Biosci. Rep. (2010) / 30 / 111–117 (Printed in Great Britain) / doi 10.1042/BSR20090012
Inhibition of brush border sucrase by polyphenols
in mouse intestine
Shiffalli GUPTA*, Safrun MAHMOOD†, Rizwan H. KHAN‡ and Akhtar MAHMOOD*1
*Department of Biochemistry, Panjab University, Chandigarh-160014, India, †Department of Biotechnology and Experimental Medicine,
Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India, and ‡Interdisciplinary Biotechnology Unit,
Aligarh Muslim University, Aligarh, India
'
$
Synopsis
The interactions of gallic acid and tannic acid with purified brush border sucrase (EC 3.2.1.48) from mouse intestine
have been studied. These findings indicate that both gallic acid and tannic acid inhibit sucrase activity, which is pH
dependent. Kinetic analysis revealed that enzyme inhibition by gallic acid is a pure V effect at pH 5.0, which changes
to mixed type at pH 7.2, and pure K effect at pH 8.5. In contrast, sucrase inhibition by tannic acid was a pure K effect
at acidic pH and uncompetitive type in the alkaline pH range. Far-CD spectroscopic analysis revealed an increase in
the helicity of the enzyme at acidic pH in the presence of tannic acid but no change at alkaline pH. Fluorescence
spectra revealed a red shift in λmax of the enzyme, suggesting that tryptophan residues come to a more hydrophilic
environment in the presence of polyphenols. These findings suggest that inhibition of mice sucrase by polyphenols
is pH dependent, and is associated with conformational modifications of the enzyme.
Key words: brush border sucrase, CD, fluorescence spectroscopy, gallic acid, tannic acid
&
INTRODUCTION
Polyphenols are a large family of natural compounds, widely
distributed in plant foods. They are mostly derivatives and/or
isomers of flavones, isoflavones, flavonols, catechins and phenolic acids, and possess diverse pharmacological properties such
as antioxidant, antiapoptosis, antiaging and anticarcinogenic [1].
They exhibit anti-inflammation, antiatherosclerosis, cardiovascular protection, improvement of the endothelial function, as well
as inhibition of angiogenesis and cell proliferation activity [2].
Phenolic monomers are potent inhibitors of plant [3] and mammalian [4–6] enzymes. Recently, we reported that plant polyphenols, gallic acid and tannic acid are potent competitive inhibitors
of brush border sucrase in rat and rabbit intestine and this inhibition is reversible [7,8]. Gallic acid is 3,4,5-trihydroxybenzoic acid
and is structurally similar to tris(hydroxymethyl) amino methane,
a well-known competitive inhibitor of brush border disaccharidases, at alkaline pH [9]. Gallic acid is richly present in the leaves
of berberry and in the roots and bark of pomegranates and gall
nuts [10]. Tea is an important source of gallic acid and contains
up to 4.5 g/kg of fresh weight [11]. Tannic acid is a penta-mdigalloyl-glucose in which each of the five hydroxy groups of the
sugar residue are esterified with a molecule of digallic acid [12].
Brush border sucrase is a heterodimeric molecule comprising
two subunits of unequal size and is membrane bound, located
%
in the vicinity of glucose transporter in intestine, since glucose
produced by the hydrolysis of sucrose has a kinetic advantage for
transport over the free glucose present in the medium [13,14].
Inhibition of brush border hydrolytic enzymes may be one of
the mechanisms by which polyphenols exert their effects and
thus play a role in the aetiology of gastrointestinal dysfunction. Whether pH-dependent enzyme inhibition by polyphenols
is associated with conformational modifications in the sucrase–
isomaltase complex is not known. Using CD and fluorescence
spectroscopic analysis, the interactions of gallic acid and tannic
acid with purified brush border sucrase from mouse intestine have
been investigated in the present study. These findings suggest that
similar to rat enzyme, sucrase activity in mouse intestine is also
inhibited by the polyphenols and is associated with conformational modifications in the enzyme protein.
MATERIALS AND METHODS
Chemicals and buffers
All the chemicals used were of analytical grade. BSA, 4aminoantipyrine, glucose oxidase (Type V), glucose peroxidase,
D-glucose and Tris were purchased from Sigma Chemical (St.
Louis, MO, U.S.A.). Buffers of various pH values (4.8, 5.0, 6.0,
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1 To
whom any correspondence should be addressed (email [email protected]).
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S. Gupta and others
Figure 1
Effect of 0.2 mM gallic acid (continuous line, 䉱) and 0.02 mM tannic acid (broken line, 䊏) on sucrase activity
in mouse intestinal brush border sucrase
Values are means (n = 4). Buffers used were as described in [15]. The skeletal formulae of gallic acid and tannic acid are
shown.
6.5, 7.0, 7.2, 7.7, 8.1 and 8.5) were prepared by mixing boric
acid, maleic acid, orthophosphoric acid and lithium hydroxide as
described earlier [15].
Protein estimation
Protein was estimated by the method of Lowry et al. [16] using
BSA as the standard.
Assay of sucrase activity
Ethical clearance
The experimental protocol was approved by the ethical committee
of the Institute on the use of laboratory animals. Experiments on
animals were performed in accordance with guidelines for the
use of laboratory animals, approved by the Indian Council of
Medical Research (New Delhi, India).
Sucrase activity was assayed by following a two-step procedure
as described by Dahlqvist [17]. After stopping the reaction with
0.5 M Tris/HCl (pH 7.2), glucose liberated by sucrose hydrolysis
was determined by the glucose–oxidase and peroxidase system
using 4-aminoantipyrene as the chromogen. The absorbance was
read at 500 nm.
Enzyme kinetic studies
Purification of brush border sucrase
Balb/c mice (8–10 weeks old) kept on standard pellet diet
(Hindustan Lever) with free access to water were used. The
entire intestine from the ligament of treitz to the caecum was
removed and used for the purification of sucrose. Brush border
sucrase was purified by following the method described earlier
[16]. Briefly, sucrase activity was solubilized by treating the microvillus membranes with papain (2 mg/10 mg of protein). The
soluble enzyme preparation was subjected to Sephadex G200
and DEAE-cellulose column chromatography. Fractions from
the DEAE-cellulose column corresponding to sucrase activity
were pooled and concentrated and the final enzyme preparation
(2.5 units/mg of protein) showed 155-fold enzyme enrichment
compared with crude membrane preparation. On SDS/PAGE, the
enzyme preparation yielded a single protein band with a molecular mass of approx. 210 kDa.
Kinetic parameters of sucrase were determined by assaying the
enzyme activity at different sucrose concentrations (8–40 mM)
in the absence and presence of 0.2 mM gallic acid or 0.02 mM
tannic acid. These concentrations of polyphenols yielded approx.
50% enzyme inhibition as described earlier [7,8]. The values of
the kinetic parameters K m and V max were calculated using a Woolf
plot as described earlier [15].
UV-CD spectroscopic studies
CD measurements were carried out with a Jasco spectropolarimeter (model J-720) equipped with a microcomputer. The instrument was calibrated with D-10-camphorsulfonic acid. All the CD
measurements were carried out at 25◦ C with a thermostatically
controlled cell holder attached to a Neslab RTE-110 water bath,
◦
with an accuracy of +
−0.1 C. Far-UV-CD spectra were measured
at a protein concentration of 2.0 μM. The path length was 1 mm.
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Polyphenol interactions with brush border sucrase
Figure 2
Far-UV-CD spectra of brush border sucrase at different pH values in the absence (continuous line) and presence
of 0.2 mM gallic acid (dotted line) or 0.02 mM tannic acid (dashed line)
(A) pH 5, (B) pH 7.2 and (C) pH 8.5. The path length used was 1 mm at a protein concentration of 2 μM.
Near-UV-CD spectra were measured between 250 and 300 nm at
a protein concentration of 6.0 μM.
RESULTS AND DISCUSSION
Fluorescence spectroscopic studies
The effect of gallic acid (0.2 mM) and tannic acid (0.02 mM)
on murine brush border sucrase as a function of pH is shown in
Figure 1. Both gallic acid and tannic acid inhibited the enzyme
activity between pH 4.8 and 8.6; however, the degree of enzyme
inhibition varied considerably. At pH 4.8, the polyphenols inhibited sucrase activity by 85–96%, which was reduced to 51 and
64% respectively at pH 7.2. However, at pH 8.5, 60 and 76% inhibition of enzyme activity was observed. The observed enzyme
inhibition was similar to that described earlier for sucrase in rat
and rabbit intestines [7,8]. The results presented indicate that
gallic acid and tannic acid are potent inhibitors of brush border
sucrase activity in mouse intestine as well. This could be of significance due to the high degree of enzyme inhibition under these
conditions.
Kinetic analysis revealed that sucrase inhibition by gallic acid
at pH 5.0 was primarily a capacity effect, where enzyme
V max (2.65 units/mg of protein in controls) was reduced to
Fluorescence measurements were performed on a Shimadzu
spectrofluorimeter (model RF-540) equipped with a data recorder
◦
DR-3. The fluorescence spectra were measured at 25 +
− 0.1 C
with a 1 cm path length cell. The excitation and emission slits
were set at 5 and 10 nm respectively. Intrinsic fluorescence was
measured by exciting the protein solution at 295 nm and emission
spectra were recorded in the range of 300–400 nm in the presence
of tannins.
Statistical analysis
Statistical analysis of the data was done using GraphPad software.
Kinetic analysis results are expressed as means +
− S.D. The kinetic parameters were calculated using a Woolf plot [15]. Significance levels P < 0.001, P < 0.01 and P < 0.05 were calculated for
n = 4.
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S. Gupta and others
Figure 3
Near-UV-CD spectra of brush border sucrase at different pH values in the absence (continuous line) and
presence of 0.2 mM gallic acid (dashed line) or gallic acid alone (dotted line)
The path length used was 1 cm at a protein concentration of 6 μM. (A) pH 5, (B) pH 7.2 and (C) pH 8.5.
Table 1 Kinetic parameters of brush border sucrase in the
absence and presence of gallic acid and tannic acid
Values are mean +
− S.D., n = 3. *P < 0.001, **P < 0.01.
K m (mM)
V max (units/mg
of protein)
130 +
− 15.8
137.5 +
− 12.5
1070 +
− 13*
2.65 +
− 0.19
0.709 +
− 0.08*
3.09 +
− 0.42
pH 5.0
Metal-ion-free buffer
Gallic acid (0.2 mM)
Tannic acid (0.02 mM)
pH 7.2
138 +
− 8.16
245 +
− 21.1**
295 +
− 25*
4.65 +
− 0.42
2.08 +
− 0.09*
1.96 +
− 0.22*
96.7 +
− 1.32
200 +
− 12*
200 +
− 5*
1.32 +
− 0.11
1.41 +
− 0.098
0.57 +
− 0.015*
Metal-ion-free buffer
Gallic acid (0.2 mM)
Tannic acid (0.02 mM)
pH 8.5
Metal-ion-free buffer
Gallic acid (0.2 mM)
Tannic acid (0.02 mM)
0.71 units/mg of protein in the presence of 0.2 mM gallic acid
with no effect on K m of the enzyme (130 mM) under these conditions (Table 1). In contrast, sucrase inhibition by tannic acid was
a pure K effect, where the K m was enhanced to 1070 mM from
130 mM in the presence of 0.02 mM tannic acid without affecting enzyme V max (2.65 units/mg of protein). However, at pH 7.2,
both gallic acid and tannic acid showed mixed type enzyme inhibition, K m was enhanced by 77–114% and V max by 55–60%. At
pH 8.5, gallic acid showed a K type of inhibition (K m increased
by 107%) but enzyme inhibition by tannic acid was of mixed
type; K m was increased by 107% and V max was reduced by 57%.
This may indicate more than one polyphenol-binding site in the
protein molecule with different affinities.
In contrast with Tris, which is a well-known inhibitor of disaccharidases at alkaline pH [9], sucrase inhibition by gallic acid
and tannic acid was optimum at pH 5 and 5.5. This is similar to
that reported for rat sucrase [7].
Both gallic acid and Tris are polyhydroxyl compounds. Vasseur et al. [9] proposed that the inhibitory activity of Tris
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Polyphenol interactions with brush border sucrase
Figure 4
Fluorescence emission spectra of brush border sucrase at different pH values in the absence (䉬) and presence
of 0.2 mM gallic acid (䊐) or 0.02 mM tannic acid ()
(A) pH 5, (B) pH 7.2 and (C) pH 8.5. Intrinsic fluorescence was measured by exciting the protein solution at 295 nm and
emission spectra were recorded in the range of 300–400 nm.
towards glycosidases is a consequence of the combined effect
of the amino group and polyhydroxy constellation of the molecule due to the presence of a deprotonated amino group next
to the free hydroxy groups. This kind of assertion, however, cannot explain the strong affinity of gallic acid/tannic acid towards
sucrase, which lacks the amino group but has an ionized carboxy
group at pH 4.4 and above.
Vasseur et al. [18] suggested that sucrase undergoes ionization
with a change in pH. The catalytically active enzyme contains
two key groups: one is deprotonated (pK 1 ≈ 5.2) and the other
protonated (pK 2 ≈ 8.4). The effects of gallic acid and tannic acid
on brush border sucrase may be related to the ionization of these
groups in the enzyme molecule. The ionization of the enzyme
at pH 5.2 may make it more sensitive to inhibition, while the
second ionization at pH 8.4 may interfere with the inhibition
process. To investigate whether observed enzyme inhibition by
polyphenols is associated with conformational changes in the
enzyme molecule, the UV-CD and fluorescence spectral studies
were also carried out.
Figure 2 shows the far-UV-CD spectra of native, tannic acid
and gallic acid at pH 5.0, 7.2 and 8.5 respectively. The enzyme
showed two resolved negative peaks at approx. 220–222 nm and
at 208 nm with greater in magnitude signal at 208 nm (Figure 2),
suggesting that the enzyme belongs to the α+β class of proteins
[19] and in the absence of polyphenols is composed of α-helixand β-sheet-rich regions in the molecular structure of the protein
molecule, indicating a high level of structural integrity. We observed almost overlapping spectra at pH 5.0 and 7.2, suggesting
that the secondary structure of the enzyme is unaltered in the
presence of gallic acid, while its helicity changed in the presence
of tannic acid. Similarly, at pH 7.2, the ellipticity at 208 nm was
low, which was enhanced around 218 nm in the presence of the
polyphenol. This may be attributed to the interaction of the indole
chromophore of tryptophan with the backbone chromophore. At
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S. Gupta and others
pH 8.5, the CD spectra were altered in the presence of gallic acid,
where the ellipticity around 208–212 and 220 nm was enhanced
compared with the control. In contrast, there was no change in
the far-UV-CD spectra in the presence of tannic acid at pH 8.5,
which suggests that tannic acid interaction with the enzyme does
not modify the enzyme secondary structure under the alkaline
conditions.
Near-UV-CD spectra provide evidence of tertiary structure
and dynamics. The shape of the spectra depends critically on the
atomic environment and closeness of packing of the aromatic
residues, including their accessibility to the solvent. The side
chain of aromatic residues interacts with the side chain amide,
carboxylate groups and peptide bonds of the main chain [20].
Figure 3 shows near-UV-CD spectra of brush border sucrase in
the absence and presence of gallic acid at pH 5.0, 7.2 and 8.5.
A negative peak centred between 268 and 297 nm was noticed.
At the latter wavelength, tyrosine and phenylalanine residues do
not contribute to the CD spectra of native protein [21,22] and
the negative band probably originates from tryptophan residues
located in a symmetrical environment [23]. The presence of
gallic acid resulted in a shift in the position of the transition from
the native to unordered state with increasing alkalinity (pH 7.2
and 8.5), suggesting loss of the fine structure. However, the nearUV-CD spectrum at pH 7.2 and 8.5 in the absence as well as in the
presence of gallic acid is featureless, indicating that the aromatic
side chains are disordered as expected, when the protein unfolds.
The CD signal in the near-UV region at pH 8.5 is lower than
that measured at pH 7.2, suggesting that the increasing alkalinity
induces a looser and more flexible environment near the aromatic
residues [22]. Rawel et al. [24] have reported similar modulation
in CD spectra of soy glycinin in the presence of plant flavanols. It
has been suggested that non-covalent binding of the polyphenol
does not affect the secondary structure but causes a significant alteration in tertiary structure of the protein, similar to that observed
with brush border sucrase in the present study. Thus polyphenol
interaction with enzyme proteins in general may affect tertiary
structure, but the precise site(s) of interaction remains unknown.
At 295 nm excitation wavelength, only tryptophan residues
are attributed to the intrinsic fluorescence of a protein [25,26].
As shown in Figure 4, fluorescence spectra revealed that when
enzyme protein was excited at 295 nm, the tryptophan emission maximum of the enzyme showed an emission maximum
at 338 nm in the native state and had an obvious red shift after
the protein had interacted with polyphenols that became more
obvious in the presence of tannic acid. Spectra of the unfolded
enzyme in the presence of gallic acid remained almost similar
in shape, while emission maximum red-shifted approx. 5–20 nm
after binding with tannic acid, with a decrease in intensity of
approx. 20% at acidic pH (pH 5), 40% at neutral pH (pH 7.2) and
25% at pH 8.5. The red-shifted spectra indicate that the tryptophan residue in the protein has been brought to a more hydrophilic
environment in the presence of the polyphenols. These findings
suggest that polyphenol inhibition of brush border sucrase in
mouse intestine is pH dependent and is associated with conformational changes in the enzyme molecule. Both CD spectra and
fluorescence analysis revealed alterations in the tertiary structure
of the enzyme in the presence of gallic acid and tannic acid,
suggesting the exposure of tryptophan residues under these conditions. In conclusion, the results presented indicate that both
gallic acid and tannic acid are potent inhibitors of brush border
sucrase in mouse intestine. The inhibition is pH dependent and
the enzyme undergoes conformational modifications on binding
to polyphenols. Because of the presence of these phenols in dietary plant foods, they may interfere in digestive functions of the
gut, in certain diseased conditions such as diabetes and obesity.
FUNDING
S. G. is the recipient of a senior research fellowship from the
University Grants Commission (New Delhi, India).
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Received 20 January 2009/1 April 2009; accepted 9 April 2009
Published as Immediate Publication 9 April 2009, doi 10.1042/BSR20090012
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