Research Journal of Recent Sciences ________________________________________________ ISSN 2277 - 2502
Vol. 1(ISC-2011), 345-347 (2012)
Res.J.Recent.Sci.

Short Communication

A Calorimetric Study on the Interaction between Vitamin-B6 and lysozyme
Barzegar L.1, Rezaei Behbehani G.1, Moosavi M.2 and Mehreshtiagh M.3
1

Chemistry Department, Azad Takestan University, Qazvin, IRAN
2
Chemistry Department, Payam Noor University, Abhar, IRAN
3
Chemistry Department, Imam Khomeini International University, Qazvin, IRAN

Available online at: www.isca.in
(Received 30th September 2011, revised 20th January 2012 , accepted 21th February 2012)

Abstract
The binding reaction between vitamin B6 (B6, pyridoxine) and lysozyme (Lys) was investigated for the first time by isothermal
titration calorimetry (ITC), at pH 7 at 27°C in tris buffer (25mmol.L-1). The enthalpies of LYS+B6 interaction are reported and
analysed in term of the extended solvation model. The thermodynamic parameters, enthalpy changes (∆H) and entropy changes
(∆S) were calculated. These data suggested that hydrophobic interaction was the predominant intermolecular forces stabilizing the
complex, which was in good agreement with the results of molecular modeling study. It was found that LYS has one noncooperative binding site for Vitamin B6.
Keywords: Vitamin B6, lysozyme, isothermal titration calorimetry.

Introduction
Lysozyme is abundant in a number of secretions, such as tears,
saliva, human milk and mucus. It is also present in cytoplasmic
granules of the polymorphonuclear neutrophils (PMN). Large
amounts of lysozyme can be found in egg white. In humans, the
lysozyme enzyme is encoded by the LYS gene. LYS is a small
globular protein, consisting of 129 amino acid residues with
four disulfide bonds. The importance of Lys relies on its
extensive use as a model system to understand the underlying
principles of protein structure, function, dynamics and folding
through theoretical and experimental studies1,2. High natural
abundance is also one of the reasons for choosing LYS as a
model protein for studying protein-ligand interaction. Another
important aspect of LYS is its ability to carry drug or biological
activity substances, and the effectiveness of them depends on
their binding ability3. Therefore, studies on the interaction
between LYS and drugs or biological activity substances are of
importance in view of realizing disposition, transportation and
metabolism of drugs or biological activity substances as well as
efficacy process. Lys contains six tryptophan (Trp) and three
tyrosine (Tyr) residues. Three of Trp residues are located at the
substrate binding sites, two in the hydrophobic matrix box,
while one is separated from the others 4. Vitamin B6
(pyridoxine) is one of the compounds that can be called vitamin
B6, along with pyridoxal and pyridoxamine. It differs from
pyridoxamine by the substituent at the '4' position. Pyridoxine
assists in the balancing of sodium and potassium as well as
promoting red blood cell production. It is linked to
cardiovascular health by decreasing the formation of
homocysteine. Pyridoxine may help balance hormonal changes
in women and aid the immune system.Lack of pyridoxine may
cause anemia, nerve damage, seizures, skin problems, and sores
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in the mouth. A very good source of pyridoxine is dragon fruit
from South East Asia.
The reports show that Vitamin B6 improved thermal stability
and biological activity of lysozyme. Vitamin B 6 has
hydrophobic cavities that prevent direct interactions on the
hydrophobic surfaces of proteins and in this way suppress
protein aggregation. Hydrophobic interactions are the most
important non-covalent forces that are responsible for different
phenomena such as structure stabilization of proteins binding of
enzymes to substrates and folding of proteins. This kind of
interaction appears when non-polar compounds are put into
water, and it is an entropy-driven process. we tried to elucidate
the effect of Vitamin B6 on lysozyme stability at 27 °C applying
the extended salvation model for the data analysis.

Material and Methods
Chicken egg white LYS (molecular weight (MW) = 14.6 kDa)
was purchased from Sigma,Vitamin B6 (pyridoxine,
MW=169.18 gr/mol) was obtained from Merck.and solutions
were made in (25 mM, pH 7) tris buffer using doubledistilled
water. The isothermal titration calorimetric experiments were
carried out on a VP-ITC ultra sensitive titration calorimeter.
The microcalorimeter consists of a reference cell and a sample
cell of 1.8mL in volume, with both cells insulated by an
adiabatic shield. All solutions were thoroughly degassed before
use by stirring under vacuum. The sample cell was loaded with
lysozyme solution (1 mM) and the reference cell contained
buffer solution. The solution in the cell was stirred at 307 rpm
by the syringe filled with Vitamin B6 solutions (15 mmol.L-1)
to ensure rapid mixing. The titration of MT with Vitamin B 6
involved 25 consecutive injections of the ligand and each
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Research Journal of Recent Sciences ____________________________________________________________ ISSN 2277 - 2502
Vol. 1(ISC-2011), 345-347 (2012)
Res.J.Recent.Sci
injection included 50 μL Vitamin B6. The calorimetric signal
was gauged by a digital voltmeter that was part of a
computerized recording system. The heat of injection was
calculated by the ‘Thermometric Digitam 3’ software program.
The heats of dilution of Vitamin B6 were evaluated except MT
was excluded. The heats of dilution of MT are negligible. The
measurements were performed at constant temperatures of 27
°C and the temperature was controlled using a poly-science
water bath.

Results and Discussion
As we have shown before the heats of the
macromolecules+ligands interactions in the aqueous solvent
systems obtained by the following equation5:
 - δ θ (x  L + x  L ) - (δ θ - δ θ )(x  L + x  L )x 
q = q max x B
B B
B A A A
B B B
A A A

(1)

q is the heats of B6+LYS interaction at certain ligand
concentrations and qmax represents the heat value upon
saturation of all LYS. The good agreement between the
experimental and calculated heats support the extended
solvation model (figure-1).

1200

1000

800

600

q / kJ.mol-1

400

200

0

-200

-400

-600

-800
0

200

400

600

800

1000

1200

1400

1600

1800

2000

p > 1 or p<1 indicate positive or negative cooperativity of
macromolecule for binding with ligand respectively; p = 1
indicates that the binding is non-cooperative. LA and LB can be
calculated from heats of dilution of Vitamin B 6 in water, qdilut,
as follows:
q
L B  qdilut  x A ( dilut )
x B

qdilut
L A  qdilut  x B (
)
(3)
x B
Consider a solution containing ligand L, and a
biomacromolecule (M) that contains "g" sites capable of
binding the ligand. If the multiple binding sites on a
macromolecule are identical and independent, the binding
parameters can be reproduced by the following equation6,7:
q
q
1 Kd
(4)
M 0  ( )L0 
q max
q
q
g
Where q  q max  q and q represents the heat value at a
certain ligand and biomolecule concentration. qmax represents
the heat value upon saturation of all biomacromolecule. K d is
the dissociation equilibrium constant for the equilibrium:
[ M ][L ]
(5)
M  L  ML K d 
[ ML ]
Table-1
Binding parameters for Vitamin B6+LYS
interaction
recovered from Eqs. 1, 4, 5 and 6. p=1 indicates that the
binding is non-cooperative in one binding site. The positive
values of δθA and δθB show that Vitamin B6 stabilizes the
LYS structure. The binding process for LYS is entropydriven.
p
1±0.02
g
1.06±0.03
Ka / M-1
201180.4±425
∆H / kJ.mol-1
1.16±0.05
∆G / kJ.mol-1
-30.45±0.13
∆S / kJ.mol-1.K-1
0.10±0.01

 Aθ
 B

[L] / mol.L-1

Figure-1
Comparison between the experimental heats, q, for the
interaction between Lysozyme and Vitamin B6 at 27 °C and
calculated data (line) and calculated data (○)via Eq. 1.
θ

θ

The parameters δ A and δ B exhibit the LYS stability in the low
and high B6 concentrations respectively. The positive values of
δθA and δθB show that Lsozyme is substantially stabilized by
vitamin B6 at 27 °C. x'B, x'A can be expressed as follows:
px B
'
'
'
xB =
x A = 1 - x B (2)
x A + px B

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0.03±0.002
0.09±0.003

If q and qmax are calculated per mole of biomacromolecule then
the molar enthalpy of binding for each binding site ( H ) will
be H  q max . The changes in the standard Gibbs free energy,
g

∆G, and in standard entropy of binding, ∆S, could be calculated
by using association equilibrium constant, K  1 , and ∆H
a
Kd
value in equations 6 and 7, respectively.
∆G=-RTLnKa
(6)

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Research Journal of Recent Sciences ____________________________________________________________ ISSN 2277 - 2502
Vol. 1(ISC-2011), 345-347 (2012)
Res.J.Recent.Sci

S 

H  G

2.

Ghosh A., Brinda K.V. and Vishveshwara S., Dynamics of
Lysozyme Structure Network: Probing the Process of
Unfolding, J.Biophys., 92(7), 2523-2535 (2007)

3.

Zhang Z., Zheng Q., Han J., Gao J., Liu J., Gong T., Gu
Z., Huang Y., Sun X. and He Q., The targeting of 14succinate triptolide-lysozyme conjugate to proximal renal
tubular epithelial cells, J.Biomaterial 30, 1372-1381
(2009)

4.

Croguennec T., Nau F., Molle D., Graet Y L. and Brule
G., Iron and citrate interactions with hen egg white
lysozyme, J. Food Chem.,68(1), 29-35 (2000)

5.

Rezaei-Behbehani G., Taherkhani A., Barzegar L.,
Saboury A A. and Divsalar A., Refolding of Lysozyme
Upon Interaction with β-Cyclodextrin, Journal of Sciences
Islamic Republic of Iran., 22(2), 117-120 (2011)

6.

Saboury A A., A Review on the Ligand Binding Studies
by Isothermal Titration Calorimetry, Journal of the
Iranian Chemical Society, 3(1), 1-21 (2006)

7.

Saboury A A., Application of a new method for data
analysis of isothermal titration calorimetry in the
interaction between human serum albumin and Ni2 , J.
Chem. Thermodynamics, 35(12),1975–1981 (2003)

(7)

T
All calculated thermodynamic parameters are summarised in

Conclusion
p=1 indicates that the binding is non-cooperative in one binding
sites. The positive values of δθA and δθB show that Vitamin B6
stabilizes the LYS structure. The binding process is
spontaneous which is only entropy driven, indicating that
hydrophobic interaction is dominant in the binding.

Acknowledgements
Financial support from Islamic Azad University of Takestan
is gratefully acknowledged.

Reference
1.

Buck M., Schwalbe H. and Dobson C.M., Characterization
of Conformational Preferences in a Partly Folded Protein
by Heteronuclear NMR Spectroscopy Assignment and
Secondary Structure Analysis of Hen Egg-White
Lysozyme in Trifluoroethanol, J.Biochemistry., 34(40),
13219-13232 (1995)

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