Cationic reverse micelles create water with super hydrogen-bond-donor capacity for enzymatic catalysis: Hydrolysis of 2-naphthyl acetate by α-Chymotrypsin

Article abstract of DOI:10.1002/chem.201000437

Reverse micelles (RMs) are very good nanoreactors because they can create a unique microenvironment for carrying out a variety of chemical and biochemical reactions. The aim of the present work is to determine the influence of different RM interfaces on the hydrolysis of 2-naphthyl acetate (2NA) by α-chymotrypsin (α-CT). The reaction was studied in water/benzyl-nhexadecyldimethylammonium chloride (BHDC)/benzene RMs and, its efficiency compared with that observed in pure water and in sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT) RMs. Thus, the hydrolysis rates of 2-NA catalyzed by α-CT were determined by spectroscopic measurements. In addition, the method used allows the joint evaluation of the substrate partition constant K_p between the organic and the micellar pseudophase and the kinetic parameters: catalytic rate constant k_cat, and the Michaelis constant KM of the enzymatic reaction. The effect of the surfactant concentration on the kinetics parameters was determined at constant W ₀= [H₂O]/[surfactant], and the variation of W₀ with surfactant constant concentration was investigated. The results show that the classical Michaelis-Menten mechanism is valid for α-CT in all of the RMs systerns studied and that the reaction takes place at both RM interfaces. Moreover, the catalytic efficiency values k_cat/K_M obtained in the RMs systems are higher than that reported in water. Furthermore, there is a remarkable increase in α-CT efficiency in the cationic RMs in comparison with the anionic system, presumably due to the unique water properties found in these confined media. The results show that in cationic RMs the hydrogen-bond donor capacity of water is enhanced due to its interaction with the cationic interface. Hence, entrapped water can be converted into "super-water" for the enzymatic reaction studied in this work.

Full text of DOI:10.1002/chem.201000437

FULL PAPER
DOI: 10.1002/chem.201000437
Cationic Reverse Micelles Create Water with Super Hydrogen-Bond-Donor
Capacity for Enzymatic Catalysis: Hydrolysis of 2-Naphthyl Acetate by a-
Chymotrypsin
[
a]
[a]
[b]
[a]
Fernando Moyano, R. Dario Falcone, J. C. Mejuto, Juana J. Silber, and
[
a]
N. Mariano Correa*
Abstract: Reverse micelles (RMs) are
very good nanoreactors because they
can create a unique microenvironment
for carrying out a variety of chemical
and biochemical reactions. The aim of
the present work is to determine the
influence of different RM interfaces on
the hydrolysis of 2-naphthyl acetate (2-
NA) by a-chymotrypsin (a-CT). The
reaction was studied in water/benzyl-n-
hexadecyldimethylammonium chloride
evaluation of the substrate partition
constant Kp between the organic and
the micellar pseudophase and the ki-
netic parameters: catalytic rate con-
tems studied and that the reaction
takes place at both RM interfaces.
Moreover, the catalytic efficiency
values k /K obtained in the RMs sys-
cat
M
stant k , and the Michaelis constant
tems are higher than that reported in
water. Furthermore, there is a remark-
able increase in a-CT efficiency in the
cationic RMs in comparison with the
anionic system, presumably due to the
unique water properties found in these
confined media. The results show that
in cationic RMs the hydrogen-bond
donor capacity of water is enhanced
due to its interaction with the cationic
interface. Hence, entrapped water can
be converted into “super-water” for
the enzymatic reaction studied in this
work.
cat
KM of the enzymatic reaction. The
effect of the surfactant concentration
on the kinetics parameters was deter-
mined at constant W =[H O]/[surfac-
0
2
tant], and the variation of W with sur-
0
factant constant concentration was in-
vestigated. The results show that the
classical Michaelis–Menten mechanism
is valid for a-CT in all of the RMs sys-
(
BHDC)/benzene RMs and, its effi-
ciency compared with that observed in
pure water and in sodium 1,4-bis-2-eth-
ylhexylsulfosuccinate (AOT) RMs.
Thus, the hydrolysis rates of 2-NA cat-
alyzed by a-CT were determined by
spectroscopic measurements. In addi-
tion, the method used allows the joint
Keywords: enzyme catalysis · hy-
drolysis · interfaces · micelles · sur-
factants
Introduction
to enhance some effects, such as hydrogen-bonding interac-
tions between peptide bonds, because in these media the
amphipathic essence of a biological membrane is pre-
served. Among the RMs formed by anionic surfactants,
the best known are those derived from sodium 1,4-bis-2-eth-
ylhexylsulfosuccinate (AOT) in different nonpolar media.
AOT has a well-known V-shaped molecular geometry and
forms stable RMs without co-surfactant. The cationic surfac-
Several biological phenomena occur at interfaces rather
than in homogeneous solution. In particular, interface/pro-
tein interactions play a key role in reactions involving mem-
brane-bound proteins. In this regard, even though reverse
micelles (RMs) are an oversimplified model, the very large
interfacial region provided by these systems can be expected
[1]
tant
benzyl-n-hexadecyldimethylammonium
chloride
(
BHDC) also forms RMs in benzene without addition of a
co-surfactant and has properties that are characteristic of
other RMs systems. They can encapsulate water in their
[
a] Dr. F. Moyano, Dr. R. D. Falcone, Prof. J. J. Silber, Dr. N. M. Correa
Departamento de Quꢀmica. Universidad Nacional de Rꢀo Cuarto
Agencia Postal # 3. C.P. X5804BYA Rꢀo Cuarto (Argentina)
E-mail: mcorrea@exa.unrc.edu.ar
polar interior, water is solubilized up to W =[water]/-
0
AHCTUNGTRENNUNG
[2–4]
0
[
b] Dr. J. C. Mejuto
of the water become similar to those of bulk water only
when the amount of water exceeds that required for surfac-
Departamento de Quꢀmica Fꢀsica, Facultad de Ciencias
Universidad de Vigo, 32004 Ourense (Spain)
Chem. Eur. J. 2010, 16, 8887 – 8893
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
8887

[2,3]
[5]
tant solvation.
Recently, we have shown that the water
Experimental Section
properties are different for water molecules sequestrated
inside anionic and cationic RM systems. This is because the
water molecules entrapped inside BHDC RMs media
appear to be non-electron-donating and more hydrogen-
bond-donating owing to their interaction with the polar
head group of the cationic surfactant. On the other hand,
water molecules sequestrated inside AOT RMs show en-
hanced electron-donor ability in comparison with bulk
water. Thus, we suggest that RMs are highly suitable for use
as nanoreactors, since the properties of water can depend
significantly on the kind of surfactant from which they are
prepared.
Materials: Benzyl-n-hexadecyldimethylammonium chloride (BHDC)
from Sigma (>99%) was recrystallized twice from ethyl acetate. The sur-
factant was kept under vacuum over P O to minimize H O absorption.
2 5 2
The absence of acidic impurities was carefully checked by using 1-
methyl-8-oxyquinolinium betaine (QB) as indicator, because such impuri-
[
2,30]
ties can significantly affect the pH of the dispersed aqueous phase.
a-Chymotrypsin (a-CT), Mw 24800, from bovine pancreas (Sigma) and 2-
naphthyl acetate (2-NA, Sigma) were used as received. Benzene from
Merck (fluorescence spectroscopy quality) was used, and ultrapure water
was obtained from a Labonco equipment model 90901-01. The pH of the
bulk water solution was maintained at 8.7 by using a 20 mm phosphate
buffer. In the case of RM media, it is known that the pH cannot be mea-
[
31]
sured inside the polar core of the aggregate. A meaningful approxima-
tion to the pH within the aqueous pseudophase of the RMs can be made
by using a pure source of BHDC and having sufficient buffering capacity
in the bulk solution. In this sense, the value of the pH inside the polar
Many studies on enzyme kinetics in RM solutions have
[
6–17]
[18]
been reported,
and recently they were reviewed by us.
[19–24]
In most of them, a-chymotrypsin (a-CT),
a hydrophilic
core is referred to the homogeneous buffer solution and it is called pHext
.
and globular enzyme that is totally associated with the mi-
celles and substrates partitioned between the micelles and
the external solvent, was used. In these cases, it was found
that the enzymatic activity is often substantially higher than
in aqueous buffer solutions, a phenomenon known as “su-
Procedures and kinetics: Solutions of BHDC in benzene were prepared
by weighing and dilution. The molar ratio between water and BHDC is
defined as W =[H O]/ ACHUTGNENRNUG[ BHDC]. The polar solvent was added to the mi-
0 2
cellar system by using a calibrated microsyringe.
Partition constants for 2-NA between benzene and water were deter-
mined by the hand-shaking method, using UV/Vis spectroscopy to record
the decrease of the absorbance of 2-NA in water after mixing with the
polar solvent at 25.0ꢁ0.18C.
[18]
peractivity”. Falcone et al. studied the kinetics of hydroly-
sis of 2-naphthyl acetate (2-NA) catalyzed by a-CT in RM
solutions formed by glycerol (GY)–water (38% v/v) mix-
ture/AOT/n-heptane by means of spectroscopic measure-
Reactions were followed spectrophotometrically by means of the increase
at the maximum of the absorption band (lmax =327 nm) of the product 2-
naphthol (2-N) at 25.0ꢁ0.58C. To start a kinetic run in homogeneous
media, a stock solution of 2-NA in aqueous buffer solution was added to
a thermostated cell containing a-CT in the same buffer. The concentra-
[17]
ments. They showed that addition of GY to the micelle
interior results in improved catalytic properties of a-CT, be-
cause GY addition to the RM media result in a decrease in
conformational mobility of a-CT, which leads to increased
ꢂ4
tions of 2-NA and a-CT in the reaction media were around 10 and
ꢂ6
10 m, respectively. In the micellar media, the stoppered cell was filled
[17,20]
with a fixed volume of BHDC in benzene with the substrate. The desire
W value was reached by adding buffer solution with a microsyringe.
0
After thermostating the cell, the enzymatic reaction was initiated by ad-
dition of a volume of a-CT dissolved in the RMs to give 3 mL of micellar
solution with the desired 2-NA and a-CT concentrations.
enzyme stability and activity.
On the other hand, there is some controversy about the
causes of enzymatic superactivity inside RMs media. It is
believed that the increased conformational rigidity of the
enzyme promoted by the surfactant layer and the increased
concentration of the substrate at the reaction site can con-
tribute to the RM effect. Also superactivity has been ex-
plained in terms of the peculiar state of water in the RMs,
The hydrolysis of 2-NA catalyzed by a-CT, which in the RM system is to-
tally incorporated in the micellar pseudophase, follows the Michaelis–
[
7,8]
Menten mechanism
[Eq. (1)].
k1
kcat
[18,25–29]
E þ SG HE-Sƒ!E þ P
ð1Þ
which mimics the status of intracellular water.
kꢂ1
The goal of our work is to determine the influence of dif-
ferent RM interfaces on the hydrolysis of 2-NA by CT in
the presence of water/BHDC/benzene RMs, and to compare
the efficiency of this reaction with that observed in pure
Applying the steady-state approximation to E–S gives the rate law of
Equation (2),
k
cat½Eꢃ½Sꢃ
[17]
v
0
¼
ð2Þ
water and in the previously studied AOT RM systems.
ðK þ ½SꢃÞ
M
The results show remarkably enhanced efficiency for a-CT
in the cationic RMs in comparison with the anionic system
and pure water. We will show that this is mainly because the
differences in the water properties at the cationic interface
make them unique for stabilization of a-CT. Thus, this water
has a “super” hydrogen bond donating capacity and can in-
teract with the enzyme at the interface. The hydrogen-bond
network that can be created around the enzyme makes it
more stable and increases the enzyme activity, probably by
decreasing the surfactant–enzyme interaction, varying its
conformation and making the active site more accessible to
the substrate, with a remarkable increase in enzymatic effi-
ciency.
ꢂ1
where v
cal enzyme and substrate concentration, respectively, kcat is the catalytic
rate constant, and K the Michaelis constant defined by Equation (3).
0
is the initial reaction rate (in ms ), [E] and [S] are the analyti-
M
K
M
¼ ðkꢂ1 þ kcatÞ=k
1
ð3Þ
Equation (2) can be rearranged into a form that is amenable to data anal-
ysis by linear regression, known as the Lineweaver–Burk equation
[
Eq. (4)]
½
Eꢃ=v ¼ ð1=kcatÞ þ ðK
0
M
=kcatÞ 1=½Sꢃ
ð4Þ
Equation (4) directly provides kcat from the reciprocal of the intercept,
and the catalytic efficiency kcat/K from the reciprocal of the slope. K is
obtained from the slope/intercept ratio.
M
M
[
7,8]
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Chem. Eur. J. 2010, 16, 8887 – 8893

Enzymatic Catalysis in Cationic Reverse Micelles
FULL PAPER
0
The absorbance was recorded as a function of time, and v was obtained
from the slope of [2-N] versus t profiles. The formation of 2-N was linear-
ly dependent on the reaction time during the first 20 min of reaction. The
v
0
values were plotted according to the Lineweaver–Burk relationship
[
Eq. (4)].
The pooled standard deviation of the kinetic data, using different sam-
ples, was less than 5%.
Results and Discussion
To the best of our knowledge the enzymatic hydrolysis of 2-
NA in presence of water/BHDC/benzene RM has not been
explored yet, and the reaction was only investigated in re-
verse micelles media made with the anionic surfactant
[17]
AOT.
Prior to studying the enzymatic reaction in the RM solu-
tions, the stability of a-CT was tested by absorption and
emission spectroscopy (results not shown), as was done pre-
Figure 1. Lineweaver–Burk plot for the a-CT-catalyzed hydrolysis of 2-
NA in water, pH 8.7 (phosphate buffer, 20 mm). [a-CT]=2ꢂ10 m.
ꢂ6
[17]
viously for other systems. The data show that the spectro-
scopic properties of the protein are very similar in homoge-
neous and micellar media, that is, encapsulation of the
enzyme in the BHDC RM medium does not significantly
alter the tertiary structure of a-CT, as was found previously
Table 1. Summary of experimental kinetic parameters of the enzymatic
reactions in homogeneous media and in water/BHDC/benzene RMs.
Parameter
Water
Water/BHDC/benzene
exp
cat
ꢂ1
ꢂ2
k
[s ]ꢂ10
3.02ꢁ0.13
24.13ꢁ0.02
exp
ꢂ4
KM [m]ꢂ10
9.07ꢁ0.09
51.12ꢁ0.23
[5,17,32,33]
exp
exp
ꢂ1 ꢂ1
in the AOT RM medium.
(k /KM ) [m
s
]
33
0.06
0.5
–
47.2
–
cat
bulk
[
[
(K
M
)
corr
]
[m]
exp
bulk
ꢂ1 ꢂ1
k
/(K
M
)
)
corr
mic
]
[m
s
]
–
Reactions in water: Although the enzymatic hydrolysis of 2-
cat
ꢂ3
[17]
((K
M
)
corr
[m]
4.48ꢂ10
NA by a-CT was previously studied,
we tested whether
exp
cat
mic
ꢂ1 ꢂ1
[
k
/(K
M
)
corr)] [m
s
]
–
53.81
under our experimental conditions the reaction is the same.
UV/Vis spectroscopic analysis showed that the hydrolysis of
2-NA catalyzed by a-CT in water, produces 2-N [Eq. (5)] in
quantitative yield.
The absorption spectra taken at different reaction times
show a clear increase in the intensity at l=335 nm (not
shown), evidencing formation of the product 2-N. Figure 1
shows the data treated according to a Lineweaver–Burk
plot. The linearity of the plot indicates that, under the con-
dition employed, the Michaelis–Menten mechanism applies
[19]
in water at pH 8.7. From the slope and the intercept of
the line in Figure 1, values of the experimental kinetic pa-
Figure 2. Representative absorbance spectrum at t=240 s for the hydrol-
ysis of 2-NA catalyzed by a-CT in the presence of BHDC RMs at W
10. [BHDC]=0.2m; [a-CT] =2ꢂ10 m; [2-NA]=7.5ꢂ10 m; pH 8.7.
t
=
exp
exp bulk
0
rameters kcat and (KM
)
were calculated (Table 1). These
ꢂ6
ꢂ3
[17,19]
values are compatible with those obtained by others
and make us confident that, under our experimental condi-
tions, the reaction follows the same mechanism considering
the different source of the employed enzyme.
termediates and/or product decomposition. As was previous-
ly found in the AOT RM system, the band at l=335 nm
[17]
corresponds to the product 2-N.
Reactions in water/BHDC/benzene reverse micelles:
Figure 2 shows a typical absorption spectrum for the hydrol-
ysis of 2-NA at reaction time t=240 s in BHDC RM media.
The absorption spectra taken at different times of reaction
show a clear isosbestic point, which evidences the lack of in-
Interestingly, in BHDC RM medium a new band appears
at l=360 nm which is absent in the AOT RMs media but it
is present in pure water at pH 8.7. Thus, we attribute this
ꢂ
band to the presence of 2-naphtholate (2-N ). It seems that
ꢂ
2-N loses its acidic proton yielding 2-N in BHDC RMs but
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8889

N. M. Correa et al.
not in the AOT RM medium. The question is why 2-N
yields 2-N in BHDC RMs, while this process is inhibited in
does not affect the kinetic parameters. In other words, the
enzymatic reaction does not occur in the water pool of the
BHDC RM medium.
ꢂ
AOT RMs, even though the pH of the water is the same.
We have demonstrated that in AOT a proton gradient exists
inside the RMs toward the interface leaving the interior
Representative results obtained for the effect of BHDC
concentration on the relationship between v0/
ACHTUNGTRENNGNU[ a-CT] and the
[31]
neutral. Thus, the absence of the band that corresponds to
analytical concentration of 2-NA ([2-NA] ) at W =10 are
T
0
ꢂ
2
-N inside AOT RMs indicates that 2-N exists exclusively
shown in Figure 4 for water/BHDC/benzene RMs. Interest-
at the AOT interface where deprotonation is not favored.
[5]
Quintana et al. have shown that water sequestered inside
anionic and cationic RM systems has different properties.
Water molecules entrapped inside AOT RMs show en-
hanced electron-donor ability in comparison with bulk
water. Thus, water at the AOT RM interface is not good for
solvating anions. Furthermore, AOT is a very good hydro-
[34–37]
gen-bond acceptor,
so 2-N can undergo this interaction
with the polar head of the surfactant. On the other hand,
water entrapped inside the BHDC RMs appear to be non-
electron-donating because of its interaction with the polar
head group of the cationic surfactant. Thus, it seems that the
positive charge of the surfactant and the unique interfacial
ꢂ
water properties favor formation of 2-N at the BHDC RM
interface, and this is an interesting property of the RMs as
nanoreactors. The distribution of the reaction product can
be modified simply by changing the properties of the inter-
facial water, which can be done by choosing the right surfac-
tant. Moreover, our results confirm the previous suggestion
that the term “pH” is not suitable for the RM field, because
Figure 4. Effect of BHDC concentration on the relationship between the
initial rate of reaction and the analytical concentration of 2-NA in water/
BHDC/benzene RMs at W
) 0.25.
0
=10. [BHDC]/m: ~) 0.10, !) 0.15, *) 0.20,
&
[31]
the proton concentration depends on the RM region.
Even though the product of the enzymatic reaction is not
the same for BHDC and AOT RMs, deprotonation of 2-N is
fast, so we still can apply Equations (2)–(4) in both micellar
media.
A representative example of the plots obtained for water/
BHDC/benzene according to Equation (4) is shown in
Figure 3 for [BHDC]=0.05m and at different W0 values.
The slope and intercept of the lines are the same at all W0
values, that is, the increased water content inside the RM
ingly, in the cationic RMs enzyme saturation with the sub-
strate is reached at lower [2-NA]T values compared to
water/AOT and water–GY/AOT RM media, and indicates a
exp mic
trend to lower values of (KM
)
(Michaelis constant deter-
mined in terms of the analytical concentration of the sub-
strate) for the BHDC RMs. Increasing the surfactant con-
centration results in a decrease in v0/ ACHUTNGRNENUG[ a-CT] over the whole
range of concentration studied.
The data shown in Figure 4 were plotted according to
Equation (4), and the results are shown in Figure 5. The lin-
earity of the plots at all [BHDC] considered indicates that
the classical Michaelis–Menten mechanism is valid for a-CT
in water/BHDC/benzene RMs, as was observed in water/
[17]
[6,9]
AOT/n-heptane
for this and other related reactions.
From Figure 5, values of the experimental kinetic parame-
ters in the microheterogeneous media at different surfactant
concentration can be obtained, and the results are listed in
Table 1 for [BHDC]=0.2m and W =10.
0
The dependence on BHDC concentration found in
Figure 5 could have two reasons: 1) 2-NA partitioning
owing to its greater solubility in benzene, which diminishes
local substrate concentration in the zone in which the reac-
tion takes place, that is, the micellar pseudophase. In other
words, this is the result expected due to simple dilution, be-
cause the local concentration is inversely proportional to the
surfactant concentration when the substrate is totally associ-
Figure 3. Lineweaver–Burk plot for a-CT-catalyzed hydrolysis of 2-NA in
water/BHDC/benzene RMs at different W . [BHDC]=0.05m. W :
0 0
*) 5,
[32,38]
ated with the micellar pseudophase.
2) Progressive inac-
&
;) 10, ~) 15, !) 20.
tivation of the enzyme by the surfactant diminishes the kcat
8890
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Chem. Eur. J. 2010, 16, 8887 – 8893

Enzymatic Catalysis in Cationic Reverse Micelles
FULL PAPER
porated into RMs per BHDC molecule, n=[2-
NA]b/
ACHTUNGTRENNUNG[ BHDC]. The proposed method is based on the as-
sumption that, at a given W value (W =10) and [enzyme]=
0
0
ꢂ6
2
ꢂ10 m, the value of v0/
concentration of the substrate in the organic solvent
Figure 5). Since equal [2-NA] implies equal n, a simple
ACHTUNGTRNENUNG[ enzyme] is determined only by the
(
f
[9]
mass balance leads to the above Equation (8). For a set of
2-NA]T and [BHDC] values corresponding to the same
v /[enzyme] value, a plot of the left-hand side of Equa-
[
AHCTUNGTRENNUNG
0
tion (8) against [BHDC] allows evaluation of n from the
slope, and Kp from the slope/intercept ratio. The plot is
shown in Figure 6, and the result obtained was K =0.75ꢁ
p
ꢂ1
0
.2m for the water/BHDC/benzene system. This value is
lower than the corresponding value obtained for water/
[17]
AOT/n-heptane RMs and thus shows greater solubility of
-NA in benzene in comparison to n-heptane.
Figure 5. Effect of BHDC concentration on the Lineweaver–Burk plot
for the a-CT-catalyzed hydrolysis of 2-NA in water/BHDC/benzene RMs
2
at W
0
=10. [BHDC]/m: ~) 0.10, !) 0.15, *) 0.20,
m. pHext 8.7.
&
) 0.25. [a-CT]=2ꢂ
ꢂ6
1
0
value as [BHDC] increases. Figure 5 shows that all the plots
have a unique intercept independent of the surfactant con-
centration. This fact argues against possibility 2), as expect-
ed for an enzyme totally incorporated in the RMs. There-
fore, the difference in the kinetic parameters obtained from
Figure 5 is due to partitioning of the substrate between the
micellar pseudophase and the external solvent (K ).
p
The partitioning of 2-NA between the micelles and the or-
ganic solvent pseudophases, can be treated within the frame-
work of the pseudophases model. According to the two-
pseudophase model, which considers the presence of two
[7,39–42]
phases
(the surrounding nonpolar solvent and the mi-
Figure 6. Experimental data from Figure 4 plotted according to Equa-
tion (8).
croaggregates) partitioning of 2-NA between the micelles
and the external solvent pseudophase, defined in Equa-
tion (6), can be expressed in terms of K [Eq. (7)],
p
We also used emission spectroscopy as independent tech-
nique to obtain the K value in the BHDC RMs medium for
comparison with the value obtained through the kinetic pro-
2
-NA þ BHDC Ð 2-NA
ð6Þ
ð7Þ
p
f
b
cedure. Thus, the partition constant K is quantified from
½
2 ꢂ NA ꢃ
p
b
K ¼
p
the changes in the 2-NA emission spectra (not shown) with
½
2 ꢂ NA ꢃ½BHDCꢃ
f
[9,39–42]
surfactant concentration.
The distribution of 2-NA be-
tween the micelles and the external solvent pseudophases
shown in Equation (7) can also be determined from the
change in the fluorescence intensity of 2-NA with surfactant
where [2-NA] is the concentration of the substrate incorpo-
b
rated in the micelles, [2-NA] the concentration of the sub-
f
[9,39–42]
concentration measured at a given wavelength.
Thus,
strate in benzene, and [BHDC] the concentration of the sur-
the fluorescence intensity observed is given by Equation (9).
factant. This equation applies at a fixed value of W and
0
when [2-NA] ![BHDC].
As was previously described, it is possible to determine
T
[15]
I ¼ I þ Ib
ð9Þ
f
the K value from the kinetic data by using Equation (8),
p
If the analytical concentration of 2-NA is kept constant
and the absorbance of the sample at the working excitation
wavelength is low, Equation (10) can be deduced,
n
Kp
½
2ꢂNAꢃ ¼
þ n½BHDCꢃ
ð8Þ
T
I ðꢀ þ ꢀ K ½BHDCꢃÞ
0
f
b
p
where [2-NA] is the total (analytical) substrate concentra-
tion, and n the average number of substrate molecules incor-
T
I ¼
ð10Þ
ð1 þ K ½BHDCꢃÞ
p
Chem. Eur. J. 2010, 16, 8887 – 8893
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8891

N. M. Correa et al.
where I is the incident light intensity, I and I are the fluo-
with the consequence that enzymatic efficiency increas-
es.
0
f
b
[18,45]
rescent intensities when 2-NA is present in the external sol-
vent and the disperse pseudophase, respectively, I is the
fluorescence intensity measured at the surfactant concentra-
tion considered, and f and f are the fluorescent quantum
To make a significant comparison of the kinetic parame-
ters obtained in BHDC RMs with the values obtained in
pure water in terms of a common thermodynamic substrate-
activity scale and not in terms of substrate concentrations,
f
b
yield of 2-NA in the organic solvent and bound to the RM
ꢂ1
[17]
interface, respectively. A K value of about 0.67ꢁ0.1m is
some correction must be applied. The simplest approach
p
obtained from a least-squares fit of Equation (10) at l
=
is to compare the rate constants by taking as reference the
external solvent of the RMs, that is, benzene. In other
words, the catalytic efficiency of the enzymatic reaction ob-
em
333 nm (result not shown), which matches the value ob-
tained kinetically [Eq. (8)].
exp
exp
exp bulk
The value of kcat , listed in Table 1, is practically constant
at all the surfactant concentrations used at constant W0;
thus, we can assume that the shape and sizes of the RMs are
not modified either by the surfactant concentration or by
tained in the bulk solvents (kcat /KM
)
must be corrected
for partitioning of the substrate between the organic and
bulk
polar solvent=Bz
[8]
bulk polar solvents K
[Eq. (12)].
[17]
enzyme incorporation inside the RMs media.
We deter-
bulk solvent
exp bulk solvent
water=benzene
mined the size of the BHDC RMs using dynamic light scat-
½ðKMÞcorr
ꢃ
¼ ðKM
Þ
=Kp
ð12Þ
tering and obtained a value of 11 nm (at W =10) with and
0
without enzyme, which corroborates our assumption.
The experimental kinetic parameters shown in Table 1
water=benzene
A Kp
value of 0.015 was obtained, and, the values
[
9,17]
were corrected using Equation (11)
by partitioning of 2-
exp
bulk
of (kcat /[(KM)corr
]
obtained for water are gathered in
ꢂ1
NA with K =0.75m obtained before, and the results are
p
Table 1. Taking into account this correction it can be seen
that the catalytic efficiency in the micellar media is almost
summarized in Table 1.
1
00 times higher than in the corresponding homogeneous
exp mic
ðK
Þ
media. Again, this can be attributed to the highly structured
polar solvents in the inner core of BHDC RMs, which make
the interfacial water a much better hydrogen-bond donor
than bulk water.
In summary, the RMs seems to be very good nanoreactors
because they create a unique microenvironment for carrying
out a variety of chemical and biochemical reactions. In par-
ticular, the water at cationic RM interfaces seems to have
enhanced hydrogen-bond donor capability, such that is
transformed into “super-water” for the enzymatic reaction
studied in this work.
mic
M
½
ðK Þcorrꢃ
¼
ð11Þ
M
ð1 þ K ½BHDCꢃÞ
p
exp
The kcat /[(KM)
mic
]
ratio obtained for water/AOT/n-hep-
corr
17]
[
tane RMs is 1.14. Thus, the ratio obtained in BHDC RMs
Table 1) is 47 times greater than that obtained in AOT
(
RMs. This result is difficult to explain and it cannot be at-
tributed solely to enzyme confinement inside RMs, because
in both systems the enzyme is in a restricted environment.
We think that other factors must be taken into account to
explain the results. Probably, the main reason for this
enzyme superactivity is the differences in the water struc-
tures at the different RMs interfaces.
Acknowledgements
Superactivity has been frequently explained in terms of
the peculiar state of water in RM media, which mimics the
status of intracellular water, especially water adjacent to
Financial support from the Consejo Nacional de Investigaciones Cientꢀfi-
cas y Tꢃcnicas (CONICET), Universidad Nacional de Rꢀo Cuarto, Agen-
cia Nacional de Promociꢄn Cientꢀfica y Tꢃcnica, and Agencia Cꢄrdoba
Ciencia is gratefully acknowledged. J.J.S., N.M.C., and R.D.F. hold a re-
search position at CONICET. F.M. thanks CONICET for a research fel-
lowship.
[18,25–29,43]
biological membranes.
As we have previously dis-
cussed, in both RM media the reaction takes place at the in-
terface and not in the water pool. We demonstrated recently
that the water molecules in BHDC RMs are less electron
donating but more strongly hydrogen bond donating than
[
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Received: February 18, 2010
1
025.
Published online: June 22, 2010
Chem. Eur. J. 2010, 16, 8887 – 8893
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8893