Influence of Amine-Based Cationic Gemini Surfactants on Catalytic Activity of α-Chymotrypsin

Article abstract of DOI:10.1002/kin.21032

The α-chymotrypsin activity was tested in aqueous media with the presence of novel cationic amine–based gemini surfactant, with different spacer chain lengths and head group size, and also compared with the cationic cetyltrimethylammonium bromide (CTAB) a

Full text of DOI:10.1002/kin.21032

Influence of Amine-Based
Cationic Gemini Surfactants
on Catalytic Activity of
α-Chymotrypsin
1
,2
2
2
SANTOSH KUMAR VERMA, BHUPENDRA KUMAR GHRITLAHRE, KALLOL K GHOSH,
RAMESHWARI VERMA,1 SHEKHAR VERMA, XIUJIAN ZHAO
,2
3
1
1State Key Laboratory of Silicate Materials for Architectures, Wuhan University of Technology, No. 122, Luoshi Road,
Wuhan, 430070, P. R. China
2School of Studies in Chemistry, Pt. Ravishankar Shukla University, Raipur, 492010 (C.G.), India
3SSTC-SSGI-Faculty of Pharmaceutical, Science, Junwani, Bhilai 490020 (C.G.), India
Received 22 December 2015; revised 4 August 2016; accepted 6 August 2016
DOI 10.1002/kin.21032
Published online in Wiley Online Library (wileyonlinelibrary.com).
ABSTRACT: The α-chymotrypsin activity was tested in aqueous media with the presence of novel
cationic amine–based gemini surfactant, with different spacer chain lengths and head group
size, and also compared with the cationic cetyltrimethylammonium bromide (CTAB) and cetylt-
riphenylphosphonium bromide (CTPB) surfactants and aqueous buffer only. The p-nitrophenyl
acetate (PNPA) hydrolysis rate was monitored in the presence of the surfactant concentration
at 30°C. Most of these gemini surfactants gave higher catalytic activity as compared to cationic
CTAB and CTPB. The highest superactivity was measured in the presence of gemini 16-12-16,
[dodecanediyl-1,12-bis(cetyldimethylammonium bromide)] surfactant at pH 7.5. The catalytic
reaction follows the Michaelis–Menten mechanism. The catalytic rate constants, kcat, show the
same profile that the catalytic affinity; KM being enhanced with increasing space chain length.
The results are favorable for considering that the amine-based gemini surfactant influences
more than both the aqueous and cationic micellar media. ꢀC 2016 Wiley Periodicals, Inc. Int J
Chem Kinet 1–6, 2016
INTRODUCTION
surfactants affect the kinetic performance of enzymatic
reactions either below or above the critical micelle con-
centration (CMC). α-CT is more active in micellar
systems than in aqueous/buffered solutions [2,3]. This
significant increase in activity of α-CT due to the pres-
ence of micellar systems is called superactivity. Many
investigations have been devoted to α-CT superactiv-
ity using surfactant micelles [4], ionic liquid [5], mi-
croemulsions [6], reverse micelles [7], polyamines [8],
Serine protease enzyme α-chymotrypsin (α-CT) has
attracted a great deal of attention in biomedical and
bioengineering fields because of its catalytic activity
under aqueous conditions [1]. The aqueous solutions of
Correspondence to: S. K. Verma; e-mail: vermasantosh08@
gmail.com.
ꢀ
2016 Wiley Periodicals, Inc.
C

2
VERMA ET AL.
Scheme 1 Systematic representation of reaction system and chemical structure of surfactants.
and organic solvents [9], here designated as micellar
enzymology.
In previous works, we have reported that the
catalytic activity of α-CT on the hydrolysis of p-
nitrophenyl acetate (PNPA) strongly depends on re-
action medium such as oil-in-water microemulsions
[6], organic cosolvents [15], cationic surfactant me-
dia [2,3], and reverse micelles [7]. We reported that
the enzyme–surfactant interaction can lead to a loss of
enzymatic activity or superactivity, effects that can be
dependent on the surfactant concentration and charac-
teristics. The activity of the α-CT can be increased by
changing the head group hydrophilicity, size, geome-
try, nature of counterion, and hydrophobic tail length
of the surfactants.
A new class of surfactants is referred as the second-
generation surfactants [10]. They consist of two sets
of polar head groups and hydrophobic alkyl chains
per molecule linked together covalently at or near
the head groups by a third moiety called a spacer,
as schematically represented in Scheme 1. Cationic
gemini surfactants derived from quaternary ammonium
salts are able to form soluble mixed aggregates; ow-
ing to their dimeric structure, they demonstrate lower
CMC, greater capability in surface tension reduction,
and well-solubilizing capacity [11,12]. Apart from
physicochemical properties, they have better applica-
tions as additives in antiseptics, hair conditioners, skin
and eye irritation-free cosmetics, and personal care
cosmetic products [13]. Therefore, because of these
significant and beneficial applications of gemini sur-
factants, herein we studied the influence on α-CT activ-
ity. The micellization and micellar growth of the gemini
surfactants in the presence of medium-chain length al-
cohols have been investigated by Moya et al. [14]. They
suggest that the presence of alcohol in the micellar so-
lution can strongly influence the micellar behavior and
decrease the average micellar aggregation.
The present novel study has been carried out to
investigate the α-CT activity in the presence of gem-
ini surfactants with varying spacer chain lengths and
head group sizes. The influence of gemini surfac-
tants on α-CT is compared with cationic cetyltrimethy-
lammonium bromide (CTAB) and cetyltriphenylphos-
phonium bromide (CTPB) surfactants. The planned
reaction scheme for the hydrolysis of PNPA cat-
alyzed by α-CT in the presence of cationic and
gemini surfactants, at pH 7.50 and 30°C, is pre-
sented in Scheme 1. The gemini surfactants used for
−
investigation are (a) 16-3-16, 2Br [propanediyl-1,
International Journal of Chemical Kinetics DOI 10.1002/kin.21032

INFLUENCE OF AMINE-BASED CATIONIC GEMINI SURFACTANTS ON α-CHYMOTRYPSIN
3
3
-bis(cetyldimethylammonium bromide)]; (b) 16-4-
−
16, 2Br [butanediyl-1,4 bis(cetyldimethylammonium
−
bromide)]; (c) 16-6-16, 2Br
[hexanediyl-1,6-
bis(cetyldimethylammonium bromide)]; (d) 16-10-16,
−
2
Br [decanediyl-1,10-bis(cetyldimethylammonium
−
bromide)]; (e) 16-12-16, 2Br [dodecanediyl-1,12-
bis(cetyldimethylammonium bromide)]; (f) 16-4-16
MEA, 2Br [butanediyl-1,4-bis (hydroxyethylmethyl-
−
Scheme 2 Synthesis of gemini surfactants.
hexadecylammonium bromide)]; and (g) 16-6-16
MEA, 2Br [hexanediyl-1,6-bis(hydroxyethylmethyl
−
hexadecyl- ammonium bromide)]. The kinetic param-
eters, catalytic rate constants, kcat, and Michaelis–
Menten, KM, were calculated and discussed. The re-
sult of the present work may help in the understanding
the influence of gemini surfactant on α-CT activity in
micellar enzymology.
pH 4.7 (0.1 M CH3COOH and 0.1 M CH3COONa)
immediately before their use for the experiments. To
improve the solubility of a substrate, the PNPA was
prepared in pure ethanol 100% (v/v). For a reaction
mixture, Tris buffer pH was fixed at 7.50 using 0.1 M
hydrochloric acid.
EXPERIMENTAL
Materials
Enzyme Kinetics Spectra
All the spectral measurements were performed us-
ing a Varian Cary 50 UV–visible spectrophotometer
equipped with a Peltier temperature controller unit at
α-CT (EC 3.4.21.1; type-II from bovine pancreas,
molecular weight 25 kDa, and an isoelectric point pl
30°C. The rate of substrate PNPA hydrolysis, catalyzed
8.8) and CTAB were procured from Sigma (St. Louis,
by α-CT, was measured in aqueous and micellar so-
lutions following the formation of p-nitrophenoxide
MO) and used without further purification. CTPB was
prepared in the laboratory of Prof. R. M. Palepu,
St. Francis Xavier University, Antigonish, Canada,
and received as a gift. The substrate (PNPA) was
obtained from Fluka (Steinheim, Switzerland). Tris–
−
−1
−1
ion (PNP ) at 400 nm (є = 12,000 M cm ). All
the reaction mixture was maintained at pH 7.50 using
10 mM Tris/HCl buffer. The initial reaction rate, V0,
−
was determined from the slope of the PNP concen-
tration on time profiles using enzyme kinetics software
(
hydroxymethyl)amino methane (Tris) (pKa 8.3) and
HCl were obtained from Qualigens Fine Chemicals
Mumbai, India).
(
Varian).
(
Synthesis of Gemini Surfactants
The cationic gemini surfactants (16-3-16, 2Br ; 16-
RESULTS AND DISCUSSION
−
−
−
−
Effect of Head Group Size, Spacer Chain
Length, and Substituted Hydroxyl Group
4
-16, 2Br ; 16-6-16, 2Br ; 16-10-16, 2Br ; 16-
−
12-16, 2Br ) used for this investigation were syn-
thesized by 48 h refluxing of tetramethylene-1,4-
bis(cetyldimethylammonium bromide) in dry ethanol
at 80°C and recrystallized by using appropriate vol-
ume of hexane/ethyl acetate mixture. The purity of
The kinetic behavior of enzymatic reaction can be af-
fected by aqueous surfactant medium having specific
condition below and above the CMC. The CMC of
monomeric and cationic gemini surfactant in aqueous
solution is reported in Table I [20–25]. In compari-
son to the Michaelis constant, KM reported in Table I
with respect to aqueous buffer, the PNPA hydrolysis is
1
the surfactants was characterized by using H NMR
13
and C NMR [16,17] (Scheme 2). The hydroxyl-
−
substituted gemini surfactants (16-4-16 MEA, 2Br ;
1
−
6-6-16 MEA, 2Br ) were prepared by the method
2.85 and 1.42 times higher for CTAB and CTPB, re-
reported by Sharma et al. [18].
spectively, whereas the catalytic constant, kcat, remains
similar for all three cases, indicating the competitive
catalysis type of enzymatic reaction. On the other hand,
the kcat/KM ratio is higher for CTPB than the CTAB,
which is due to the specific interactions between the
surfactant and the α-CT as well as increased head group
size of the CTPB. Here, the head group size of CTPB
Solution Preparation
The solution preparation and reaction were carried out
by following our previous report [19]. In brief, the
α-CT solution was freshly prepared in acetate buffer
International Journal of Chemical Kinetics DOI 10.1002/kin.21032

4
VERMA ET AL.
Table I Comparison of Results of α-CT Catalyzed Hydrolysis of PNPA in Micellar Medium
kcat (s 1)
−
10 KM (M)
−5
10 kcat /KM (M
3
−1 −1
)
s
S. No.
Surfactant
CMC (mM)
1
2
3
4
5
6
7
8
9
Aqueous buffer
CTAB
-
0.3 ± 0.1
0.3 ± 0.3
0.3 ± 0.1
0.5 ± 0.1
0.5 ± 0.1
0.5 ± 0.1
0.5 ± 0.1
0.6 ± 0.1
0.3 ± 0.1
0.3 ± 0.1
7 ± 1
20 ± 6
10 ± 1
9.5 ± 2
10 ± 1
10 ± 10
12 ± 2
20 ± 2.8
10 ± 6
15 ± 1
4.2
1.5
3
5.2
5
5
4
3
3
a
1.00
0.16
0.03
0.045
0.045
0.028
0.022
0.0025
a
CTPB
16-3-16, 2Br
16-4-16, 2Br
16-6-16, 2Br
16-10-16, 2Br
16-12-16, 2Br
−
a
−
b
−
−
−
c
d
e
−
−
f
16-4-16 MEA, 2Br
16-6-16 MEA, 2Br
f
1
0
0.0037
2
Reaction condition: [α-CT] = 13 μM, [PNPA] = (0.026, 0.065, 0.13, 0.196 and 0.26) mM, [surfactant] = 1 mM (for S. No. 2-10),
Tris/HCl] = 10 mM, pH 7.50, ε = 12,000 M cm , Temperature = 30°C.
From [20]; From [21]; From [22]; From [23]; From [24]; From [25].
−
1
−1
[
a
b
c
d
e
f
greatly enhances its interfacial area and also increased
space between two head groups as compared to the
CTAB, further indicating the higher concentration of
α-CT and PNPA inside the interfacial region [2]. This
interfacial enhancement plays a crucial role in higher
catalytic activity in the case of CTPB.
and in the presence of cationic surfactant CTAB and
CTPB as reference to compare the activity of gemini
surfactant. The result measured under α-CT catalyzed
hydrolysis of PNPA in the presence of gemini surfac-
tants is plotted as a function of initial PNPA concentra-
tion, which is hyperbolic in nature, shown in Fig. 1a.
The enzymatic reaction follows Michaelis–Menten
type kinetics using Lineweaver–Burk treatment, which
was fairly linear (Fig. 1b), and shows competitive catal-
ysis with surfactants. The data presented in Figs. 1a and
1b show that the increase in the spacer chain length en-
hances the rate of the reaction over all the PNPA con-
centration range considered. In the similar sense, the
data obtained employing PNPA as a substrate follow
the same pattern for aqueous buffer.
Cationic gemini surfactants hold two heads and two
tail groups connected by a spacer. As the spacer chain
length increases (s = 3–12), it twists toward the alkyl
chains, further showing a strong hydrophobic inter-
face between the alkyl tails of the surfactants lead-
ing to boost the micelle formation, therefore lowering
the CMC values [23]. Although difference between
the spacer chain length modifies the molecular shape
and physicochemical parameter such as CMC of gem-
ini micellar aggregates, these changes affect the cat-
alytic rate constants for the PNPA hydrolysis. Ghosh
et al. [26] reported that the aggregation numbers and
dimensions of micelles decrease when spacer chain
length increases. From the above interesting character-
istics of micelles, catalytic behavior significantly en-
hances with increasing spacer chain length.
As clearly revealed in Table I, in general the
Michaelis constant, KM, of α-CT has been observed
to slightly increase with the increasing spacer chain
−
−
length from 16-3-16, 2Br to 16-12-16, 2Br of gem-
ini surfactants whereas the catalytic activity, kcat, re-
mains unchanged. Similar trends of α-CT activity are
−
stated for 16-4-16 MEA, 2Br and 16-64-16 MEA,
2Br head group of gemini surfactants. In compar-
−
The substituted hydroxyl groups of cationic gemini
surfactants affect the micellization and micellar proper-
ties with noticeable changes in micellar size, polar head
group size, and its counterion [27]. The hydroxyl group
ison to the aqueous buffer, the micelle-bound α-CT
showed higher catalytic affinity in case of the spacer
chain length and head group size of gemini surfac-
tant as well as for CTAB and CTPB. The results are
the most prominent in the spacer chain length from
−
present in gemini surfactant (16-3-16 MEA, 2Br and
1
−
6-4-16 MEA, 2Br ) enhances the solubility of the
− −
16-3-16, 2Br to 16-12-16, 2Br , and its KM value in-
surfactant, leading to the greater tendency to form mi-
celles on the way to improve the catalytic behavior of
surfactant.
−
5
−5
creases from 9.5 ± 2 × 10 M to 20 ± 2.8 × 10 M,
respectively. On the other hand, the catalytic efficiency,
3
3
−1 −1
s
kcat/KM decreases from 5.2 × 10 to 3 × 10 M
−
−
for 16-3-16, 2Br to 16-12-16, 2Br gemini surfac-
tants, respectively. The same trend was followed in the
Influence of Gemini Surfactants on the
α-CT Activity
−
case of head group 16-4-16 MEA, 2Br and 16-6-16
MEA, 2Br . As seen in Table I, the smaller values of
KM (16-3-16, 2Br to 16-12-16, 2Br ) indicate that the
−
The rate of hydrolysis of PNPA catalyzed by α-CT was
measured initially in aqueous buffer (10 mM Tris–HCl)
−
−
International Journal of Chemical Kinetics DOI 10.1002/kin.21032

INFLUENCE OF AMINE-BASED CATIONIC GEMINI SURFACTANTS ON α-CHYMOTRYPSIN
5
Figure 1 (a) The relationship between the initial reaction rate (per enzyme) and the analytical concentration of PNPA in the
presence of gemini surfactant to varying spacer chain length. (b) Lineweaver–Burk plot for the α-CT catalyzed hydrolysis of
PNPA: ◦, aqueous; +, 16-3-16; ꢀ, 16-4-16; ♦, 16-6-16; •, 16-10-16; ꢀ, 16-12-16.
enzyme and substrate are tightly bound and form the
enzyme-substrate (ES) complex more quickly than the
spacer chain length 16-12-16, 2Br gemini surfactant
having higher KM values. From the overall analysis
of Table I, the values of KM affects only and not kcat,
since high concentration of substrate PNPA displaces
surfactant from the α-CT. On the other hand, the surfac-
tant concentration overcome by increasing the PNPA
concentration, in this case the PNPA outcompetes the
surfactant in binding to the α-CT.
fect is that the enzyme–surfactant interactions depend
on surfactant head group size, hydrophobic content,
and protein conformation [2,19]. The increase in α-CT
activity is strongly related to the spacer alkyl chain
length as well as the hydrophobic tail part of surfac-
tant. Thus, the dependence on the α-CT superactivity
on the PNPA is credited to hydrophobic nonspecific in-
teractions between the surfactants and the α-CT active
site, which created additional hydrophobic microenvi-
ronment, and resulting in an increase in nucleophilicity
in the catalytic active site [30].
−
Bianconi et al. [28] reported that the enzyme–
micelle interaction takes place due to an increase
in the number of micelles in solution and therefore
the decrease in the concentration of free substrate.
The dependence on kcat and KM with concentration
of surfactant remained analogous to that of the cat-
alytic efficiency. Apart from that the catalytic effi-
ciency increases above the higher concentration of
CMC and also with longer tail of surfactants, respec-
tively [2,6,15]. Larger values of kcat denote a higher
activity of the enzyme reported in Table I. The KM val-
ues represent the affinity with the α-CT for the PNPA.
Smaller values of KM indicate that the enzyme and sub-
strate are tightly bound and form the ES complex more
quickly. Larger values of the KM constant indicate that
the components are loosely bound and form the ES
complex more slowly. Apart from that the enzyme, α-
CT and substrate PNPA are water soluble; therefore,
the enzyme–substrate reaction takes place exclusively
in the aqueous phase.
As the head group size of gemini surfactant (16-3-
−
−
16 MEA, 2Br and 16-4-16 MEA, 2Br ) increases,
the results show higher catalytic affinity than aqueous
buffer because of increasing interfacial area of surfac-
tant enhances the space between two heads groups.
This free space between two head group permits the
solubilization of α-CT at the interfacial region [2].
Apart from that, the solubility of gemini surfactant
−
−
(16-3-16 MEA, 2Br and 16-4-16 MEA, 2Br ) in-
creases in the presence of the hydroxyl group. Owing to
above-mentioned interfacial enhancement, the contact
between α-CT concentration and the available PNPA
increases, showing higher catalytic activity. The 16-
−
4-16 MEA, 2Br shows the best results than 16-3-16
MEA, 2Br in terms of catalytic efficiency. A recent
−
study on α-CT-gemini surfactant systems shows the
significant catalytic efficiency of enzyme.
Enzyme displays duality because of the alkyl chain
length and hydrophilic properties of the amino acids,
which cause enzyme to interact with amphiphilic
molecules such as gemini surfactants [29]. Based on
the results presented in Table I, the influence of gem-
ini surfactant on α-CT is the most prominent on the
spacer chain length as well as head group than aque-
ous and cationic micellar media. The reason for this ef-
CONCLUSION
In conclusion, the effect of amine-based gemini surfac-
tant on the α-CT activity catalyzed hydrolysis of PNPA
is prominent than reference aqueous buffer, CTAB and
CTPB. All the catalytic result shows the Michaelis–
Menten competitive type of enzymatic reaction. Super-
activity is observed in the presence of all the gemini
International Journal of Chemical Kinetics DOI 10.1002/kin.21032

6
VERMA ET AL.
surfactants used and highest catalytic affinity, KM is
observed for 16-12-16, 2Br surfactant. The proposed
13. Shukla, D.; Tyagi, V. K. J Oleo Sci 2006, 55, 381–390.
14. Graciani, M. M.; Rodr ´ı guez, A.; Mart ´ı n, V. I.; Moya,
M. L. J Colloid Interface Sci 2010, 342, 382–391.
−
investigation may be helpful in designing a surfactant
to manipulate α-CT activity for biomedical and bio-
engineering application. Therefore, it is essential to
study the α-CT–surfactant interactions at a molecular
level.
1
1
1
1
5. Verma, S. K.; Ghosh, K. K. Indian J Chem, Sect A 2010,
49, 1041–1046.
6. Moore, S. A.; Glenn, K. M.; MacDonald, A. M.; Palepu,
R. M. Colloid Polym Sci 2007, 285, 543–552.
7. Rodriguez, A.; Moreno-Vargas, M. D. G.; Moya, M. L.
J Phys Chem B 2008, 112, 11942–11949.
8. Sharma, V.; Borse, M.; Aswal, V. K.; Pokhriyal, N. K.;
Joshi, J. V.; Goyal, P. S.; Devi, S. J Colloid Interface Sci
Financial support of this work by Council of Scientific and
Industrial Research, New Delhi (CSIR Project Ref. No. 01
2004, 277, 450–455.
(2143)/07/EMR-II) is gratefully acknowledged. Authors are
1
2
2
2
9. Verma, S. K.; Ghosh, K. K.; Verma, R.; Xiang, W.; Li,
N.; Zhao, X. Colloids Surf A 2015, 470, 188–193.
0. Verma, S. K.; Ghosh, K. K. J Surfactants Deterg 2011,
also grateful to UGC SAP Project of Pt. Ravishankar Shukla
University, Raipur, India.
14, 347–352.
1. Tikariha, D.; Ghosh, K. K.; Barbero, N.; Quagliotto, P.;
Ghosh, S. Colloids Surf A 2011, 381, 61–69.
2. Mohammad, R.; Schulz, P. C.; Khan, I. A.; Kabir-ud-Din
J Solution Chem 2013, 42, 2310–2328.
BIBLIOGRAPHY
1
2
. Sintra, T. E.; Ventura, S. P. M.; Coutinho, J. A. P. J Mol
Catal, B: Enzym 2014, 107, 140–151.
. Ghosh, K. K.; Verma, S. K. Int J Chem Kinet 2009, 41,
23. Kumar, B.; Tikariha, D.; Ghosh, K. K.; Quagliotto, P. J
Mol Liq 2012, 172, 81–87.
3
77–381.
24. Tikariha, D.; Kumar, B.; Ghosh, S.; Tiwari, A. K.;
Saha, S. K.; Barbero, N.; Quagliotto, P.; Ghosh, K. K. J
Nanofluids 2013, 2, 316–324.
3
4
. Verma, S. K.; Ghosh, K. K. Kinet Catal 2011, 52, 6–10.
. Ghosh, K. K.; Verma, S. K. Indian J Biochem Biophys
2
008, 45, 350–353.
. Kumar, A.; Venkatesu, P. Chem Rev 2012, 112, 4283–
307.
. Verma, S. K.; Ghosh, K. K. Colloids Surf A 2010, 368,
54–158.
. Verma, S. K.; Ghosh, K. K. J Chem Sci 2013, 125, 875–
82.
25. Tikariha, D.; Singh, N.; Satnami, M. L.; Ghosh, K. K.;
Quagliotto, P.; Barbero, N. Colloids Surf A 2011, 411,
1–11.
26. Ghosh, K. K.; Kolay, S.; Bal, S.; Satnami, M. L.;
Quagliotto, P.; Dafonte, P. R. Colloid Polym Sci 2008,
286, 293–303.
27. Kuperkar, K. C.; Mata, J. P.; Bahadur, P. Colloids Surf
A 2011, 380, 60–65.
28. Celej, M. S.; Dandrea, M. G.; Campana, P. T.; Fidelio,
G. D.; Bianconi, M. L. Biochem J 2004, 378, 1059–
1066.
5
6
7
8
9
4
1
8
. Kurinomaru, T.; Kameda, T.; Shiraki, K. J Mol Catal, B:
Enzym 2015, 115, 135–139.
. Secundo, F.; Barletta, G. L.; Parini, G.; Roda, G. J Mol
Catal, B: Enzym 2012, 84, 128–131.
1
1
0. Zana, R. Adv Colloid Interface Sci 2002, 97, 205–253.
1. Menger, F. M.; Keiper, J. S. Angew Chem, Int Ed 2000,
29. Miller, R.; Fainerman, V. B.; Makievski, A. V.; Kragel,
J.; Grigoriev, D. O.; Kazakov, V. N.; Sinyachenko,
O. V. Adv Colloid Interface Sci 2000, 86, 39–82.
30. Morrisett, J. D.; David, J. S. K.; Pownall, H. J.; Gotto,
A. M. Biochemistry 1973, 12, 1290–1299.
3
9, 1906–1920.
2. Han, Y.; Wang, Y. Phys Chem Chem Phys 2011, 13,
939–1956.
1
1
International Journal of Chemical Kinetics DOI 10.1002/kin.21032