutions (0.05-0.5 mg/mL) for the extrinsic PRODAN assay. Intrinsic and extrinsic
fluorescence data were collected with a Fluoro Max-3 fluorometer (Jobin Yvon Horiba


Spex, Edison, NJ) and data-conversion software (DATAMAX and GRAMS/32P P, Jobin
Yvon Horiba Spex, Edison, NJ).
Surface Hydrophobicity
The extrinsic aromatic uncharged hydrophobic PRODAN fluorescent probe is
unlike other probes used to determine hydrophobicity of proteins. Unlike PRODAN,

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fluorescent probes such as ANS or CPA are anionic and may contribute charged
interactions on the determination of surface hydrophobicity at various pH values
(Alizadeh-Pazdar and Li-Chan 2000). PRODAN, on the other hand, has no charge,
eliminating possible electrostatic contributions, and hence the PRODAN probe was used
to assay the hydrophobic nature of BLG.
Surface hydrophobicity of BLG was determined using the PRODAN probe. A


stock solution of 1.53 x 10P PM PRODAN was prepared in methanol, sealed with Parafilm
to prevent evaporation of methanol, covered with aluminum foil to avoid exposure to


light, and stirred for 5 hr. The PRODAN stock solution was stored in the freezer (-11P PC)
until the day of the assay, and held on ice throughout the experiment. The concentration
of PRODAN in the stock solution was spectrophotometrically determined using a molar
absorption coefficient of εB360B
the assay.

-1 -1

= 1.8 x 104 MP




(Chakrabarti 1996) before performing

Intrinsic tryptophan fluorescence determinations were performed using
excitation/emission wavelengths of 295 nm/338 nm and slits were set at 5 nm/5 nm to
avoid excitation of tyrosine. Even though tyrosine has a high quantum yield, the tyrosine
emission of most proteins is small and undetectable due to quenching by the presence of
nearby charged or uncharged amino groups (Lakowicz 1983). The fluorescence emission
of tryptophan is often studied for its solvent sensitivity and characterization of the protein
structure during denaturation as evidenced by its blue or red shifts (Lakowicz 1983).
Extrinsic fluorescence determination of PRODAN assay hydrophobicity of BLG was
performed with excitation/emission wavelengths of 365 nm/465 nm and slits were set at 5
nm/5 nm. A 10 µL aliquot of the PRODAN stock solution was added to 4 mL of diluted

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