Effect of dioxane on the binding of competitive inhibitor proflavin and catalytic activity of bovine pancreatic α-chymotrypsin

Article abstract of DOI:10.1134/S0036024407070278

The binding of competitive inhibitor proflavin by α-chymotrypsin in water-dioxane mixtures over the entire range of thermodynamic activities of water a _w was studied. The data on the degree of binding of proflavin were compared to the results on the catalytic activity of the enzyme preliminary incubated in water-dioxane mixtures. An analysis of the behavior of the concentration dependences of these characteristics demonstrated that, at low a _w values, the behavior of the interprotein contacts in the enzyme formed during its drying largely governs its functional properties, while at high a _w values, they are determined by the interaction of the enzyme with the organic solvent. Interplay of these two factors is responsible for the observed complex shape of the isotherm of binding of proflavin, with the maximum degree of binding being attained at moderate a _w values.

Full text of DOI:10.1134/S0036024407070278

ISSN 0036-0244, Russian Journal of Physical Chemistry A, 2007, Vol. 81, No. 7, pp. 1160–1164. © Pleiades Publishing, Ltd., 2007.
Original Russian Text © V.A. Sirotkin, T.A. Mukhametzyanov, Yu.V. Karmanova, 2007, published in Zhurnal Fizicheskoi Khimii, 2007, Vol. 81, No. 7, pp. 1318–1323.
BIOCHEMICAL
CHEMISTRY
Effect of Dioxane on the Binding of Competitive Inhibitor Proflavin
and Catalytic Activity of Bovine Pancreatic a-Chymotrypsin
V. A. Sirotkin, T. A. Mukhametzyanov, and Yu. V. Karmanova
Butlerov Chemical Institute, Kazan State University, ul. Kremlevskaya 18, Kazan, Tatarstan, 420008 Russia
e-mail: vsir@mail.ru
Received May 15, 2006
Abstract—The binding of competitive inhibitor proflavin by α-chymotrypsin in water–dioxane mixtures over
the entire range of thermodynamic activities of water aw was studied. The data on the degree of binding of
proflavin were compared to the results on the catalytic activity of the enzyme preliminary incubated in water–
dioxane mixtures. An analysis of the behavior of the concentration dependences of these characteristics dem-
onstrated that, at low aw values, the behavior of the interprotein contacts in the enzyme formed during its drying
largely governs its functional properties, while at high aw values, they are determined by the interaction of the
enzyme with the organic solvent. Interplay of these two factors is responsible for the observed complex shape
of the isotherm of binding of proflavin, with the maximum degree of binding being attained at moderate aw val-
ues.
DOI: 10.1134/S0036024407070278
This work is a continuation of our systematic studies binding of the competitive inhibitor may prove useful
[1–3] aimed at solving a topical problem—elucidating for understanding the nature of the intermolecular
the physicochemical regularities of the functioning of forces that determined the state of the active site of the
proteins in aqueous–organic media with low water con- enzyme in low-water organic media.
tent. This problem is of considerable interest for non-
These circumstances motivated us to continue stud-
aqueous enzymology, an innovation-promising scien-
ies on the binding of competitive inhibitor proflavin by
tific direction [4–6]. The use of organic solvents as a
reaction medium makes it possible to successfully con-
α-chymotrypsin. Dioxane, one of the solvents widely
used in nonaqueous enzymology, was chosen as the
duct enzymatic reactions with hydrophobic compounds
model organic solvent [4–6]. Data on the binding of
poorly soluble in water. Nonaqueous organic media
proflavin were compared to the results on the effect of
provide the possibility of conducting industrially
dioxane on the catalytic activity of the enzyme. The aim
important reactions of synthesis that do not occur in
of the present work was to study the concentration
aqueous media, for example, the synthesis of peptides
dependences of the efficiency of binding of the compet-
and esterification. The enzymatic catalysis in organic
itive inhibitor and the enzymatic activity chymotrypsin
solvents is competitive and cost-saving technology for
in order to elucidate what intermolecular processes pro-
producing substances with a high optical purity [4–6].
duce the main effect on the state and functioning of the
enzyme at high and low activities of water in organic
media and to demonstrate how common are the regular-
ities observed for acetonitrile [2], another proton-
acceptor solvent.
On the other hand, it is clear that studying the regu-
larities of biocatalysis in organic media makes it possi-
ble not only to optimize various biotechnological pro-
cesses but also substantially extent fundamental knowl-
edge on the stability of protein macromolecules and on
the intramolecular forces maintaining the catalytically
active conformation of enzymes under conditions of
low water content.
EXPERIMENTAL
Materials. Bovine pancreatic α-chymotrypsin
(EC 3.4.21.1, Sigma, C-4129, Type II) and proflavin
(P-2508, Sigma) were used without additional puri-
fication. The catalytic activity of α-chymotrypsin in
the preparation was 52 units per g of substance. The
organic solvent was purified and dried as described
in [11].
At present, there is solid evidence that reactions cat-
alyzed by the α-chymotrypsin enzyme in water and
anhydrous organic media proceed via a single mecha-
nism [7]. The classical competitive inhibitors of reac-
tions catalyzed by α-chymotrypsin are aromatic com-
pounds, including proflavin (3,6-diaminoacridine) [8–
10]. One advantage of proflavin is its ability to form a
Determination of the enzymatic activity. The model
1 : 1 complex with the active site of the α-chymotrypsin process was the hydrolysis of N-acetyl-L-tyrosine ethyl
molecule. Therefore, studying the regularities of the ester (ATEE) catalyzed by α-chymotrypsin. The mea-
1160

EFFECT OF DIOXANE ON THE BINDING OF COMPETITIVE INHIBITOR PROFLAVIN
1161
Reaction yield, %
surements were performed on a Hiranuma Comitite-
101 potentiometric titrator (Japan) in the pH-static
mode at pH 8.0 and 25°ë. The concentration of the sub-
strate was 4.0 × 10–3 mol/l. The constant level of pH
was maintained by adding a titrant (a known concentra-
tion solution of potassium hydroxide), which neutral-
ized the N-acetyl-L-tyrosine acid formed by the hydrol-
ysis reaction. The kinetic curve was plotted as the time
dependence of the amount of the reactant spent for titra-
tion of the acid released. Kinetic curves for each given
set of conditions were measured twice at least.
100
80
60
40
20
0
5
1
2
3
4
The reaction mixture was prepared as follows.
A lyophilized preparation of the enzyme with an initial
humidity of 8.6 wt % (g water/g protein) was placed
into an aqueous–organic mixture of required composi-
tion and incubated at 25°ë for 1 h. The content of the
enzyme in the mixture was 1 mg/ml. Next, adding a
100-ml aqueous–organic aliquot containing the
enzyme, we initiated the enzymatic reaction. In all
cases, the content of dioxane in the final reaction mix-
ture was not less than 0.5 vol % (with a water activity
of aw > 0.99).
0
2
4
6
8
10
t × 10–2, s
Fig. 1. Typical kinetic curves for the hydrolysis of N-acetyl-
L-tyrosine ethyl ester catalyzed by bovine pancreatic α-
chymotrypsin preliminary incubated in water–dioxane mix-
tures with various thermodynamic activities of water:
(1) 0.01, (2) 0.6, (3) 0.8, (4) 0.9, and (5) 1.0.
In special experiments, we checked the reliability of
the method. For this purpose, using the linearized
Michaelis–Menten integral equation, we calculated the
kinetic parameters of the enzymatic reaction in water at
pH 8.0 and 25°ë: Km = 1.1 × 10–3 å–1 and Vmax/[E]0 =
209 s–1. Under similar conditions (pH 8.2, 25°ë), the
authors of [12] obtained Km = 1.2 × 10–3 M–1 and
Vmax/[E]0 = 155 s–1, a result indicative of the reliability
of the method we used.
In the regions of low and moderate water activities,
where the enzyme is insoluble, the spectra feature a dif-
ferent pattern. In this case, the binding of proflavin by
chymotrypsin gives rise to a minimum in the difference
spectra. In these experiments the concentration of the
enzyme in the mixture was constant, 1.9 × 10–4 mol/l.
Thermodynamic activity of water in dioxane. The
activity of water in dioxane was calculated by the for-
mula
UV spectrophotometric measurements. The degree
of binding of proflavin by chymotrypsin in water–
organic solvent mixtures was developed by us in [2].
The measurements were conducted on a Perkin-Elmer
Lambda 35 double-beam scanning spectrometer at
25°C. In all the experiments, the initial concentration of
proflavin was 1.0 × 10–5 mol/l, the concentration of
Tris-HCl-buffer was 0.05 mol/l, and the pH of the aque-
ous solution was 8.0. The enzyme used in the experi-
ments with proflavin was a solid preparation with a
humidity of 8.6 wt % (g water/g dry enzyme), which
was measured on a Setaram MGDTD-17S microther-
moanalyzer by drying the sample at 25°ë and 0.1 Pa to
constant mass.
aw = γwxw,
(1)
where xw is the mole fraction of water in the solution
and γw is the activity coefficient of water (mole fraction
scale; the standard state, pure substance). The water
activity coefficients were calculated from the published
data on the vapor–liquid equilibrium characteristics for
water–dioxane mixtures [13] by the formula
γw = ywpt/xwp0,
(2)
where yw is the mole fraction of water in the gas phase,
pt is the total pressure, p0 is the saturation vapor pres-
sure over pure water at the same temperature, and xw is
the mole fraction of water in the liquid phase.
This method is based on the following principle.
The interaction of proflavin with the active site of the
enzyme results in shift of the spectrum of the dye
(λmax = 444 nm in water) towards the longwave region.
Recording the difference spectrum of a proflavin solu-
tion with respect to the same solution but containing the
enzyme makes it possible to determine the extent of
formation of the enzyme–proflavin complex. The opti-
cal density of the difference spectrum at the maximum
(λmax = 465 nm in water) is proportional the concentra-
tion of proflavin bound in the complex. The concentra-
RESULTS AND DISCUSSION
Figure 1 shows kinetic curves for the hydrolysis of
ATEE catalyzed by a α-chymotrypsin preparation pre-
liminary incubated in water–dioxane mixtures. The cat-
alytic activity was characterized by the ratio of the
200-s reaction yield after incubation of the enzyme in
an aqueous organic mixture to that after its incubation
in pure water (Fig. 2, curve 1).
As can be seen from Fig. 2, dioxane influences the
tion of the enzyme in these experiments was varied catalytic activity of the biocatalyst in a complicated
from 6.2 × 10–6 to 1.9 × 10–4 mol/l.
way. At water activities from 0 to 0.5, the catalytic
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 81 No. 7 2007

1162
SIROTKIN et al.
catalytic activity increases, approaching the level corre-
sponding to pure water.
arel, %
100
β, %
100
Spectra of proflavin in water–dioxane mixtures. Fig-
ure 3a shows spectra of proflavin in water–dioxane
mixture. As can be seen, the organic solvent produces
an appreciable effect on the shape of the proflavin spec-
trum. For example, at high water activities, the shape of
the spectra and the position of the maximum are similar
to those observed in pure water at pH 8.0. At aw < 0.5,
however, the shape of the proflavin spectrum is mark-
edly different: a new shortwave absorption band
appears, the intensity of which increases with the
organic solvent concentration, while the intensity of the
longwave band concurrently decreases. This behavior
was interpreted as reflecting the coexistence of two
forms of proflavin, protonated and deprotonated. As the
water content in the water–dioxane solution decreases,
the equilibrium shifts towards the deprotonated form.
This conclusion is supported by the results of the fol-
lowing experiment. Figure 4 displays the spectra of
proflavin in water at various pH values. As seen, as in
the case of water–dioxane mixtures, variations in the
pH are accompanied by the redistribution of the abun-
dances of the protonated (predominant at low pH val-
ues) and deprotonated (predominant at high pH values)
forms of proflavin.
1
2
3
90
80
80
60
40
20
0
~
~
20
10
0
0.2
0.4
0.6
0.8
1.0
aw
Fig. 2. Relative catalytic activity a of α-chymotrypsin in
rel
(1) the hydrolysis ofATEE and (2) solid-phase hydrolysis of
N-succinyl-L-phenylalanine p-nitroanilide according to [14]
and (3) the fraction β of bound proflavin as functions of the
water activity a in an aqueous–organic mixture in which
w
the enzyme was preliminary incubated.
D
4
(a)
0.4
Binding of proflavin in water–dioxane mixtures.
Figure 3b shows the difference spectrum of proflavin in
the presence of α-chymotrypsin with respect to the ini-
tial solution of proflavin in an aqueous–organic mixture
with aw = 0.12 (0.5 vol % water). As can be seen, the
presence of the enzyme produces no effect on the
proflavin spectrum, which means that, in this mixture,
no binding of proflavin by the enzyme occurs. Similar
spectra were obtained for mixture with aw = 0.24, 0.33,
and 0.8.
3
1
2
0.2
0
~
(b)
(c)
~
0
–0.05
~
~
Figure 3c displays the difference spectrum of profla-
vin in the presence of α-chymotrypsin with respect to
the initial solution of proflavin in an aqueous–organic
mixture with aw = 0.5 (3.7 vol % water). Within this
range of water activities, α-chymotrypsin is insoluble,
and, therefore, the dip in the difference spectra was
attributed to a decrease in the concentration of proflavin
due to its binding by the enzyme. Similar spectra were
obtained for mixtures with aw = 0.45, 0.6, 0.65, and 0.7.
0
–0.05
–0.10
350
400
450
500
550
λ, nm
The process of binding of proflavin in water–diox-
ane mixtures was qualitatively characterized by the
absorbance at the isobestic point (at 414 nm, Fig. 4).
This was motivated by the following circumstances. On
the other hand, it is unknown what form of proflavin,
protonated or deprotonated, is bound by the enzyme in
aqueous–organic mixtures. On the one hand, at the
isobestic point the absorption coefficients of both forms
coincide. Correspondingly, by measuring changes in
the absorbance at 414 nm, one can determine changes
in the concentration of the competitive inhibitor in the
solution irrespective of the form in which it is bound.
Fig. 3. (a) Typical spectra of proflavin in water–dioxane
mixtures with various thermodynamic activities of water
((1) 0.12, (2) 0.24, (3) 0.5, and (4) 0.8)) and (b, c) difference
spectra of proflavin in the presence of α-chymotrypsin in
water–dioxane mixtures at a = (b) 0.12 and (c) 0.5. The
w
initial concentrations of proflavin and enzyme were 1.0 ×
–5
–4
10 and 1.9 × 10 mol/l, respectively. The Tris-HCl-buffer
concentration was 0.05 mol/l.
activity remains virtually constant, equal to that
observed after incubation in pure water. As the water
activity increases from 0.5 to 0.9, the catalytic activity
decreases sharply. At aw > 0.9 (34 vol % dioxane), the The ratio of the absorbance at the 414-nm point of the
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 81 No. 7 2007

EFFECT OF DIOXANE ON THE BINDING OF COMPETITIVE INHIBITOR PROFLAVIN
1163
difference spectrum to the absorbance at the isobestic
point of the spectrum of the initial solution is a measure
of the fraction of bound proflavin at a given concentra-
tion of the enzyme. The degree of binding of proflavin
by α-chymotrypsin at a constant concentration of the
latter (1.9 × 10–4 mol/l) as a function of the water activ-
ity is displayed in Fig. 2.
D
0.2
0.1
0
1, 2
3
4
5
Effect of dioxane on the catalytic activity of α-chy-
motrypsin and its ability to bind proflavin. The results
on the catalytic activity of α-chymotrypsin and its abil-
ity to bind proflavin were interpreted within the frame-
work of the model proposed in [1]. According to this
model, the dehydration of proteins results in the forma-
tion of firm intermolecular contacts via the establish-
ment of hydrogen bonds and/or ionic bridges between
side polar groups of the protein (carboxy, alcohol,
amido, and amino groups). As a result, the rigidity of
the protein structure increases while an appreciable
fraction of the protein polar groups of the dry protein,
those involved in the formation of interprotein contacts,
becomes incapable of acting as sorption sites. These
changes manifest themselves through sorption hystere-
sis [1]. Correspondingly, if these groups enter into the
composition of the active site of the enzyme, they
become incapable of interacting with molecules of the
substrate or competitive inhibitor. As a result, α-chy-
motrypsin shows no catalytic activity in reactions of
solid-phase hydrolysis at low water activities (aw < 0.4)
in the absence of an organic solvent [14] (Fig. 2).
6
7
350
400
450
500
550
λ, nm
Fig. 4. Typical absorption spectra of proflavin in water at
various pH values: (1) 3.0, (2) 5.9, (3) 7.8, (4) 8.5, (5) 8.9,
(6) 9.2, and (7) 10.5.
incubation of α-chymotrypsin in the aqueous-organic
solvent at aw < 0.4 (Fig. 2).
Only above the threshold protein humidity (h ~
0.1 g water/g protein or aw ~ 0.4–0.5), the mobility of
protein macromolecules increases markedly, the cata-
lytic activity rises sharply, and the state of the second-
ary structure approaches that of the native protein [1,
14, 16, 17]. Note that, in our experiments, the signifi-
cant increase in the degree of binding of the competitive
inhibitor and the catalytic activity of the enzyme in the
reaction of solid-phase hydrolysis are also observed at
aw = 0.4–0.5. According to the proposed model, at low
water activities, water molecules penetrate into the
structure of the dry enzyme, break interprotein con-
tacts, and hydrate the polar groups of these contacts. At
low water activities, interprotein contacts play a nega-
tive role, hindering the formation of the active form of
the enzyme. Thus, the stage of breaking interprotein
contacts plays a key role in the behavior of proteins in
organic media.
On the other hand, it was demonstrated that the sta-
bility and structure of dry proteins are substantially
dependent on the ability of the organic solvent to form
hydrogen bonds [15]. Considerable structural changes
and exothermic effects were observed only in solvent
capable of forming strong hydrogen bonds. Conse-
quently, knowledge of the tradeoff between the proton-
donor and proton-acceptor properties of the solvent is
important for predicting the possible influence of
organic molecules on the functional characteristics of
the protein. Indeed, when the interprotein contact
formed by hydrogen bonds is broken, a solvent (water
or dioxane) molecule is to choose with which fragment
to interact, proton-donor or proton-acceptor.
At aw > 0.5, the catalytic activity of the enzyme
(Fig. 2) and its ability to bind proflavin vary in a similar
way, both passing through a minimum at aw ~ 0.8. On
the one hand, the enzymatic activity in the absence of
the organic solvent shows no minimum at high water
activities. This suggests that, at a high degree of hydra-
tion of the enzyme, when most of the interprotein con-
tacts are already broken, the interaction with the
organic solvent determines the functional characteris-
tics of the enzyme. It was demonstrated [1, Fig. 2] that,
in this range of water activities, the enzyme is denatur-
ated by dioxane with the formation of intermolecular
β-structures.
Water molecules can solvate both proton-donor and
proton-acceptor groups of the protein. By contrast, pro-
ton-acceptor dioxane molecules will predominantly
solvate the proton-donor group of the broken contact
(the remaining proton-acceptor group will be more
effectively solvated by water molecules). Thus, dioxane
molecules are incapable of breaking interprotein con-
tacts on their own, in the absence of water. Correspond-
ingly, the interaction of dry α-chymotrypsin with anhy-
drous dioxane is not accompanied by significant heat
At moderate water activities, the degree of binding
effects or structural changes [15]. This means that, at of proflavin (Fig. 2) passes through its maximum,
low water activities, dioxane produces no appreciable reflecting the interplay between the following pro-
effect on the state of the enzyme in the form of a low- cesses. On the other hand, the hydration of the enzyme
humidity solid preparation. Therefore, no noticeable is already high enough, so that its conformation is close
decrease in the catalytic activity was observed upon to the native one. On the one hand, some interprotein
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 81 No. 7 2007

1164
SIROTKIN et al.
4. A. M. Klibanov, Nature (London) 409, 241 (2001).
contacts remain unbroken, a factor that plays a positive
role by hindering the denaturation of the enzyme by the
organic solvent.
5. G. Carrea and S. Riva, Angew. Chem., Int. Ed. Engl. 39,
2226 (2000).
6. M. N. Gupta, in Methods in Non-Aqueous Enzymology,
Note also that, as follows from Fig. 2 and the data
reported in [2], the shapes of the isotherms of binding
of proflavin by α-chymotrypsin and the positions of the
maximum degree of binding in dioxane and acetonitrile
are similar. This means that the formation of the
enzyme competitive inhibitor complex in moderate-
strength proton-acceptor solvents, such as dioxane and
acetonitrile, exhibits similar regularities.
Ed. by M. N. Gupta (Birkhäuser, Basel, 2000).
7. A. M. Klibanov, Trends Biochem. Sci. 14, 141 (1989).
8. K. Martinek, A. V. Levashov, and I. V. Berezin, Mol.
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9. A. L. Fink, Biochemistry 13, 277 (1974).
10. S. A. Bernhard, B. F. Lee, and Z. H. Tashjian, J. Mol.
Biol. 18, 405 (1966).
11. D. D. Perrin, W. L. F. Armarego, and D. R. Perrin, Puri-
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1980).
ACKNOWLEDGMENTS
12. M. R. Eftink, R. E. Johnson, and R. L. Biltonen, Anal.
The authors are grateful to B.N. Solomonov for
stimulating discussions of the results and help in per-
forming experiments.
Biochem. 111, 305 (1981).
13. V. B. Kogan, B. N. Fridman, and V. V. Kafarov, Equilib-
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