Activity of α-Chymotrypsin in Cationic and Nonionic Micellar Media: Ultraviolet and Fluorescence Spectroscopic Approach

Article abstract of DOI:10.1002/kin.20972

α-Chymotrypsin (α-CT) activity was measured in aqueous buffer with the following alkyltriphenylphosphonium bromide surfactants in the series cetyl, tetradecyl, and dodecyl as a tail length. For the sake of comparison with mixed micellar investigation on a

Full text of DOI:10.1002/kin.20972

Activity of α-Chymotrypsin
in Cationic and Nonionic
Micellar Media: Ultraviolet
and Fluorescence
Spectroscopic Approach
SANTOSH K. VERMA, KALLOL K. GHOSH, RAMESHWARI VERMA, SHEKHAR VERMA,3
1
,2
2
1,2
4
1
GIRISH H. N., XIUJIAN ZHAO
1State Key Laboratory of Silicate Materials for Architectures, Wuhan University of Technology, Wuhan, 430070, People’s
Republic of China
2School of Studies in Chemistry, Pt. Ravishankar Shukla University, Raipur, India
3Faculty of Pharmaceutical Sciences, Shri Shankaracharya Group of Institutions, Bhilai, India
4State Key Laboratory of Advance Technology for Material Synthesis and Processing, Wuhan University of Technology,
Wuhan, 430070, People’s Republic of China
Received 7 August 2015; revised 9 November 2015; accepted 10 November 2015
DOI 10.1002/kin.20972
Published online 14 December 2015 in Wiley Online Library (wileyonlinelibrary.com).
ABSTRACT: α-Chymotrypsin (α-CT) activity was measured in aqueous buffer with the following
alkyltriphenylphosphonium bromide surfactants in the series cetyl, tetradecyl, and dodecyl
as a tail length. For the sake of comparison with mixed micellar investigation on activity of
α-CT, cationic cetyltriphenylphosphonium bromide (CTPB) and nonionic surfactant Triton X-
1
00, Brij-56, Brij-35, Tween 20, and Igepal Co-210 have been used. The p-nitrophenyl acetate
(PNPA) hydrolysis rate was determined at the surfactant concentration of both cationic and
mixed micellar systems by a UV–vis spectrophotometer. The catalytic reaction follows the
Michaelis–Menten mechanism, and the catalytic efficiency (kcat/KM) was evaluated for both
homogeneous and mixed-micellar media. The maximum catalytic efficiency was observed at
−
1
−1
5
mM concentration of CTPB, but the highest catalytic efficiency, 572 M
s , was measured
in the presence of mixed micellar (7.5 mM CTPB + 2.5 mM Tween-20). The fluorescence (FL)
spectra showed the differences of α-CT conformations in the presence of cationic surfactants.
The FL results suggest that the influence of cationic surfactant on proteolysis arises from the
interaction with the α-CT. The binding constant, ksv, of α-CT with cationic aggregates was
Correspondence to: Santosh K. Verma; e-mail: vermasantosh08
@
gmail.com.
ꢀ
2015 Wiley Periodicals, Inc.
C

8
0
VERMA ET AL.
determined in the buffer using the Stern–Volmer equation by the fluorescence spectroscopic
approach. ꢀC 2015 Wiley Periodicals, Inc. Int J Chem Kinet 48: 79–87, 2016
INTRODUCTION
proteins, as well as affording methods to study associ-
ation reactions and denaturation [11,12]. The FL arises
uniquely from its tryptophan residues when selectively
excited at 280 nm. Analysis of the effect of quencher
concentration upon protein FL (Stern–Volmer plots)
can supply information on quenching constants and
estimation of tryptophan accessibility [13]. Celej
et al. [14] reported that the catalytic behavior of α-
CT affected with the increasing of CTAB concentra-
tion showing bell-shaped profile. It is clearly indicating
that the micelle-bound enzyme reacts with the free sub-
strate. The α-CT interaction with CTAB changes the
tertiary structure of enzyme shows red shift in tryp-
tophan fluorescence spectrum, signifying the termina-
tion of internal quenching and a more polar location of
tryptophan residues.
The activity and stability of water-soluble enzymes
in aqueous micelles is closely related to the chemical
nature of the surfactant. The kinetics and some confor-
mational investigation have already been reported for
proteins by fluorescence [1] and UV spectroscopy [2].
The activity of enzyme can be improved by interac-
tion of the α-chymotrypsin (α-CT) with the micelles
using surfactants at concentrations above the critical
micellar concentration (CMC) [3]. In fact, the inter-
action between surfactants and proteins in aqueous
solutions can be rather specific, the residual enzyme
activity being dependent on the nature of both sur-
factant and enzyme [4]. Alfani et al. [5,6] have car-
ried out numerous studies on the α-CT–catalyzed hy-
drolysis of N-glutaryl-L-phenylalanine p-nitroanilide
in the presence of cetyltrimethylammonium bromide
In previous works, we reported that the enzymatic
activity of α-CT on the hydrolysis of p-nitrophenyl
acetate (PNPA) strongly depends upon the reaction
medium such as oil-in-water microemulsions [15],
organic cosolvents [16], cationic surfactant media
(CTAB) surfactants with different alkyl groups, which
indicate that the α-CT activity increases with increas-
ing size of the alkyl head groups. Nevertheless, the
changes in the head group size of surfactant can alter
the micellar surface area and the electrostatic interac-
tions of α-CT/surfactant and subsequently affect the
degree of ionization and the surroundings of microen-
vironment. However, an alternative approach has been
carried out to minimize above factors to modify the
alkyl tail length of surfactant, which can vary the char-
acteristics of the micellar interface [7].
The most important function of nonionic surfac-
tants is to stimulate the catalytic activity of most
enzymes. For example, Brij 35 stimulated 1.5-fold
cytoplasmic glycerol-3-phosphate dehydrogenase [8],
deoxycholate, and many bile salts activated four- to
sevenfold alcohol dehydrogenase from rat liver [9],
Tween 20 or micellization of substrates resulted in a
three- to sixfold increase in the activity of mitochon-
drial carnitine palmitoyltransferase [10]. Furthermore,
it is necessary to study the catalytic activity of α-CT in
the presence of cationic and nonionic mixed surfactants
system.
The fluorescent probe (e.g., tryptophan, DNA
strands) has been used to study the catalytic activity
measurement and conformation changes in enzyme.
The tryptophan fluorescence (FL) is very sensitive to
solvent polarity for the protein–surfactant complex for-
mation. Thus, the emission spectrum of tryptophan
residues in proteins is strongly influenced by their en-
vironment. This information can be useful in provid-
ing an indication of the location of these residues in
[17–19], and reverse micelles [20]. Recently, we re-
ported surface and conformational behavior of protein–
surfactant interaction by using trypsin and α-CT in
cetyltriphenylphosphonium bromide (CTPB) micellar
media [21]. We noticed that the tryptophan probe and
CTPB molecules adsorb in specific sites on the protein
and probably change in micelle-like cluster, which is
due to the ionic nature of both protein and surfactant.
The present study was undertaken to investigate the
protein and surfactant interaction activity in micellar
media and its conformational behavior upon alkyltriph-
enylphosphonium bromide (cetyl, tetradecyl, dodecyl)
surfactants. The proposed reaction scheme for the hy-
drolysis of PNPA catalyzed by α-CT in the presence
of cationic and nonionic surfactants is presented in
Scheme 1. Special emphasis has been given in this
investigation on the α-CT activity in the cationic sur-
factants and mixed micellar prepared by a fixed molar
ratio of CTPB and nonionic surfactants (Triton X-100,
Brij-56, Brij-35, Tween 20, and Igepal Co-210). The
kinetic parameters were calculated and discussed. The
FL measurements were carried out to study the confor-
mational analysis of α-CT upon the cationic surfactant
(CTPB, TTPB, and DTPB). The results of the present
study may be of productive use in understanding the in-
teraction of α-CT with cationic (CTPB, TTPB, DTPB)
and nonionic surfactants for expanding the mixed mi-
cellar application.
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ACTIVITY OF α-CHYMOTRYPSIN IN CATIONIC AND NONIONIC MICELLAR MEDIA
81
Scheme 1 α-CT–catalyzed hydrolysis of PNPA and chemical structure of surfactants.
EXPERIMENTAL
PNPA was prepared in pure acetonitrile (CH3CN). The
presence of acetonitrile does not modify the enzyme–
substrate interaction. For a reaction mixture, Tris
buffer pH was fixed at 7.70 using 0.1 M hydrochloric
acid.
Materials
α-CT (type-II, from bovine pancreas, molecular mass
2
5 kDa and an isoelectric point pI 8.8) was pro-
cured from Sigma (St. Louis, MO) and used with-
out further purification. The cationic surfactant, i.e.
alkyltriphenylphosphonium bromide (R = C16, C14,
C12), was obtained from Prof. R. M. Palepu, St. Fran-
cis Xavier University, Antigonish, Canada, as a gift.
The nonionic surfactant, i.e., Triton X-100, Igepal
CO-210, Tween-20, Brij-35, Brij-56, were procured
from Sigma. The substrate PNPA was procured from
Sisco (Mumbai, India). Enzyme and substrate solu-
tions were freshly prepared in the appropriate buffer
immediately, before their use in the experiment. Tris-
Enzyme Kinetics Measurements
All the spectral measurements were performed using
a Varian Cary 50 UV–vis spectrophotometer equipped
with a Peltier temperature controller unit at 25°C. The
hydrolytic activity of α-CT toward PNPA was moni-
tored by following the increase in the absorbance at
400 nm due to the formation of p-nitrophenolate ion
−
(PNP ) at pH 7.70. All kinetic reaction has been car-
ried out with maintaining 0.065 to 0.52 mM and 13 µM
concentrations of PNPA and α-CT, respectively. The
obtained p-nitropenolate ion product extinction coeffi-
(
hydroxymethyl)aminomethane (Tris) (pKa 8.3) and
HCl were obtained from Qualigens (Mumbai, India).
−
1
−1
cient was 12,000 M cm . The initial reaction rate,
−
V0, was determined from the slope of the PNP con-
Solution Preparation
centration versus time profiles using enzyme kinetics
software (Varian). The rates of all enzyme–catalyzed
reactions were corrected for the rate of sponta-
neous nonenzymatic hydrolysis (buffer/surfactants)
determined under identical conditions. The Kinetic
The α-CT solution was freshly prepared in ac-
etate buffer pH 4.7 (0.1 M CH3COOH and 0.1 M
CH3COONa) immediately before their use in the ex-
periments. To improve the solubility of a substrate, the
International Journal of Chemical Kinetics DOI 10.1002/kin.20972

8
2
VERMA ET AL.
Table I Effect of Chain Length of Cationic Surfactants on α-CT Catalyzed Reaction of PNPA
2
10 kcat(s–1)
3
–1 –1
Surfactant
CMC (mM)
[Surfactant] (mM)
10 KM (M)
kcat/KM (M s )
Buffer
CTPB
–
1.0
0.51 ± 0.02
2.03 ± 0.07
4.28 ± 2.04
5.39 ± 0.26
0.72 ± 0.03
0.59 ± 0.10
1.21 ± 0.12
1.46 ± 0.17
1.52 ± 0.06
1.14 ± 0.06
0.59 ± 0.11
1.09 ± 0.06
1.20 ± 0.08
1.24± 0.04
0.95 ± 0.10
0.58 ± 0.07
0.20 ± 0.10
0.80 ± 0.01
0.76 ± 0.40
0.70 ± 0.01
0.10 ± 0.01
0.30 ± 0.01
0.25 ± 0.02
0.23 ± 0.01
0.20 ± 0.01
0.22 ± 0.01
0.24 ± 0.01
0.40 ± 0.01
0.24 ± 0.02
0.20 ± 0.01
0.28 ± 0.04
0.30 ± 0.01
23.1
25.3
56.3
77.0
72.0
19.6
48.4
63.4
76.0
51.8
24.5
27.2
50.0
62.0
33.9
19.3
0.16 ± 0.01
3
5
.0
.0
1
2
0
0
TTPB
DTPB
0.55 ± 0.01
1.81 ± 0.02
1.0
3
5
.0
.0
1
2
0
0
1.0
3
5
.0
.0
1
2
0
0
[
*
α-CT] = 0.02 mM, [Tris/HCl] = 10 mM, temperature = 25°C, pH 7.70, [PNPA] = 0.02–0.20 mM.
CMC values are taken from [24].
parameters, catalytic constant, kcat, and Michaelis con-
stant, KM, in micellar solution were obtained by the
linear regression analysis of Lineweaver–Burk plot
RESULTS AND DISCUSSION
Hydrophobicity of Cationic Surfactants on
the Enzymatic Activity of α-CT
(Eq. (1)).
α-CT activity was measured initially in aqueous buffer
(10 mM Tris–HCl, pH 7.70) and in the presence of
cationic surfactants at different concentrations above
the CMC value (CMC values are taken from [24]). The
enzyme kinetic study was undertaken to investigate
the effect of cationic surfactants CTPB, TTPB, and
DTPB on the α-CT activity in aqueous system, which
is enhanced the hydrolysis rate of PNPA (Table I).
As clearly manifested in Table I, the catalytic effi-
ciency (kcat/KM) of α-CT has been observed to increase
with the increasing surfactant from 1 to 5 mM concen-
tration but readily falls at higher concentrations up to
ꢀ
ꢀ
ꢁ
ꢀ
ꢂ
−
1
[
α − CT] V0 = 1 (kcat) + KM kcat [PNPA]
(1)
FL Measurements
The FL emission spectra of a tryptophan residue in α-
CT–surfactant systems were measured using a Varian
Carry Eclipse FL spectrophotometer with a slit width
of 0.5 cm. The excitation wavelength was 295 nm with
bandwidths of 5 nm for excitation and emission. The
spectra of 0.54 µM α-CT were obtained at different
incubation times of the protein in 20 mM Tris/HCl, pH
20 mM in case of all cationic surfactants. In com-
7
.70 at 25°C, in the absence or presence of increas-
parison with the aqueous buffer, the micelle-bound α-
CT showed higher catalytic efficiency for all the three
cationic surfactants. The results are most pronounced
ing surfactant concentrations. Slow changes in the FL
spectrum of α-CT were observed up to 20 min incu-
bation with alkyltriphenylphosphonium bromide (C16,
C14, C12) micelles. The binding constant of enzyme-
surfactant interaction were estimated by the FL mea-
surements using the Stern–Volmer equation (Eq. (2))
−
2
in CTPB, kcat increases from 2.03 × 10 to 5.39 ×
−
2
−1
s
1
0
0
for 1–5 mM and further decreases 0.59 ±
2
−
−1
.10 × 10
s for 20 mM of the surfactant concen-
tration. On the contrary, KM values decrease from 1 to
mM and further rises up to 20 mM of the surfactant
[22,23].
5
concentration.
ꢀ
I0 I = 1 + Ksv[Surfactant]
(2)
Similar trends of α-CT activity are observed for
TTPB and DTPB. The catalytic activity for micelle-
bound α-CT was raised up to 10.56-, 2.98-, and 2.43-
fold for CTPB, TTPB, and DTPB, respectively, than
the aqueous buffer. Here, the increase in the catalytic
activity of α-CT with increasing hydrophobicity of
where I0 and I are the FL intensities without and
with surfactants in the presence of α-CT and Ksv
is the α-CT surfactant binding constant in protein
medium.
International Journal of Chemical Kinetics DOI 10.1002/kin.20972

ACTIVITY OF α-CHYMOTRYPSIN IN CATIONIC AND NONIONIC MICELLAR MEDIA
83
surfactants, Brij-35, Brij 56, Tween-20, Triton X-100,
and Igepal CO-210 using 10 mM Tris/HCl buffer (pH
7.70) at 25°C. Mixtures of cationic and nonionic sur-
factants form mixed micelle aggregates. The properties
of mixed surfactant systems are quite different from
those of a single individual surfactant system. The re-
sults presented in Table II show that, irrespective of
the nature of the nonionic surfactants, α-CT entrapped
in aqueous buffer of mixed micellar concentration in
the ratio (7.5 + 2.5) mM showed 1.02, 2.07, 1.49, and
1.59 times higher catalytic activity for system 2, 6, 10,
and 14, respectively, and for mixed micellar system
8 exhibited a less improved α-CT activity (0.90-fold)
1
compared to system 1 containing only CTPB. The in-
teractions between cationic and nonionic surfactants
in a mixed micelle result in synergistic effects on the
micellization of mixed surfactant systems. Thus, it is
quite clear that the nonionic surfactants definitely play
an important role in regulating the α-CT activity. The
increasing amounts of nonionic surfactants possibly
help in reducing the positive surface charge density
at the micellar interface. As a consequence, the α-CT
activity was boosted up in the mixed micellar systems
compared to CTPB [26].
Figure 1 Dependence of the catalytic rate constant on the
surfactant concentration. Symbols indicate (ꢀ), CTPB; (ꢁ),
TTPB; (•), DTPB.
surfactant is due to the enhancing the micellar inter-
face. Further increased interface strongly affects the
enzyme activity and providing enhanced microenvi-
ronment to aggregate higher concentration of enzyme
at the micellar surface [25].
In the presence of CTPB aggregates, the α-CT activ-
ity depends markedly on the surfactant concentration:
Bell-shaped curves of kcat versus surfactant concentra-
tion were obtained. For all surfactant concentrations,
the addition of TTPB and DTPB surfactants leads to a
decrease in α-CT activity. The full line in Fig. 1 shows
reasonable agreement with the experimental data. The
α-CT–micelle interaction leads to an increase in both
the kcat and the affinity for the substrate PNPA, in-
dicating higher catalytic efficiency for micelle-bound
α-CT. Figure 1 also shows the effect of the surfactant
concentration on the catalytic activity of α-CT. The α-
CT–micelle interaction leads to an increase in both kcat
and catalytic efficiency for the substrate PNPA, indi-
cating higher catalytic efficiency for bound α-CT. The
bell-shaped profile of α-CT activity with increasing
surfactant (CTPB, TTPB, and DTPB) concentration
suggests that the micelle-bound α-CT reacts with the
free PNPA [14]. These results confirm that the depen-
dence of kcat on the surfactant concentration is similar
to that of the catalytic efficiency, being the increase
above the CMC higher than the surfactant having the
shorter tail length.
Conformational Analysis of α-CT upon
Cationic Surfactants
The crystallographic study of atomic structure of tosyl-
[27] and native-α-CT [28] confirmed that two trypto-
phan residues (Trp-27 and -29) are located in the in-
terior area of the α-CT molecule and the rest of them
(Trp-51, -141, -172, -207, -215, and -237) are located
at the surface of the α-CT. In the present investigation,
the FL study has been done to explore the conforma-
tional changes in α-CT in the presence of the cationic
surfactant at pH 7.70. The emission spectrum of the
native α-CT upon excitation at 295 nm showed a peak
at 339 nm for CTPB and 333 nm for both TTPB and
DTPB, indicating that the fluorophore of α-CT such as
tryptophan at 295 nm was noticeably shielded from the
cationic micelles in the native conformation. The least
part of the surface area tryptophan residues is accessi-
ble to the external solvent [27]. Consequently, it may
be presumed that when the surfactant composition is
changing the emission wavelength of α-CT has been
affected by both conformational changes of the α-CT
and changes in surfactant polarity.
Winter and co-workers analyzed using FT-IR spec-
troscopy and synchrotron small-angle X-ray diffrac-
tion that the protein aggregation occurs at relatively
low temperatures and at low α-CT concentration [29].
Furthermore, the incorporation of α-CT in aqueous so-
lutions of micelles is less ordered or partially ordered
Enzymatic Efficacy of α-CT on Cationic and
Nonionic Mixed Micellar System
The activity of α-CT was estimated in several
mixed micelles prepared from CTPB and nonionic
International Journal of Chemical Kinetics DOI 10.1002/kin.20972

8
4
VERMA ET AL.
Table II Summary of α-CT Catalyzed Hydrolysis of PNPA in Cationic and Nonionic Mixed Micellar Media
2
10 kcat(s–1)
3
–1 –1
System
Surfactant
[Surfactant] (mM)
10 KM (M)
kcat/KM (M s )
1
2
3
4
5
6
7
8
9
CTPB
CTPB + TX-100
10
3.59 ± 0.01
3.68 ± 0.80
1.49 ± 0.04
1.04 ± 0.06
0.91 ± 0.05
7.43 ± 0.66
5.53 ± 0.24
4.57 ± 0.23
1.87 ± 0. 06
5.35 ± 0.13
4.13 ± 0.22
4.21 ± 0.20
2.35 ± 0.50
5.72 ± 0.16
2.64 ± 0.17
1.49 ± 0.06
1.14 ± 0.03
3.24 ± 0.09
2.91 ± 0.10
0.99 ± 0.01
0.87 ± 0.05
0.20 ± 0.01
0.23 ± 0.30
0.29 ± 0.01
0.34 ± 0.01
0.40 ± 0.01
0.27 ± 0.10
0.43 ± 0.01
0. 60 ± 0.02
0.68 ± 0.02
0.20 ± 0.01
0.32 ± 0.02
0.44 ± 0.01
0.57 ± 0.03
0.10 ± 0.01
0.24 ± 0.05
0.32 ± 0.02
0.46 ± 0.01
0.40 ± 0.02
0.43 ± 0.02
0.49 ± 0.01
0.63 ± 0.01
180
160
51.3
30.5
22.7
200
7.5 + 2.5
5.0 + 5.0
2.5 + 7.5
0 + 10
CTPB + Brij-35
7.5 + 2.5
5.0 + 5.0
2.5 + 7.5
0 + 10
128
76.1
27.5
268
10
11
12
13
14
15
16
17
18
19
20
21
CTPB + Brij-56
7.5 + 2.5
5.0 + 5.0
2.5 + 7.5
0 + 10
129
95.6
41.2
572
CTPB + Tween-20
CTPB + Igepal CO-210
7.5 + 2.5
5.0 + 5.0
2.5 + 7.5
0 + 10
110
46.5
24.7
81.0
67.6
20.2
13.8
7.5 + 2.5
5.0 + 5.0
2.5 + 7.5
0 + 10
[α-CT] = 0.02 mM, [Tris/HCl] = 10 mM, temperature. = 25°C, pH 7.70, [PNPA] = 0.02–0.20 mM.
structure conformation. The influence of α-CT incor-
poration on the cationic micellar system resulted in
a redshift of the FL emission maximum by a magni-
tude about the 15 nm from 339 to 354 nm for CTPB,
whereas 11 nm from 333 to 344 nm for TTPB and 4 nm
from 333 to 337 nm for DTPB at pH 7.70 (Figs. 2a–2c).
This structural shifting suggested that the tryptophan
for 295 nm was more exposed to the micellar environ-
ment in the unfolded aggregation-competent species
of the α-CT [30]. Figures 2a–c show that the larger
spectral widths of emission spectra also specified the
higher heterogeneity of the tryptophan environments
in the unfolded structures of α-CT. The influence of α-
CT incorporation on a cationic surfactant, a 1.25- and
α-CT-CTPB as compared to the pure α-CT with PNPA.
This is due to the binding of cationic CTPB to high
affinity sites of tryptophan residues on the surface of
α-CT. The significant change in the absorbance value
of α-CT-CTPB mixture is nearly two times higher than
the absorbance of pure α-CT, denoting the structural
change or denaturation of the α-CT [31].
Fluorescence Binding Studies of α-CT
To active conformation of water-soluble protein,
α-CT, a fraction of the hydrophobic side chains is typ-
ically buried in the interior of the molecule. However,
one of the difficult aspects of the study of protein–
surfactant interaction is the determination of the struc-
ture of the protein–surfactant complex. For obtain-
ing a molecular-based understanding, it is important
to establish how the different regions of the binding
isotherm relate to protein conformational changes in-
duced by the surfactant. According to Turro et al. [32],
surfactant binding to protein generally displays four
characteristics regions, binding is to specific high-
energy sites on the protein. Consequently, a decrease
in pH shifts the binding isotherm to a higher concen-
tration for cationic surfactants [33].
1
.54-fold increase in the FL intensity was observed for
the TTPB and DTPB in comparison the CTPB. The FL
emission spectral shift is further evidenced for the pro-
tein conformation and interaction of surface of α-CT
with the micellar interface.
The UV spectroscopic technique can be applied to
examine binding and folding/unfolding of α-CT ini-
tiated by cationic surfactant, CTPB. Figure 3 shows
the absorption spectra of α-CT-catalyzed hydrolysis of
PNPA in aqueous buffer at 400 nm due to the formation
−
of p-nitrophenolate ion (PNP ). The absorption spec-
tra slightly shifted (ꢀ8 nm) in the presence of 1 mM of
CTPB of the same solution. Apart from that the spectra
show a sharp increase in absorbance with interaction of
Incorporation of molecular probes into aqueous mi-
celles and proteins is affected by parameters such as the
CMC, degree of water penetration into these surfactant
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ACTIVITY OF α-CHYMOTRYPSIN IN CATIONIC AND NONIONIC MICELLAR MEDIA
85
Figure 3 UV–visible absorption spectra of α-CT and
α-CT-CTPB micellar interaction in aqueous solution of
Tris/HCl buffer at 25°C. [α-CT] = 0.13 µM, [PNPA] =
26 µM, [CTPB] = 1 mM.
Table III Binding Parameter of α-CT-Surfactant
Interaction at 25°C
λexc
(nm)
λemiss
(nm)
λmax
(nm)
Ksv
Surfactant
(M–1)
CTPB
TTPB
DTPB
290
290
290
337
332
332
342
337
337
76718 ± 321
16168 ± 189
3818 ± 175
aggregations, and local polarity of the microenviron-
ment of the binding sites of proteins [34]. Tryptophan is
a novel molecular reporter with potential advantage for
following surfactant-induced protein unfolding as well
as micelle formation. Thus, FL spectral intensity signa-
tures from a location of the probe in the bulk solvent as
well as within the various microenvironments of a sur-
factant aggregation and protein are discernible. Both
the polarity-dependent spectral shift and FL intensity
variation enable the FL contributions from multiple
populations of probe molecules to be easily resolved.
The FL quenching of α-CT in cationic micellar
solutions and water was carried out, and the Stern–
Volmer plots I0/I against the surfactant concentration
are found to be linear. A representative Stern–Volmer
plot is given in Fig. 4. The binding constant, ksv, of
enzyme–surfactant interaction has been determined by
the FL intensity data using the Stern–Volmer equation
(Eq. (2)), which is reported in Table III. We assume
this method can estimate the binding constant for the
association of the α-CT with the micelles because the
FL intensity of the probe is significantly different from
both the environment water and micellar. The bind-
ing constant obtained for CTPB with α-CT is higher
Figure 2 Florescence emission spectra of α-CT and in-
creasing concentration of CTPB (a), TTPB (b), and DTPB
(
2
c) at pH 7.70, Temperature 25°C. [α-CT] = 0.54 µM, λexc =
90 nm, and λemiss = 337–342 nm. Note: Fig. 2a has been
taken from our recently published paper [24].
International Journal of Chemical Kinetics DOI 10.1002/kin.20972

8
6
VERMA ET AL.
show that with increasing hydrophobicity of alkylt-
riphenylphosphonium bromide (R = C16, C14, C12),
the catalytic activity of α-CT was enriched due to the
enhancing micellar interface which strongly boosts en-
zyme activity. The α-CT activity was boosted up in the
mixed micellar systems, showing that the nonionic sur-
factant plays a significant role in regulating the α-CT
activity. Information on the nature of α-CT-surfactant
binding can be obtained from FL probe studies such as
tryptophan residues. This study revealed that emission
spectra shifted to higher wavelength in the presence of
cationic surfactants (CTPB, TTPB, and DTPB). The
FL result is confirmed by considering the hydropho-
bicity of the surfactants in different concentrations and
its association with the enzyme.
Figure 4 Stern–Volumer plot for α-CT interaction with
CTPB (◦), TTPB (ꢁ), and DTPB (ꢀ).
Financial support of this Work by the Council of Scientific
and Industrial Research, New Delhi, India (CSIR project ref.
no. 01 (2143)/07/EMR-II) is gratefully acknowledged. Au-
thors are also grateful to UGC SAP Project, Pt. Ravishankar
Shukla University, Raipur for providing financial support.
compared to those for TTPB and DTPB. This is due to
the electrostatic attraction between the α-CT and the
charged surface of the cationic micelles, which creates
a very high quencher concentration in the vicinity of
the probe and causes an efficient quenching [7].
The number of specific binding site increases
with hydrocarbon chain length for the interac-
tion between α-CT and a homologous series of
n-alkyltriphenylphosphonium bromide; the specific
binding may occur at lower surfactant concentration
with increasing chain length. The order of the bind-
ing constant for α-CT with a surfactant has been as-
sumed to follow the following trend: CTPB > TTPB
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