P. K. Das et al.
hydrophobicity and steric rigidity because of the ring struc-
ture) as its end group. A striking improvement in the perox-
etry (MS) data were acquired by the electron-spray ionization (ESI)
technique on a Q-tof-Micro Quadruple mass spectrophotometer from
1
À1 À1
Micromass. H NMR spectra were recorded on
a
Bruker Avance
idase activity of cyt c (k /K =3.8 mm s ) was observed
cat
M
3
00 MHz spectrometer.
in the presence of 10 nm GNP525-7 (Table 2) in a CTAB w/o
microemulsion. This remarkably improved catalytic activity
was 3.6-fold higher than the efficiency in the CTAB-only
system and 200-fold greater than the cyt c activity in water
Synthesis of amphiphiles 1–5 and 7:
CTAB (1) was procured and crystallized thrice by using methanol and di-
ethyl ether. Recrystallized CTAB was without minima in its surface ten-
sion plot.
(
Table 2 and Figure 4). The increase in the emission intensi-
Amphiphiles 2 and 3 were synthesized by using previously reported pro-
tocols.
[
17]
ty of Trp59 in the presence of GNP-7 (relative to that with
GNP-1) revealed further unfolding of the protein (Fig-
ure 5a). Similarly, the fluorescence of Na-Fl was quenched
further because the heme part of the protein was exposed
more effectively due to a greater loss of tertiary structure of
the protein upon interaction with the rigid cholesterol
moiety (Figure 5b). Thus, the greater is the hydrophobicity
and steric crowding around cyt c at the interface of reverse
micelles, the higher is the peroxidase activity of the protein.
Amphiphiles 4 and 5 were prepared by quaternizing dodecylamine and
octylamine, respectively, with iodomethane. The long-chain amine
(
1 equiv) was quaternized with excess iodomethane by using 2.2 equiva-
lents of anhydrous potassium carbonate and a catalytic amount of
18]crown-6 ether in dry dichloromethane for 2 h. The reaction mixture
[
was taken up in ethyl acetate and washed with aqueous thiosulfate solu-
tion and water, respectively. The concentrated ethyl acetate phase was
subjected to ion exchange on a bromide ion-exchange resin to yield the
corresponding bromide salts, 4 and 5. These amphiphilic bromides were
1
crystallized from methanol and diethyl ether. Amphiphile 4: H NMR
(
300 MHz, CDCl
3
, tetramethylsilane (TMS)): d=0.82 (t, 3H), 1.19–1.32
(
br, 18H), 1.69–1.72 (m, 2H), 3.38 (s, 9H), 3.51–3.54 ppm (m, 2H); ESI-
+
MS: m/z calcd for C15
): 228.2686; found: 228.3449; elemental analysis: calcd (%) for
34NBr: C 58.43, H 11.11, N 4.54; found: C 58.61, H 11.43, N 4.72.
H34N (the quaternary ammonium ion, 100%, [M
Conclusion
]
15
C H
A new strategy has been developed to remarkably increase
the peroxidase activity of the mitochondrial protein cyt c by
tuning the hydrophobicity of surface-functionalised GNPs in
compartmentalized systems like reverse micelles. This study
clearly delineates the crucial influence of hydrophobic/steric
crowding in the vicinity of the cyt c on its unfolding and,
thereby, on the catalytic activity of the protein. Incorpora-
tion of hydrophobically surface-modified GNPs into the
water pool of the reverse micelles enhanced the extent of
perturbation of the tertiary structure of cyt c. Inclusion of
GNPs capped with a cholesterol-based amphiphile resulted
in an approximately 200-fold increase in the peroxidase ac-
tivity of cyt c. Thus, our present investigation establishes a
correlation between the peroxidase activity of cyt c and the
modulated hydrophobicity in its vicinity within the reverse
micelle. This study will help in understanding the peroxida-
tive function of cyt c in different cellular processes and also
creates new avenues for using cyt c as an industrial catalyst.
1
Amphiphile 5: H NMR (300 MHz, CDCl
1.37 (br, 10H), 1.65–1.75 (m, 2H), 3.45 (s, 9H), 3.57–3.60 ppm (m, 2H);
ESI-MS: m/z calcd for C11 26N (the quaternary ammonium ion, 100%,
M ]): 172.2060; found: 172.2394; elemental analysis: calcd (%) for
26NBr: C 52.38, H 10.39, N 5.55; found: C 52.54, H 10.43, N 5.31.
3
, TMS): d=0.87 (t, 3H), 1.26–
H
+
[
11
C H
Amphiphile 7 was synthesized by first coupling cholesteryl chloroformate
with one end of the diamine (2,2’-(ethylenedioxy)bis(ethylamine)) in dry
dichloromethane with catalytic amounts of dry triethylamine. The di-
chloromethane solution of the cholesteryl chloroformate was added drop-
wise to the diamine solution under cold conditions (0–58C). After com-
plete addition, the solution was stirred for 8 h at room temperature and
then washed with water and brine to remove the excess base and excess
diamine. The dichloromethane part was evaporated on a rotary evapora-
tor and a solid product was obtained. The product was purified by using
column chromatography with chloroform/methanol as the eluent. The
primary amine (1 equiv) obtained was quaternized with excess iodome-
thane by using 2.2 equivalents of anhydrous potassium carbonate and a
catalytic amount of [18]crown-6 ether in dry dichloromethane for 2 h.
The reaction mixture was taken up in ethyl acetate and washed with
aqueous thiosulfate solution and brine, respectively. The concentrated
ethyl acetate part was subjected to ion exchange on a bromide ion-ex-
1
change resin to yield the pure bromide. Amphiphile 7: H NMR
(
300 MHz, CDCl
3
, TMS): d=0.67–2.33 (m, 43H), 3.32 (s, 9H), 3.40–3.66
(
m, 12H), 3.99 (m, 1H), 5.35 ppm (br, 1H); ESI-MS: m/z calcd for
Experimental Section
+
37 67 2 4
C H N O (the quaternary ammonium ion, 100%, [M ]): 603.5095;
found: 603.4586; elemental analysis: calcd (%) for C37
67 2 4
H N O Br: C
Materials: Cytochrome c (oxidized) from horse heart, all amino acids,
silica gel of 60–120 mesh, n-hexadecyl amine, n-dodecyl amine, n-octyl
amine, N,N-dicyclohexylcarbodiimide, 4-N,N-(dimethylamino)pyridine,
64.99, H 9.88, N 4.01; found: C 65.13, H 10.06, N 4.38.
Preparation of different sized GNPs capped by 1:
Different sized GNPs were synthesized according to the previously re-
N-hydroxybenztriazole, iodomethane, NaBH
4
, triethylamine, solvents and
(30 wt% solu-
[11d]
ported protocol.
all other reagents were procured from SRL, India. HAuCl
4
’
Synthesis of GNP510: An aqueous solution (5 mL) containing 0.5 mm
tion), hemin, cholesteryl chloroformate and 2,2-(ethylenedioxy)bis(ethyl-
amine) were purchased from Sigma–Aldrich chemical company, USA.
Milli-Q water was used throughout the study. Analytical grade CTAB
was purchased from Spectrochem, India. Pyrogallol was obtained from
Qualigens Fine Chemical Company, India. Trisodium citrate was ob-
tained from Merck, India. Hydrogen peroxide (30%, w/v solution) was
purchased from Ranbaxy, India. Thin layer chromatography was per-
formed on Merck precoated silica gel 60-F254 plates. Amberlyst A-26 bro-
mide ion-exchange resin was obtained from Sigma–Aldrich chemical
company. A Perkin–Elmer Lambda 25 spectrophotometer was used to
record UV/visible absorption spectra. Fluorescence spectra were record-
ed on a Varian Cary Eclipse luminescence spectrometer. Mass spectrom-
HAuCl
NaBH
4
and 100 mm CTAB (1) was prepared. Ice-cold, freshly prepared
solution (0.1m, 500 mL) was added to this solution with stirring to
4
attain a final NaBH
turned pink after the addition of NaBH
tion. The GNP solution showed a surface plasmon resonance at 510 nm
GNP510) and the diameter (d) of the nanoparticles was found to be 3–
nm from TEM images.
Synthesis of GNP525: An aqueous solution (5 mL) containing 0.5 mm
4
concentration of 10 mm. The solution immediately
4
, which indicated GNP forma-
(
5
HAuCl
NaBH
4
and 100 mm CTAB (1) was prepared. Ice-cold, freshly prepared
solution (0.1m, 150 mL) was added to this solution with stirring to
4
4
attain a final NaBH concentration of 1.5 mm. The SPR peak of the solu-
&
8
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ÝÝ
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