Hydrophilic ionic liquids as reaction media for the determination of guaiacol using horseradish and soybean peroxidases

Article abstract of DOI:10.1016/j.mencom.2011.03.013

The advantages of hydrophilic ionic liquids - 1-butyl-2-methylimidazolium and N-butyl-3-methylpyridinium tetrafluoroborates - over conventional polar solvents (DMSO and acetonitrile) for the enzymatic determination of 0.05-3 mm guaiacol in the reaction media containing 20-40 vol% H₂O are shown as the result of a comparative study of the kinetics of guaiacol oxidation by Bu^tOOH catalyzed by native horseradish and soybean peroxidases.

Full text of DOI:10.1016/j.mencom.2011.03.013

Mendeleev
Communications
Mendeleev Commun., 2011, 21, 97–98
Hydrophilic ionic liquids as reaction media for the determination
of guaiacol using horseradish and soybean peroxidases
Svetlana V. Muginova,* Anna Z. Galimova, Aleksei E. Polyakov and Tatiana N. Shekhovtsova
Department of Chemistry, M. V. Lomonosov Moscow State University, 119991 Moscow, Russian Federation.
Fax: +7 495 939 4675; e-mail: sveta_muginova@mail.ru
DOI: 10.1016/j.mencom.2011.03.013
The advantages of hydrophilic ionic liquids – 1-butyl-2-methylimidazolium and N-butyl-3-methylpyridinium tetrafluoroborates –
over conventional polar solvents (DMSO and acetonitrile) for the enzymatic determination of 0.05–3 mm guaiacol in the reaction
media containing 20–40 vol% H2O are shown as the result of a comparative study of the kinetics of guaiacol oxidation by ButOOH
catalyzed by native horseradish and soybean peroxidases.
Ionic liquids (ILs) are solvents widely used in chemical analysis.1
In biotechnology, hydrophobic ILs are mainly applied for carrying
out organic synthesis employing lipases.2 The kinetic studies of
reactions catalyzed by native hem-containing proteins (hemo-
globin and myoglobin) and oxidases (peroxidase, laccase and
tyrosinase) in hydrophilic ILs have been reported.2,3 Note that a
significant drawback of molecular polar organic solvents is their
denaturing effect on enzymes,4 especially native peroxidases,5
which are commonly used in chemical analysis. This fact limits
the applicability of biochemical methods to the analysis of aqueous-
organic solutions, organic extracts and samples with low water
concentrations.
Guaiacol is a classic peroxidase substrate, which is poorly
soluble in water but readily soluble in polar organic solvents.
The development of rapid and simple procedures for the deter-
mination of guaiacol in various samples is an important and
promising challenge.
The aim of this work was to study the analytical possibilities
of hydrophilic ILs {1-butyl-2-methylimidazolium ([bmim]) and
N-butyl-3-methylpyridinium [bmpy] tetrafluoroborates}, and polar
organic solvents (DMSO, acetonitrile) as reaction media for the
determination of a model phenolic substrate (guaiacol) using its
oxidation with ButOOH catalyzed by horseradish (HRP) and
soybean (SBP) peroxidases (Scheme 1), and to develop enzymatic
procedures for the determination of guaiacol in organic solutions.
It was reasonable to compare the efficiency of guaiacol trans-
formation in ILs and polar organic solvents under the influence of
commercial cationic HRP and anionic SBP (EC 1.11.1.7).Aceto-
nitrile and DMSO are often applied as solvents for phenolic com-
pounds6 and diluents for their extraction from different samples.7
To solve the above problems, we studied the dependence of
the rate of the indicator reaction of guaiacol oxidation catalyzed
by peroxidases on the volumetric concentration of an organic
solvent in the mixture. The reaction rate was controlled by spectro-
photometry (see Online Supplementary Materials). The optimal
order of the introduction of the components into the indicator
system was IL–ButOOH–buffer solution–guaiacol–enzyme
because the dynamic viscosity of IL is higher than that of DMSO
or acetonitrile.
To oxidize guaiacol in the presence of H25 vol% IL, organic
buffer solutions (Table 1) should be added to the indicator reaction.
The components of a phosphate buffer solution, which provides
the high catalytic activity of peroxidases in aqueous solutions
and mixtures with DMSO (acetonitrile), get salted out in IL. The
same situation was observed in the reaction catalyzed by HRP in
a phosphate buffer solution in the presence of hydrophilic ILs
of a different nature.8 The proper choice of an optimal buffer solu-
tion, which is a co-solvent of IL, allowed us to study the kinetics
of guaiacol oxidation catalyzed by peroxidase in the presence of
H60 vol% of hydrophilic IL.
The indicator reaction catalyzed by HRP did not proceed in
the presence of 60 vol% [bmpy][BF4] in all of the buffer systems,
while the relative activity of SBP was high and decreased in the
following order of buffer solutions: imidazole–HCl H collidine–
HCl > Tris–HCl (Table 1). In imidazole–HCl and collidine–HCl
buffer mixtures, the values of SBP relative activity were com-
parable; however, the reproducibility of the results in the col-
lidine–HCl buffer was better (sr G 0.11, n = 3) as compared to
the imidazole–HCl buffer (sr G 0.26; n = 3). In the solution of
[bmim][BF4]–imidazole–HCl (80:20 vol%), the relative catalytic
activity of SBP was 2.5 times higher than that in [bmim][BF4]–
Table 1 Relative catalytic activity (a/a0)a of HRP and SBP in the guaiacol
oxidation with ButOOH depending on the nature of a buffer solution –
co-solvent of IL (concentrations: SBP, 8.0 nmol dm–3; HRP, 9.0 nmol dm–3
;
ButOOH, 120 mmol dm–3; guaiacol, 1 mmol dm–3; 0.05 m buffer solutions,
OH
MeO
O
pH 7.0).
OMe
HRP or SBP
+
ButOOH
a/a0 (%)
O
System (vol%)
Enzyme
Imidazole– 2,4,6-Collidine– Tris–
OMe
HCl
HCl
HCl
Reaction solution:
hydrophilic IL ( 60 vol%) + aqueous buffer solution
[bmpy][BF4]–buffer HRP
solution (60:40) SBP
—
39
—
34
—
12
BF4
BF4
or
N
N
N
[bmim][BF4]–buffer HRP
solution (80:20) SBP
6
23
8
9
3
—
[bmim][BF4]
[bmpy][BF4]
aa/a0 is the ratio between peroxidase catalytic activities in the presence and
absence of organic solvent, respectively.
Scheme 1
© 2011 Mendeleev Communications. All rights reserved.
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Mendeleev Commun., 2011, 21, 97–98
2,4,6-collidine–HCl and higher than the relative catalytic activity
of HRP in the collidine–HCl buffer by a factor of 4. In the indicator
reaction in the presence of [bmpy][BF4] and [bmim][BF4], the
components of optimal buffer solutions (collidine–HCl and imid-
azole–HCl, respectively) contained the structural elements of IL.
In the absence of IL, the catalytic activity of the plant peroxidases
increased in the following order of buffer solutions: Tris–HCl <
collidine–HCl < imidazole–HCl according to the decrease of
Table 2 Optimal conditions for the guaiacol oxidation with ButOOH
catalyzed by HRP and SBP in water–organic media.
Concentration
ButOOH/
System (vol%)
Enzyme/
Guaiacol/
nmol dm–3 mmol dm–3 mmol dm–3
[bmpy][BF4]–0.05 m 2,4,6-col-
lidine–HCl, pH 7.0 (60:40)
60 (SBP) 145
1
3
9
pKa of the major components of these buffers [Tris (8.08) >
[bmim][BF4]–0.05 m imidazole– 45 (SBP) 170
2,4,6-collidine (7.43) > imidazole (6.95)].
HCl, pH 7.0 (80:20)
Regardless of IL, SBP was found to have the greatest catalytic
activity (Table 1) and substrate specificity towards guaiacol char-
acterized by the effective rate constant keff, which was calculated
according to the ‘ping-pong’ mechanism.10 The values of keff in
the presence of 80 vol% [bmim][BF4] and 60 vol% [bmpy][BF4]
were 2.6×103 and 2.2×103 dm3 mol–1 s–1, respectively. The effi-
ciency of guaiacol transformation in aqueous DMSO and aceto-
nitrile (their concentrations were 20 and 25 vol%, respectively),
was higher in the case of SBP by factors of 2 and 1.5, respectively,
as compared to HRP. In our opinion, the high catalytic activity
of SBP at pH 2–11 (against HRP, which is active at pH 4–8)11
along with its high conformational flexibility and thermal stability
in aqueous solutions12,13 are responsible for more efficient catalysis
with SBP in the presence of IL along with DMSO and acetonitrile,
as compared to HRP.
DMSO–0.1 m phosphate buffer, 180 (HRP) 290
15
9
pH 6.0 (20:80)
MeCN–0.1 m phosphate buffer, 180 (HRP) 145
pH 6.0 (25:75) 180 (SBP) 220
180 (SBP) 220
9
9
(Omega, Russia) which contained guaiacol, phenol, formaldehyde
and dexamethasone. The guaiacol concentration in 100 g of the
sample found by the addition method was (31 2) g (certified value,
30 g; n = 5, P = 0.95).
The results demonstrate the applicability of [bmim][BF4] and
[bmpy][BF4] to the determination of guaiacol using SBP. In our
opinion, the application of hydrophilic IL expands the possibi-
lities of enzymatic methods for the analysis of samples sparingly
soluble and insoluble in water.
In the presence of H30 vol% DMSO and acetonitrile in the
indicator system, the enzymatic reaction did not proceed. At the
same time, in the presence of IL and optimum buffer solutions,
the relative catalytic activity of SBP remained at a level of about
20–30%. Thus, the use of hydrophilic IL instead of DMSO and
acetonitrile provided efficient peroxidase catalysis in the pres-
ence of the polar solvents. The polarity was characterized by
the logarithm of octanol–water partition coefficient logP {logP
increased in the following order: acetonitrile (–0.33)4 < DMSO
(–1.30)4 < [bmim][BF4] (–2.44)14 < [bmpy][BF4] (–2.64)15}. Note
that the thermodynamic parameters (Johns–Doul B-coefficients
of viscosity and the structural volumes) characterizing the ability
of cosmotropic cations [bmim]+ and [bmpy]+ towards hydration
did not differ essentially from each other.16
Under the optimal conditions (concentrations of IL, organic
solvents and buffer solutions), the rate of the indicator reaction
(2–3 units of tga×102) could be precisely determined spectro-
photometrically; the residual catalytic activity of peroxidases was
at least 10% of its value in water. The optimal concentrations of
the enzymes, ButOOH, and guaiacol (Table 2) were ascertained
as a result of the consecutive study of the concentration depend-
ence of the reaction rates.
This work was supported by the Russian Foundation for
Basic Research (grant no. 09-03-00823-a) and the RF Ministry
of Education and Science (contract no. P991, 2010).
Online Supplementary Materials
Supplementary data associated with this article can be found
in the online version at doi:10.1016/j.mencom.2011.03.013.
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in the presence of [bmpy][BF4] and [bmim][BF4], respectively
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to dry IL once a day before carrying out the experiments.
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Received: 21st September 2010; Com. 10/3594
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