Interaction of milk a- and b-caseins with tea polyphenols

Imed Hasni, Philippe Bourassa, Saber Hamdani, Guy Samson, Robert Carpentier, Heidar-Ali Tajmir-Riahi

Dpartement de Chimie-Biologie, Universit du Qubec Trois-Rivires, C.P. 500, Trois-Rivires, Qubec, Canada G9A 5H7
a r t i c l e i n f o
Article history:
Received 25 June 2010
Received in revised form 27 October 2010
Accepted 12 November 2010
Keywords:
Milk
Tea
Casein
Polyphenol
Secondary structure
FTIR
UVvisible
CD
Fluorescence spectroscopy
a b s t r a c t
The interaction of a- and b-caseins with tea polyphenols (+)-catechin (C), ()-epicatechin (EC), ()-epi-
gallocatechin (EGC) and ()-epigallocatechin gallate (EGCG) was examined at a molecular level, using
FTIR, UVvisible, CD and uorescence spectroscopic methods as well as molecular modelling. The poly-
phenol binding mode, the binding constant and the effects of polyphenol complexation on casein stability
and conformation were determined. Structural analysis showed that polyphenols bind casein via both
hydrophilic and hydrophobic interactions with overall binding constants of K
Ca-cas
= 1.8
(0.8) 10
3
M
1
, K
ECa-cas
= 1.8 (0.6) 10
3
M
1
, K
EGCa-cas
= 2.4 (1.1) 10
3
M
1
and K
EGCGa-cas
= 7.4
(0.4) 10
3
M
1
, K
Cb-cas
= 2.9 (0.3) 10
3
M
1
, K
ECb-cas
= 2.5 (0.6) 10
3
M
1
, K
EGCb-cas
= 3.5
(0.7) 10
3
M
1
and K
EGCGb-cas
= 1.59 (0.2) 10
4
M
1
. The number of polyphenol bound per protein
molecule (n) was 1.1 (C), 0.9 (EC), 1.1 (EGC), 1.5 (EGCG) for a-casien and 1.0 (C), 1.0 (EC), 1.1 (EGC)
and 1.5 (EGCG) for b-casein. Structural modelling showed the participation of several amino acid residues
in polyphenolprotein complexation with extended H-bonding network. Casein conformation was
altered by polyphenol with a major reduction of a-helix and b-sheet and increase of random coil and turn
structure suggesting further protein unfolding. These data can be used to explain the mechanism by
which the antioxidant activity of tea compounds is affected by the addition of milk.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Tea is one of the most popular beverages in the world. In recent
years, much has been said about the added health benet or re-
duced benet of adding milk to tea (Ho, Lin, & Shahidi, 2009; Stan-
ner, 2007). The dual effects of milk on the antioxidant capacity of
different tea polyphenols were recently reported (Dubeau, Samson,
& Tajmir-Riahi, 2010). However, the mechanism by which the anti-
oxidant activity of tea polyphenols affected by milk is not yet
known. Caseins are the major phosphoproteins of mammalian milk
and exist as micelles made of polypeptides known as a-, b- and j-
caseins (Uversky, 2002). The three casein components are almost
similar in size, molecular weight (24 kDa) and net negative charge
but differ in their degree of unfoldedness (Farrell et al., 2004; Fox &
McSweeny, 1998; Kumosinski, Brown, & Farell, 1993). Caseins be-
long to the rapidly growing family of unstructured proteins that
have lately attracted much interest due to their unique unfolded
structure under native conditions, brought about by a combination
of high net charge and low intrinsic hydrophobicity (Phadungath,
2005; Uversky, 2002). a-Casein contains two tryptophan (Trp),
whilst b- and j-caseins have one Trp residue (Kumosinski et al.,
1993). Plant polyphenolic compounds show strong interaction
with globular proteins and can cause protein unfolding. In solution
polyphenols such as catechins (Scheme 1) can form insoluble com-
plexes with milk proteins including b-casein (Liang & Xu, 2003).
The binding afnity of polyphenols to protein is size dependent
and increases with their molecular size (De Fretias & Mateus,
2001). Larger polyphenols like those in black tea are most likely
to form complexes with milk proteins. This binding can affect the
electron donation capacity of catechins by reducing the number
of hydroxyl groups available in the solution. Studies in the past
have shown the effects of milk protein on the antioxidant capacity
of tea polyphenols, whilst the effect of polyphenol complexation
on the stability and conformation of milk proteins has not been ad-
dressed (Aguie-Beghin, Sausse, Meudec, Cheynier, & Doullard,
2008; Alexandropoulou, Komaitis, & Kapsokefalou, 2006; Jobstl,
Howse, Fairclough, & Williamson, 2006; Kartsova & Alekseeva,
2008; Shukla, Narayanan, & Zanchi, 2009; Yan, Hu, & Yao, 2009).
Therefore, the structural characterisation of the interaction be-
tween milk proteins and polyphenols is a major step in elucidating
the induced effect of polyphenols on milk protein structure and on
the antioxidant activity of tea compounds.
Fluorescence quenching is considered as a useful technique for
measuring binding afnities. Fluorescence quenching is the
decrease of the quantum yield of uorescence from a uorophore
0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.11.087
Abbreviations: cas, casein; C, catechin; EC, epicatechin; EGC, epigallocatechin;
EGCG, epigallocatechin gallate; FTIR, Fourier transform infrared; CD, circular
dichroism.

Corresponding author. Tel.: +1 819 376 5011x3310; fax: +1 819 376 5084.
E-mail address: tajmirri@uqtr.ca (H.-A. Tajmir-Riahi).
Food Chemistry 126 (2011) 630639
Contents lists available at ScienceDirect
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j our nal homepage: www. el sevi er . com/ l ocat e/ f oodchem
induced by a variety of molecular interactions with the quencher
molecule (Lakowicz, 1999; Tayeh, Rungassamy, & Albani, 2009).
Therefore, it is possible to use quenching of the intrinsic trypto-
phan uorescence of Trp-37, Trp-66 in a
s1
-casein and Trp-193,
Trp-109 in a
s2
-casein as well as Trp-143 of b-casein (Kumosinski
et al., 1993) as a tool to study the interaction of polyphenol with
caseins in an attempt to characterise the nature of tea catechin
casein complexation.
In this contribution, spectroscopic analysis and docking studies
of the interaction of a- and b-caseins with several tea polyphenols
catechin, epicatechin, epigallocatechin and epigallocatechin gallate
(Scheme 1) in aqueous solution under physiological conditions,
using constant protein concentration and various polyphenol con-
tents are presented. Structural analysis regarding catechin binding
mode and the effects of catechincasein complexation on the pro-
tein stability and secondary structure is also reported.
2. Materials and methods
2.1. Materials
The compounds a- and b-caseins (with purity of 70% and 98%)
and tea catechins were purchased from SigmaAldrich Chemical
Co. (St. Louis, MO) and used as supplied. Other chemicals were of
reagent grade and used without any further purication.
2.2. Preparation of stock solutions
Casein was dissolved in aqueous solution (8 mg/ml for a-casein
and 11.8 mg/ml for b-casein to obtain 0.5 mM protein content)
containing 10 mM TrisHCl buffer (pH 7.4). Polyphenol (1 mM)
was prepared in TrisHCl and diluted to various concentrations
in TrisHCl (0.5, 0.25 and 0.125 mM). The protein concentration
was determined spectrophotometrically using the extinction coef-
cients of 11,000 M
1
cm
1
(b-casein, MW = 24,000) and
15,000 M
1
cm
1
(a-casein, MW = 23,600) at 280 nm (Fox &
McSweeny, 1998; Thorn et al., 2005).
2.3. FTIR spectroscopic measurements
Infrared spectra were recorded on a FTIR spectrometer (Impact
420 model, Digilab, Mississauga, ON, Canada), equipped with deu-
terated triglycine sulphate (DTGS) detector and KBr beam splitter,
using AgBr windows. A Solution of polyphenol was added dropwise
to the protein solution with constant stirring to ensure the forma-
tion of homogeneous solution and to reach the target polyphenol
concentrations of 0.125, 0.25 and 0.5 mM with a nal protein con-
centration of 0.25 mM. Spectra were collected from hydrated lms
after 2 h incubation of casein with polyphenol solution at room
temperature. Interferograms were accumulated over the spectral
range of 4000600 cm
1
with a nominal resolution of 2 cm
1
and
100 scans. The difference spectra [(protein solution + polyphenol
solution) (protein solution)] were generated using the water
combination mode around 2300 cm
1
as standard (Dousseau,
Therrien, & Pezolet, 1989). When producing difference spectra, this
band was adjusted to the baseline level in order to normalise the
difference spectra.
2.4. Analysis of protein conformation
Analysis of the secondary structure of caseins and their poly-
phenol complexes was carried out on the basis of the procedure al-
ready reported (Byler & Susi, 1986). The protein secondary
structure was determined from the shape of the amide I band, lo-
cated at 16601650 cm
1
. Fourier self-deconvolution and second
derivative resolution enhancement were applied to increase the
spectral resolution in the region of 17001600 cm
1
. The second
derivatives were obtained using a point convolution of 11 or 13.
The resolution enhancement resulting from self-deconvolution
and the second derivative is such that the number and the position
of the bands to be tted are determined. In order to quantify the
Scheme 1. Chemical structure of tea polyphenols.
I. Hasni et al. / Food Chemistry 126 (2011) 630639 631
area of the different components of amide I contour revealed by
self-deconvolution and second derivative, a least-square iterative
curve-tting was used to t the Gaussian line shapes to the spectra
between 1700 and 1600 cm
1
. The details of spectral manipulation
regarding curve-tting have previously been reported (Beauche-
min et al., 2007). The curve-tting analysis was performed using
the GRAMS/AI Version 7.01 software of the Galactic Industries
Corporation.
2.5. Circular dichroism
CD Spectra of caseins and their polyphenol complexes were re-
corded with a Jasco J-720 spectropolarimeter (Jasco Inc., Easton,
MA, USA). For measurements in the far-UV region (178260 nm),
a quartz cell with a path length of 0.01 cm was used in nitrogen
atmosphere. Protein concentration was kept constant (12.5 lM),
whilst varying each polyphenol concentration (0.125, 0.25 and
0.5 mM). An accumulation of ve scans with a scan speed of
50 nm per minute was performed and data were collected for each
nm from 260 to 180 nm. Sample temperature was maintained at
25 C using a Neslab RTE-111 (Jasco Inc., Easton, MA, USA) circulat-
ing water bath connected to the water-jacketed quartz cuvettes.
Spectra were corrected for buffer signal and conversion to the
Mol CD (De) was performed with the Jasco Standard Analysis soft-
ware. The protein secondary structure was calculated using
CDSSTR, which calculates the different assignments of secondary
structures by comparison with CD spectra measured from different
proteins for which high quality X-ray diffraction data are available
(Johnson, 1999; Sreerama & Woddy, 2000). The program CDSSTR is
provided in CDPro software package which is available at the web-
site: http://lamar.colostate.edu/~sreeram/CDPro.
2.6. Absorption spectroscopy
The absorption spectra were recorded on a Perkin-Elmer Lamb-
da 40 spectrophotometer (Woodbridge, ON, Canada). Quartz cuv-
ettes of 1 cm were used and the absorption spectra recorded for
free protein (100 lM) and for its polyphenol complexes with each
polyphenol (10200 lM).
The binding constants of the polyphenolcasein complexes
were calculated as reported (Connors, 1987; Stephanos, 1996). It
is assumed that the interaction between the ligand L and the sub-
strate S is 1:1 (mol/mol); for this reason a single complex SL (1:1) is
formed. It was also assumed that the sites (and all the binding
sites) are independent and nally the Beers law is followed by
all species. A wavelength is selected at which the molar absorptiv-
ities e
S
(molar absorptivity of the substrate) and e
11
(molar absorp-
tivity of the complex) are different. Then at total concentration S
t
of
the substrate, in the absence of ligand and the light path length is
b = 1 cm, the solution absorbance is
A
o

S
bS
t
1
In the presence of ligand at total concentration L
t
, the absor-
bance of a solution containing the same total substrate concentra-
tion is
A
L

S
bS
L
bL
11
bSL 2
where [S] is the concentration of the uncomplexed substrate, [L] the
concentration of the uncomplexed ligand and [SL] is the concentra-
tion of the complex) which, combined with the mass balance on S
and L, gives
A
L

S
bS
t

L
bL
t

11
bSL 3
where De
11
= e
11
e
S
e
L
(e
L
molar absorptivity of the ligand). By
measuring the solution absorbance against a reference containing
ligand at the same total concentration L
t
, the measured absorbance
becomes
A
S
bS
t
D
11
bSL 4
Combining Eq. (4) with the stability constant denition K
11
= [SL]/
[S][L], gives
DA K
11
D
11
bSL 5
where DA = A A
o
. From the mass balance expression S
t
= [S] + [SL]
we obtain [S] = S
t
/(1 + K
11
[L]), which is Eq. (5), giving Eq. (6) that
provides the relationship between the observed absorbance change
per centimetre and the system variables and parameters:
DA
b

S
t
K
11
De
11
L
1 K
11
L
6
Eq. (6) is the binding isotherm, which shows the hyperbolic depen-
dence on free ligand concentration.
The double-reciprocal form of plotting the rectangular hyper-
bola
1
y

f
d

1
x

e
d
, is based on the linearisation of Eq. (6) according
to the following equation,
b
DA

1
S
t
K
11
De
11
L

1
S
t
De
11
7
Thus the double reciprocal plot of 1/DA versus 1/[L] is linear and the
binding constant can be estimated from the following equation
K
11

intercept
slope
8
2.7. Fluorescence spectroscopy
Fluorometric experiments were carried out on a Perkin-Elmer
LS55 Spectrometer (Woodbridge, ON, Canada). Stock solutions of
polyphenol 1 mM were prepared at room temperature
(24 1 C). Various solutions of polyphenol (10200 lM) were
prepared from the above stock solutions by successive dilutions
also at 24 1 C. Solution of casein (100 lV) in 10 mM TrisHCl
(pH 7.4) was also prepared at 24 1 C. The above solutions were
kept in the dark and used soon after. Samples containing 0.4 ml
of the above casein solution and various polyphenol solutions were
mixed to obtain nal polyphenol concentration of 10200 lV with
constant casein content 100 lV. The uorescence spectra were re-
corded at k
exc
= 280 nm and k
em
from 287 to 500 nm. The intensity
at 350 nm (tryptophan) was used to calculate the binding constant
(K) according to previous literature reports (Bi et al., 2004; Dufour
& Dangles, 2005; He et al., 2005; Tang, Qi, & Chen, 2005).
2.8. Molecular modelling
The docking studies were performed with ArgusLab 4.0.1 soft-
ware (Mark A. Thompson, Planaria Software LLC, Seattle, WA,
http://www.arguslab.com). The protein structures were obtained
from Kumosinski et al. (1993) and the polyphenol three dimen-
sional structures were generated from PM3 semi-empirical calcu-
lations using Chem3D Ultra 6.0. The whole protein was selected
as a potential binding site since no prior knowledge of such site
was available. The docking runs were performed on the ArgusDock
docking engine using regular precision with a maximum of 1000
candidate poses. The conformations were ranked using the Ascore
scoring function, which estimates the free binding energy. Upon
location of the potential binding sites, the docked complex confor-
mations were optimised using a steepest decent algorithm until
convergence, with a maximum of 20 iterations. Amino acid resi-
dues within a distance of 3.5 relative to the polyphenol were in-
volved in the complexation.
632 I. Hasni et al. / Food Chemistry 126 (2011) 630639
3. Results and discussion
3.1. FTIR spectra of caseinpolyphenol complexes
The polyphenolcasein complexation was characterised by
infrared spectroscopy and its derivative methods. Since there was
no major spectral shifting for the casein amide I band at 1656
1652 cm
1
(mainly C@O stretch) and amide II band at 1544
1540 cm
1
(CN stretching coupled with NH bending modes)
(Beauchemin et al., 2007; Krimm & Bandekar, 1986) upon polyphe-
nol interaction, the difference spectra [(protein solution + polyphe-
nol solution) (protein solution)] were obtained, in order to
monitor the intensity variations of these vibrations and the results
are shown in Figs. 1 and 2. Similarly, the infrared self-deconvolu-
tion with second derivative resolution enhancement and curve-t-
ting procedures (Byler & Susi, 1986) were used to determine the
protein secondary structures in the presence of polyphenols
(Fig. 3).
At low polyphenol concentration (0.125 mM), an increase in
intensity was observed for the protein amide I at 1656 and amide
II at 1544 cm
1
(a-casein) and 1652 and 1540 cm
1
(b-casein) in
the difference spectra of the polyphenolcasein complexes (Figs.
1 and 2, diffs., 0.125 mM). The positive features are located in the
difference spectra for amides I and II bands at 1648, 1540 (Ccase-
in), 1650, 1542 (ECcasein), 1654, 1541 (EGCcasein) and at 1651,
1542 cm
1
(EGCGcasein) for a-casein polyphenols and at 1645,
1548 (Ccasein), 1655, 1548 (ECcasein), 1652, 1549 (EGCcasein)
and 1647, 1549 cm
1
(EGCGcasein) for b-caseinpolyphenol com-
plexes (Figs. 1 and 2, diffs., 0.125 mM). These positive features are
related to increase of the intensity of the amides I and II bands
upon polyphenol complexation. The increase in intensity of the
amides I and II bands is due to polyphenol binding to protein
C@O, CN and NH groups (hydrophilic interaction). Additional
evidence to support the polyphenol interaction with CN and N
H groups comes from the shifting of the protein amide A band at
3295 cm
1
(NH stretching mode) in the free caseins to 3285
3280 cm
1
upon polyphenol interaction (spectra not shown).
As polyphenol concentration increased to 0.5 mM, a decrease in
the intensity of the amides I and II bands was observed with neg-
ative features in the difference spectra for amides I and II bands at
1654, 1530 (Ccasein), 1654, 1530 (ECcasein), 1658, 1538 (EGC
casein) and at 1650, 1537 cm
1
(EGCGcasein) for a-casein and
Fig. 1. FTIR spectra of hydrated lms (pH 7.4) in the region of 1800600 cm
1
for free a-casein (0.25 mM), free C (A) (0.5 mM), free EC (B) (0.5 mM), free EGC (C) (0.5 mM) and
free EGCG (D) (0.5 mM) with difference spectra (diff.) of a-caseinpolyphenol complexes (bottom two curves) obtained at different poylphenol concentrations (indicated in
the gure).
I. Hasni et al. / Food Chemistry 126 (2011) 630639 633
at 1651, 1539 (Ccasein), 1655, 1539 (ECcasein), 1655, 1538
(EGCcasein) and 1654 and 1532 cm
1
(EGCGcasein) for b-casein
upon polyphenol complexation (Figs. 1 and 2, diffs., 0.5 mM). The
decrease in the intensity of the amide I band at 16561652 cm
1
in the spectra of the polyphenolcasein complexes suggests a ma-
jor reduction of protein a-helical structure at high polyphenol con-
centrations. Similar infrared spectral changes were observed for
protein amide I band in several ligandprotein complexes where
major protein conformational changes occurred (Ahmed Ouameur
et al., 2006).
A quantitative analysis of the protein secondary structure for
the free a- and b-caseins and their polyphenol adducts in hy-
drated lms has been carried out and the results are shown in
Fig. 3. The free a-casein has 36% a-helix (1658 cm
1
), 20% b-sheet
(1632 and1618 cm
1
), 19% turn structure (1675 cm
1
), 4% b-anti-
parallel (1688 cm
1
) and random 21% coil (1644 cm
1
) (Fig. 3A).
The free b-casein contains 32% a-helix (1658 cm
1
), 24% b-sheet
(1632 and 1618 cm
1
), 18% turn structure (1674 cm
1
), 4% b-anti-
parallel (1686 cm
1
) and random 23% coil (1644 cm
1
) (Fig. 3B).
These results are consistent with spectroscopic studies of caseins
previously reported (Curley, Kumosinski, Unruh, & Farrell, 1998;
Malin et al., 2001). Upon polyphenol interaction, a major decrease
of the a-helix and b-sheet occurred, whilst an increase of turn
and random structure was observed in the polyphenolcasein
complexes (Fig. 3A and B). The conformational changes observed
were more pronounced for ECG and EGCG than C and EC com-
plexes. This is indicative of larger perturbations of casein second-
ary structure by larger and bulkier polyphenols. This is also
consistent with the extra stability of casein complexes with larger
polyphenols, which will be discussed with uorescence and UV
data further on.
3.2. CD spectroscopy
CD spectroscopy was also used to analyse the protein conforma-
tion in the polyphenolcasein complexes. The CD results exhibit
marked similarities with those of the infrared data. The protein
conformational analysis based on CD data suggests that free a-
casein has a-helical 35%, b-sheet 12%, turn 20% and random coil
33%, whilst free b-casein contains a-helical 33%, b-sheet 16%, turn
17% and random coil 34%. These data for free b-casein are
consistent with the literature report (Chakraborty & Basak, 2007).
Upon polyphenol interaction, major reduction of a-helix was ob-
served from 35% to 33% in free caseins (a and b) to 3224% in
Fig. 2. FTIR spectra of hydrated lms (pH 7.4) in the region of 1800600 cm
1
for free b-casein (0.25 mM), free C (A) (0.5 mM), free EC (B) (0.5 mM), free EGC (C) (0.5 mM) and
free EGCG (D) (0.5 mM) with difference spectra (diff.) of b-caseinpolyphenol complexes (bottom two curves) obtained at different polymer concentrations (indicated in the
gure).
634 I. Hasni et al. / Food Chemistry 126 (2011) 630639
the polyphenolcasein complexes. The decrease in a-helix was
accompanied by an increase in the turn and randomcoil structures.
The major reduction of the a-helix with an increase in the turn and
random structures is consistent with the infrared results that
showed reduction of a-helix with increase in random coil and turn
structure due to a further protein unfolding.
3.3. Hydrophobic interactions
The spectral changes of the casein CH
2
antisymmetric and sym-
metric stretching vibrations
,
in the region of 30002800 cm
1
were
monitored in order to locate the presence of hydrophobic contact
in the polyphenolcasein complexes. The CH
2
bands of the free
Fig. 3. Second derivative resolution enhancement and curve-tted amide I region (17001600 cm
1
) for free a-casein (A) and free b-casein (B) with their polyphenol
complexes (0.5 mM polyphenol and 0.25 mM protein concentrations at pH 7.4).
I. Hasni et al. / Food Chemistry 126 (2011) 630639 635
a-casein at 2957, 2933, and 2879 cm
1
shifted to 2959, 2935 and
2873 cm
1
(Ccasein), to 2960, 2935 and 2875 cm
1
(ECcasein),
to 2961, 2936, and 2877 cm
1
(EGCcasein) and to 2959, 2938
and 2874 cm
1
(EGCGcasein), upon polyphenol interaction (Fig-
ure not shown). Similarly, the CH
2
bands of the free b-casein at
2957, 2933, and 2879 cm
1
shifted to 2959, 2934 and 2877 cm
1
(Ccasein), to 2958, 2934 and 2875 cm
1
(ECcasein), to 2959,
2933 and 2874 cm
1
(EGCcasein) and to 2958, 2931 and
2875 cm
1
(EGCGcasein), upon polyphenol interaction (gure
not shown). The shifting of the protein antisymmetric and sym-
metric CH
2
stretching vibrations suggests the presence of hydro-
phobic interactions via polyphenol rings and hydrophobic
pockets in caseins, which is consistent with uorescence spectro-
scopic results discussed below.
3.4. Fluorescence spectra and stability of polyphenolcasein complexes
a-Casein (mixture of a
s1
- and a
s2
-caseins) has two tryptophan
residues Trp-66, and Trp-37 (in a
s1
-casein) and Trp-109 and Trp-
193 (in a
s2
-casein), whilst b-casein contains one tryptophan Trp-
143 with intrinsic uorescence. These tryptophan residues are lo-
cated in the protein surfaces (Kumosinski et al., 1993). Tryptophan
emission dominates casein uorescence spectra in the UV region.
When other molecules interact with casein, tryptophan uores-
cence may change depending on the impact of such interaction
on the protein conformation (Lakowicz, 1999; Tayeh et al., 2009).
On the assumption that there are (n) substantive binding sites
for quencher (Q) on protein (B), the quenching reaction can be
shown as follows:
nQ B ()Q
n
B 9
The binding constant (K
A
), can be calculated as:
K
A
Q
n
B=Q
n
B 10
where [Q] and [B] are the quencher and protein concentration,
respectively, [Q
n
B] is the concentration of non uorescent uoro-
phorequencher complex and [B
0
] gives total protein concen-
tration:
Q
n
B B
0
B 11
K
A
B
0
B=Q
n
B 12
The uorescence intensity is proportional to the protein concentra-
tion as described:
B=B
0
/ F=F
0
13
Results from uorescence measurements can be used to estimate
the binding constant of polyphenolprotein complex from Eq. (14):
logF
0
F=F log K
A
nlogQ 14
The accessible uorophore fraction (f) can be calculated by the
modied SternVolmer equation:
F
0
=F
0
F 1=fKQ 1=f 15
where F
0
is the initial uorescence intensity and F is the uores-
cence intensities in the presence of quenching agent (or interacting
molecule). K is the SternVolmer quenching constant, [Q] is the mo-
lar concentration of quencher and f is the fraction of accessible uo-
rophore to a polar quencher, which indicates the fractional
uorescence contribution of the total emission for an interaction
with a hydrophobic quencher (Lakowicz, 1999). The plot of F
0
/
(F
0
F) vs 1/[Q] yields f
1
as the intercept on y axis and (fK)
1
as
the slope. Thus, the ratio of the ordinate and the slope gives K.
The decrease of uorescence intensity of casein is monitored at
340 nm for polyphenolcasein systems (Fig. 4AH shows represen-
tative results for each system). Representative plots of F
0
/(F
0
F) vs
1/[polyphenol] are shown in Fig. 4A
0
H
0
. Assuming that the ob-
served changes in uorescence come from the interaction between
polyphenols and casein, the quenching constant can be taken as the
binding constant of the complex formation. The K values given here
are averages of three-replicate runs for polyphenol/casein systems,
each run involving several different concentrations of polyphenol
(Fig. 4). The binding constants obtained were K
Ca-cas
= 1.8 (0.8)
10
3
M
1
, K
ECa-cas
= 1.8 (0.6) 10
3
M
1
, K
EGCa-cas
= 2.4 (1.1)
10
3
M
1
and K
EGCGa-cas
= 7.4 (0.4) 10
3
M
1
, K
Cb-cas
= 2.9
(0.3) 10
3
M
1
, K
ECb-cas
= 2.5 (0.6) 10
3
M
1
, K
EGCb-cas
= 3.5
(0.7) 10
3
M
1
and K
EGCGb-cas
= 1.59 (0.2) 10
4
M
1
(Fig. 4A
0

H
0
). The binding constants calculated for the polyphenolcasein
suggest low afnity polyphenolcasein interaction compared to
the other strong ligandprotein complexes (Kragh-Hansen, 1990;
Kratochwil, Huber, Muller, Kansy, & Gerber, 2002; Nsoukpo-Kossi
et al., 2007). However, similar binding constants (10
3
M
1
10
4
M
1
)
were also reported for several ligandprotein complexes using uo-
rescence spectroscopic methods (Bi et al., 2004; Liang, Tajmir-Riahi,
& Subirade, 2008; Sulkowska, 2002). The binding constants of the
larger polyphenolcasein complexes are bigger than those of smal-
ler polyphenolcasein adducts, which can be due to the presence of
more OH groups associated with the bulkier polyphenols. However,
the stability of polyphenols with b-casein is larger than those of the
corresponding a-casein complexes. This can be attributed to the
more hydrophobic nature of b-casein than a-casein due to the pres-
ence of ve phosphoserine residues in b-casein (Phadungath, 2005).
The number of polyphenol bound per protein (n) is calculated
from log [(F
0
F)/F] = log K
S
+ n log [polyphenol] for the static
quenching (Charbonneau & Tajmir-Riahi, 2010; Froehlich, Jen-
nings, Sedaghat-Herati, & Tajmir-Riahi, 2009; Jiang, Gao, & He,
2002; Jiang et al., 2004; Liang et al., 2008; Mandeville & Tajmir-
Riahi, 2010). The n values from the slope of the straight line are
for a-casein 1.1 (C), 0.9 (EC), 1.1 (EGC), 1.5 (EGCG) and for b-casein
1.0 (C), 1.0 (EC), 1.1 (EGC) and 1.5 (EGCG).
The f values obtained for polyphenolcasein complexes suggest
that polyphenols interact with uorophore via hydrophobic inter-
actions. As a result, we predict that polyphenols bind mainly with
the uorophores located on the surface of caseins. This argument is
based on the fact that the emissions, k
max,
of Trp-214 (HSA) and
Trp-212 (BSA) are at 340 nm, which is the emission region of hid-
den tryptophan molecules, whilst uorescence emission of ex-
posed tryptophan molecule is at a higher wavelength (350 nm)
due to solvent relaxation (Liang et al., 2008; Sulkowska, 2002; Tay-
eh et al., 2009). The tightening of protein structure through intra-
molecular interactions, such as hydrogen bonds, seems to bury
tryptophan in a more hydrophobic environment. The changes in
uorescence intensity of tryptophan in both a- and b-casein in
the presence of polyphenol exhibited two different patterns. In
the catechin and epicatechin complexes of a- and b-caseins, the
emission band of the free protein at 350 nm (a-casein) and at
348 nm (b-casein) shifted towards a lower wavelength at 346 nm
(Ccasein) and 348 nm (EC) for a-casein, and at 334 nm (Ccasein)
and 343 nm (ECcasein) in b-casein complexes (Fig. 4A, B, E and F).
On the other hand, the emission band of the free caseins shifted to-
wards a higher wavelength at 351 nm (EGCcasein) and 365 nm
(EGCGcasein) for a-casein, and at 349 nm (EGCcasein) and
367 nm (EGCGcasein) in b-casein complexes (Fig. 4C, D, G and
H). The upward shift of the casein emission band was more pro-
nounced in the case of the larger and bulkier EGCG molecule with
more OH groups (Fig. 4D and H). The downward shift of the emis-
sion band of casein in the catechin and epicatechinprotein com-
plexes is due to the tightening of protein structure through
intramolecular interactions, such as hydrogen bonds. This seems
to change tryptophan in a more hydrophobic environment. How-
ever the upward shift of the protein emission bands observed in
the spectra of EGCcasein and EGCGcasein is related to more
636 I. Hasni et al. / Food Chemistry 126 (2011) 630639
exposure of tryptophan residue and unfolding of protein structure.
This is consistent with our infrared results that show major sec-
ondary structural changes for caseins upon EGC and EGCG
complexation.
3.5. UV spectra and stability of polyphenolcasein complexes
The polyphenolcasein binding constants were also determined
using UVvisible spectroscopic method (described in Section 2).
The double reciprocal plot of 1/DA versus 1/[L] is linear and the
binding constant can be estimated from the intercept to slope.
One binding site was observed for each polyphenolprotein com-
plex with the overall binding constants of K
Ca-cas
= 1.3
(0.2) 10
3
M
1
, K
ECa-cas
= 1.1 (0.7) 10
3
M
1
, K
EGCa-cas
= 2.3
(0.9) 10
3
M
1
and K
EGCGa-cas
= 3.2 (1.4) 10
3
M
1
, K
Cb-
cas
= 1.2 (0.2) 10
3
M
1
, K
ECb-cas
= 2.3 (0.9) 10
3
M
1
, K
EGCb-
cas
= 4.1 (1.2) 10
3
M
1
and K
EGCGb-cas
= 6.1 (4.0) 10
3
M
1
.
The association constants calculated for the polyphenolcasein ad-
ducts from UV spectroscopy are consistent with those from uo-
rescence spectroscopy with K
Ca-cas
= 1.8 (0.8) 10
3
M
1
, K
ECa-
cas
= 1.8 (0.6) 10
3
M
1
, K
EGCa-cas
= 2.4 (1.1) 10
3
M
1
and
K
EGCGa-cas
= 7.4 (0.4) 10
3
M
1
, K
Cb-cas
= 2.9 (0.3) 10
3
M
1
,
K
ECb-cas
= 2.5 (0.6) 10
3
M
1
, K
EGCb-cas
= 3.5 (0.7) 10
3
M
1
and K
EGCGb-cas
= 1.59 (0.2) 10
4
M
1
(Fig. 4A
0
H
0
). The stability
of casein adducts with catechin and epicatechin are very similar
with the same number of OH groups. However, a larger stability
was observed for EGCGcasein than EGCcasein complexes due
to the extra OH groups associated with the bulkier EGCG molecule.
Fig. 4. Fluorescence emission spectra of polyphenolcasein systems in 10 mM TrisHCl buffer pH 7.4 at 25 C for (A) (Ca-casein) (1) free a-casein 100 lM, (212) C at 10, 20,
40, 60, 80, 100, 120, 140, 160, 180, 200 lM; (B) (ECa-casein) (1) free a-casein 100 lM, (212) EC with similar concentrations as catechin in A; (C) (ECGa-casein) (1) free a-
casein 100 lM, (212) ECG with similar concentrations as catechin in A; (D) (EGCGa-casein) (1) free a-casein 100 lM, (212) EGCG with similar concentrations as catechin
in A; (E) (Cb-casein) (1) free b-casein 100 lM, (212) C with similar concentrations as catechin in (A); (F) (ECb-casein) (1) free b-casein 100 lM, (212) EC with similar
concentrations as catechin in A; (G) (ECGb-casein) (1) free b-casein 100 lM, (212) ECG with similar concentrations as catechin in A; (H) (EGCGb-casein) (1) free b-casein
100 lM, (212) EGCG with similar concentrations as catechin in A. The plot of F
0
/(F
0
F) as a function of 1/polyphenol concentration. The binding constant K being the ratio of
the intercept and the slope for polyphenol complexes with both a- and b-caseins (A
0
H
0
).
I. Hasni et al. / Food Chemistry 126 (2011) 630639 637
3.6. Docking studies
Our results from FTIR, UVvisible, CD and uorescence spectro-
scopic methods are accompanied by docking experiments in which
the EC and EGCG molecules were docked to a- and b-caseins to
determine the preferred binding sites on these proteins. The stereo-
viewof the dockings of EC and EGCGare shown in Fig. 5 and Table 1.
The models show that EC is surrounded by Gln-30, Phe-23, Phe-24,
Phe-28, Phe-32 and Val-31, whilst EGCG is in the vicinity of Asp-43,
Gln-30, Glu-63, Glu-77, His-80, Ile-65, Ile-71, Ile-81, Lys-42, Phe-32
and Ser-41 residues in a-casein complexes (Fig. 5 and Table 1). In b-
casein complexes, the EC is surrounded by Ala-177, Gly-203, Leu-
208, Leu-171, Leu-191, Leu-192, Leu-198, Phe-190, Pro-204, Tyr-
193, Val-178 and Val-197, whilst EGCG is in the vicinity of Ala-
177, Gly-203, Ile-208, Leu-191, Leu-192, Leu-198, Phe-190, Pro-
179, Pro-204, Tyr-180, Try-193, Val-178 and Val-197 residues
(Fig. 5 and Table 1). The binding energy (DG) shows more stable
polyphenol complexes formed with b-casein than a-casein, which
is consistent with our spectroscopic data and the binding constants
obtained for the polyphenolcasein complexes.
4. Conclusions
Based on our spectroscopic results and docking studies pre-
sented here, tea polyphenols weakly bind to both a-casein and
b-casein in solution. The order of binding increases as the number
of OH group increased with C EC > EGC > EGCG. b-Casein forms
stronger complexes with tea polyphenols than a-casein, due to
more hydrophobic nature of b-casein. Polyphenolcasein interac-
tion is more hydrophobic than hydrophilic. Polyphenol binding al-
ters casein secondary structure with major decrease in a-helix and
b-sheet accompanied by an increase in random coil and turn struc-
tures, leading to more protein unfolding. The casein structural
changes can be a major factor in the effect of milk on the antioxi-
dant activity of tea polyphenols.
Acknowledgments
The nancial support of the Natural Sciences and Engineering
Research Council of Canada (NSERC) to H.A. Tajmir-Riahi and R.
Carpentier is highly acknowledged.
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