Effect of Alcohols and Organic Solvent on
Neocarzinostatin and its stability
Christopher G. Sudhahar, Der-Hang Chin*
Department of Chemistry, National Chung Hsing University, Taichung,
Taiwan, R.O.C.
Introduction:
Neocarzinostatin (NCS) is a potent enediyne antitumor
antibiotic complex containing non-protein chromophore (NCS-Chr), which
is tightly1 (kd = 7.0 X 10-10 M), non-covalently1,2 and specifically3 bound to
an apoprotein (apo NCS, 113 amino acids having an all -sheets
polypeptide). The photo4- and thermal labiality of chromophore with an
unusual structure is responsible for DNA cleavage and its greatly stabilized
by binding with its apoprotein. Though the crystal structure and secondary
structure is known in the literature, the binding and the type of interactions
between the apo-NCS and NCS-Chr are not thoroughly studied and the
interactions which leads to stability is an interesting aspects in terms of
molecular recognition and protein transport. Apo-NCS forms a hydrophobic
cluster consisting of the side chains of Trp39, Leu45, Phe52, Phe76 and
Phe78, Which are present inside the barrel.6 Based on the NOE’s
observation, the interaction between the hydrobophic cluster and NCS-Chr
with two acetylenic functions and the reactive epoxide group, which result
the protection and stability of the chromophore. A hydrophobic pocket with
approximate dimensions of 12 x 9 x 9 Å is formed by the four-stranded βsheet and two loops7. ApoNCS accepts the naphthoate moiety of NCS-Chr
deeply into this pocket, which may be essential for specific and strong
binding and this is in consistent with the binding of β –napthol1 to apoNCS8
and release in the presence of protein denaturants, such as SDS, Urea and 2propanol are reported in the literature1 and no other report is available on
interaction between NCS-Chr and apoNCS. A pre-requiste to this is to have
some studies. The literature is scant with respect to such model studies due
to the problem associated with the light and heat labiality of the
chromophore. In this context, we proposed to perform the studies which has
been known for some time the protein stability is affected by certain salt
species primarily at higher concentration in the same order as the lyotropic
or Hofmeister series. For example strongly hydrated anions such as SO42- are
the mostly stabilizing whereas weakly hydrated anions such as ClO4- and
CNS- are destabilizing with Cl- and Br-. These salt effects on protein stability
are generally taken as evidence for hydrophobic interaction10. In addition,
several DNA- intercalating drugs and α-tocopherol strongly inhibits the
invitro reaction, whereas alcohols and organic solvents greatly stimulate the
invitro reaction.12 In an attempt to understand interaction between the
chromophore and apoNCS, we have studied the effects of several alcohols,
organic solvents and salts in the release of chromophore and it also leads to
control its release for interaction with the target DNA, which opens up a
brighter scope for sustained drug delivery system and also . Our perivious
results say that chromophore release does not require major conformational
change. Which indicate that the carrier apoNCS should be stable for the
extreme conditions for its action. Most bacteria including Streptpomyces
cannot survive at very high temperature and the chromophore too. Thus it
seems not necessary for the nature to build up a heat stable protein to protect
the chromophore. To protect the chromophore from this extreme
environment, apoNCS need to be more stable in this denaturants and organic
solvents. ApoNCS is stable probably against chemical reduction of disulfide,
unfolding by hydrophobic interactions, etc. Here the Stability refers to the
maintenance of a defined functional state under extreme condition. The
problem of protein stability is closely related to that of protein denaturation,
because it is necessary to destroy the native structure of the protein in order
to obtain information concerning its stability. Thus, many different organic
solvents have been used for studying the denaturation of apo NCS proteins.
In order to understand the stability of apo NCS, we have studied the
effects of several alcohols, other organic solvents and denaturants on the
stability of the apo protein, which was compared to other reported other
proteins. Our results suggest that the stability of the apo NCS was quiet high,
which make more stable environment for the chromophore and its function.
MATERIALS AND METHODS:
Neocarzinostatin was obtained as a gift from Kayaku Laboratories Ltd
(Tokyo) Japan. methanol, ethanol, isopropanol, acetonitrile and 1-anilino-8napthalene sulphonic acid were obtained from Merck Co., Germany and
Sigma Co., USA. All protein solutions were prepared in Milli-Q water and
the pH of the unbuffered protein solutions were adjusted to 5.0 before
making all spectroscopic measurements. The holoNCS samples were
prepared in 5mM ammonium acetate buffer. The apoNCS samples were
prepared in 2.67mM ammonium acetate, 20mM sodium acetate buffer. All
the measurements were made at 252C and the concentration of alcohols
and organic solvent solutions are reported as percentage volume by volume.
Circular Dichroism :
All circular dichroism (CD) measurements were carried out on a Jasco
spectropolarimeter, model J715, equipped with an interfaced personal
computer. The instrument was calibrated with ammonium d-10campoursulphonate. . The results are expressed as mean residue ellipticity
[], which is defined as []=[]obs / 113 x l x c, where obs is the observed
ellipticity in degrees, c is the concentration in dmol / l, l is the length of the
light path in cm and constant 113 is the number of amino acid residues in the
protein. All CD spectra were measured with appropriate concentrations
using a 1 mm and 10 mm quartz cell. All samples were corrected for
appropriate solvent blanks. All protein solutions were made in milli-Q water.
Fluorescence Spectroscopy :
A Hitachi model F-4500 fluorescence spectrofluorimeter was used for
measuring the effect of alcohols on the tryptophan fluorescence of NCS. A
set of solutions (total volume, 250µl) containing requisite amounts (v/v) of
alcohols were mixed with 10-20µm (final concentration) apoNCS. For all
these measurements a rectangular micro cell (4x10mm) was used. The
fluorescence emission spectra were recorded at the light path of 10mm
between 290 and 450nm with an excitation wavelength of 280nm. The
solvent and buffer backgrounds were used to correct all the spectra. All the
measurements were made at 252C
Anilinonapthalene Sulphonic Acid Binding studies:
Anilinonapthalene Sulphonic Acid (ANS) binding studies were
carried out on a Hitachi F-4500 spectrofluorimeter. A set of solutions (total
volume, 300l) was prepared as described previously except that apoNCS
concentration was increased to 50µm due to lower sensitivity. ANS was
added at 10:1 molar ratio to apoNCS. The fluorescence spectra were
recorded between 375 and 600 nm using an excitation wavelength of 355
nm. Appropriate background corrections were made in all spectra
Kinetic Study:
The kinetic studies were performed on a Hitachi-F4500
spectrofluorimeter for studying the release of NCS-Chr from the holo-NCS.
The reactions were performed in the presence of 2.5-5m of holoNCS (final
Concentration) in 100mM (final Concentration) of Tris-HCl (pH 7.0) buffer
was 5mM (final Concentration) Glutathione. The rate of release of NCS-Chr
was initiated by adding alcohols and organic solvents. All the measurements
were made at 252C. Excitation was set at 340nm at the 4mm path length;
the emission was recorded at 440nm at the 10mm light path. A repetitive
Wavelength Scan Mode with the shutter closed between each run was used
to avoid the light induced degradation of NCS-Chr. The wavelength interval
was set at the minimum (10nm, from 434 to 444nm) with the scan rate of
1000nm per min. The total light exposure time to the light-sensitive NCS
sample was thus minimized to 36 s for an experiment that recorded data per
min and monitored for 1 hr. Measurement continued until the change of
emission at 440nm reached a plateau, which indicates that the reaction of the
released chromophore has gone to completion.
The change in [holoNCS] with time was determined by increase of the
fluorescent emission resulting from the thiol-induced drug product. Because
the calibration curve shows a linear relation in the drug concentration range
studied, the measured fluorescent emission in mV was directly converted
into the concentration of the remaining intact chromophore that was bound
to the protein by Equation 1:
[holoNCS] = [holoNCS]0 – (Ft / (F – F0 )) x [holoNCS]0
(Eq. 1)
Where Ft is the measured Fluorescent emission in mV at 440nm at
time t; F0 is the initial reading after alcohol were added; F is the plateau
reading after the reaction was completed.
Hydrophobic measurements :
To studying the relation between the hydrophobic nature and rate of
release chromophore was observed by vapor pressure and Synder’s polarity
index. These values are standard value obtained from the Merck index and
Material Safety Data Sheet (MSDS). Unfortunately, the relative dielectric
constants of the alcohol/water mixer are mostly unavailable. We assumed a
linear dependency of the relative dielectric constant on the concentration of
alcohols (v/v) and estimated by relative dielectric constant of alcohol/water
mixer. Such a simple apporoximation seems to be valid within the
experimental error.22
RESULTS:
Circular Dichroism:
We know that the stability of the protein is a function of
conformational properties of the folded and the denatured states. To study
the conformational property of apo NCS, we tried far UV CD experiment
with wide range of alcohols and organic solvents concentrations. CD spectra
of native apoNCS showed a broad negative signal from 300 to 250nm which
is centered at 270nm and followed by a positive peak around 222nm and a
negative peak centered at 212nm indicative of the secondary structure being
predominantly β-sheet. CD spectra in the far UV region, represent the
secondary structural transitions of the apoNCS. Addition of alcohol is
known to stabilize the helical structure, but to destabilize the tertiary
structure of the protein. In the case of methanol, ethanol and isopropanol up
to 55 to 60% (v/v) appears to have no effect on the backbone conformation.
At 50% acetonitrile concentration the secondary structure starts melting.
Mean Ellipticity Value at 215nm([θ]215) (implies the conformation of
backbone structure) showed that the β-sheet signal obtained from CD spectra
of apoNCS after induction with alcohols and organic solvents has not been
modified. Methanol, ethanol and isopropanol have induced helix structure
for 80,70 and 70 percentage and finally it forms aggregation. But in case of
acetonitrile, it induces only helix in less intensity when compare with other
alcohols and then leads to aggregation. Even at high percentage of alcohols
and organic solvent, apoNCS shows some residual interaction. ApoNCS
does not reach the completely unfolded state at higher concentration of
alcohols and organic solvent. Due to the lower concentration of apoNCS the
aggregation was not observed when higher concentration of isopropanol &
acetonitrile was used. But the CD ellipticity values are random or scattered.
Fluorescence studies
Upon addition of methanol, ethanol, isopropanol and acetonitrile the
change in the tryptophan emission was studied by fluorescence
spectroscopy. When excited at 280nm, the emission maximum occurs at
346nm with slight red shift at higher concentration of methanol, ethanol,
isoporpanol and acetonitrile. Unlike the trifluroethanol, apoNCS emission
maximum increases upon increasing the concentration of alcohols and
organic solvent. Contrasting to the CD spectra, the tryptophan fluorescence
emission increased even at lower percentage of alcohols and organic solvent,
which indicates that the lower percentage of methanol, ethanol, isopropanol
and acetonitrile altered the tryptophan environment by hydrophobic
interaction with the aromatic side chain of the apoNCS but not with the
secondary and tertiary structure of the apoNCS. Which is enough to release
the chromophore from the binding packet.
Anilinonapthalene Sulphonic Acid Binding studies:
It is well known that the ANS is a dye, a fluorescent hydrophobic
probe, which has a stronger affinity for the molten globule like intermediate
than for the protein in the native or fully unfolded state. We therefore carried
out ANS-binding studies with various concentrations of methanol, ethanol,
isopropanol, acetonitrile and untreated sample. Up to 20 to 25% of organic
solvent and alcohols the emission intensity doesn’t change much. But while
using greater than 25% of organic solvent and alcohols, characteristic
changes in the emission intensity by upto 85% was observed. The
wavelength maximum emission (λmax) of the ANS in the presence of the
untreated apoNCS was 506nm. Like the emission intensity, up to 20 to 25%
of methanol, ethanol, isopropanol and acetonitrile the wavelength maximum
emission (λmax) did not change considerably. When we increased the
concentration of organic solvent and alcohols the wavelength shift to 20nm
indicates that the wavelength was shift to the higher energy region. This blue
shift in the ANS emission maximum, accompanied by the steep increase in
its emission intensity, suggests increased accessibility of hydrophobic sites
for ANS binding in the apoNCS treated with alcohols and organic solvents.
Even at 85%, the apoNCS shows greater binding with the ANS, which
proves that even at higher percentage of alcohols the apoNCS has the partial
unfolded state; such a feature is characteristic of the molten globule state.
Kinetic Study:
Upon attack by a thiol (GSH), the enediyne nucleus of chromophore is
cycloaromatized into stable thiol adducts. Further it is known that the free
chromophore (not in the bound form) from holoNCS can react with
glutathione which forms a high fluorescent active product, which has a
characteristic HPLC profile and fluorescence emission (excitation at 340nm
and emission at 440nm). We utilized these findings to carry out few kinetic
studies for the release of chromophore from the holoNCS using different
concentrations of methanol, ethanol, isopropanol and acetonitrile. In the
present study, we monitored the relative fluorescence emission from the
“product 1” to indicate the release of chromophore and found that the rate of
release reached maximum at the higher percentage of alcohols and organic
solvent. Figure 2 shows that the release of NCS-Chr starts from 20% of
ethanol, isopropanol and acetonitrile. But in the case of methanol, the release
of NCS-Chr begins above 40%. The intensity of release was followed the
order given below.
Isopropanol > Ethanol > Acetonitrile > Methanol
Further to study the interaction with the NCS-Chr, we tried some salt
induced kinetic release studies. For that we choose the Na2SO4 and NaClO4
for chromophore release reaction. In NaClO4, the release was occurring
above 5M NaClO4 concentrations. In the case of Na2SO4, NCS-Chr release
was not occur because we already prove that the release of chromophore
from the holo NCS does not require the major conformational changes. 7
Na2SO4 normally stabilize the protein even though the release was not
decrease.
Hydrophobic measurement :
Further to prove the hydrophobic interaction is the major factor not in
the ionic interaction. We choose dielectric constant, vapor pressure and
polarity to explain the hydrophobic nature of the solvent. To studying the
relation between the hydrophobic nature and rate of release chromophore
was observed by vapor pressure, dielectric constant and Synder’s polarity
index. These values are standard value obtained from the Merck index and
Material Safety Data Sheet (MSDS). These physical parameters are constant
for methanol, ethanol, isopropanol and acetonitrile. Vapor pressure and
polarity were indirectly proportional to the hydrophobicity. When polarity
and vapor pressure increase the rate of release decrease, which was shown in
the Fig.5.
Discussion:
One important pharmacological function is that the protein regulates
the drug by proper release of its biologically potent chromophore. To
understand the physiological mechanism of the natural drug delivery system,
it is essential to study how the apoprotein bind to the chromophore and how
the protein releases the tightly bound chromophore to the potential target
DNA. For these many of the NMR7, 13 and X-ray crystal studies14speculate
that the binding was hydrophobic interaction between the chromophore and
hydrophobic packet, which was three aromatic residues, Trp 39, Phe 52 and
Phe 78 at the chromophore- binding site, Trp39 and Phe52 formed a specific
binding pocket for the naphthoate of the chromophore.15 Binding studies
with tritium-labeled proteins revealed that CM-NCS had a diminished ability
to bind to cell surfaces, which indicates that the acidic groups affects the
affinity of the protein for the chromophore.16 Other than this some model
studies of chromophore interaction also interpret this interaction. A release
of the chromophore from NCS has been observed in a substrate-specific
manner as well as in a pH-dependent manner1. β-Napthol completely
released the chromophore at 3mM, while only 3% release was detected at
the same concentration on α-naphthoic acid. There was also a large
difference between D-galactosamine and D-galactose for the chromophore
release at 10-500 mM, indicating the importance of the amino group in the
N-methylfucosamine moiety of the chromophore for the chromophore –
apoprotein interaction.17 Other than this two-dimensional 1H NMR
spectroscopy has been used to investigate the binding site, binding
interactions, and the conformation of 1:1 complex of apoNCS with ethidium
bromide in solution. It may derive from the numerous hydrogen-bonding and
hydrophobic interactions that occur between amino acid residues of the
protein and various functional groups on the chromophore.18
Comparison with apo NCS stability and NCS-Chr release:
As we studied in TFE, the conformational stability of the apo NCS
doesn’t affect the release of the chromophore. Which also observed in the
alcohols and organic solvent. Apo NCS more stable even in the higher
percentage of the alcohols and organic solvent. In methanol, the release was
starts in 40-43% but until the 68-70% methanol the protein maintain its
compactness. The same effects were observed in other alcohols and organic
solvent, which indicate that release the first step then the conformational
change. It clearly indicates that the release of the chromophore is not due to
the denaturation effect. So what should be the major factor to release the
chromophore from its holoprotein ?
Is Hydrophobicity the major factor to release the chromophore?
In our present studies, we further envisage the fact that the interaction
between chromophore and apoNCS is hydrophobic. Which is proved by the
chromophore release kinetic studies using various ranges of organic solvents
and salts. The fact that the methanol which is the highly polar solvent in our
experiment is need in higher percentage (40% Methanol) to elicit the
chromophore release reaction compared to the less polar other organic
solvents like acetonitrile, ethanol and isopropanol, which release the
chromophore at lower percentage (20-25% of organic solvents). The
intensity of release was followed the order given below.
Isopropanol > Ethanol > Acetonitrile > Methanol
This indicates that if the hydrophobicity of denaturant increases the
rate of release will decrease. These results show that the more hydrophobic
nature of the solvent higher is the percentage of release. Luo and Baldwin
studied the mechanism of the helix-induction by TFE and concluded that the
strengthening of the hydrogen bonds is responsible for TFE effects of the
helix formation of the short peptides. It’s highly correlate these results with
effect of alcohols in NCS, which also stablize the ionic interaction
(hydrogen bond). On the other hand the chromophore has released out from
the apo NCS. Which clearly indicate that the stabilization of ionic interction
doesn’t affect the release mechanism. The strength of experiments was
further proved by salt induced chromophore release kinetic data. In the
Hofmeister series, the well known denaturant like NaClO4 which release the
chromophore from its protein counter part in its higher concentration (5M
NaClO4) indicates that the release is not from the ionic interaction but
primarily from its denaturing effects. When a highly polar salt like Na2SO4 is
used in the release reaction, there is a much evident decrease in the release
rate. This is due the fact that plays a role in the stabilization of holoNCS.
This fact strengthens that the binding between the NCS-Chr and apoNCS
was major hydrophobic interaction.
Alcohol-induced chromophore release has been considered to arise
from the low polarity of the solvent, which decreases the hydrophobic
interactions. The same things were observed in the proteins and peptides,
which shows the highly correlation between the extent of conformational
transition with the relative dielectric constant of the several organic solvents
and concluded the importance of the solvent polarity. On the other hand, the
polarity of the solvent is an important factor determining the alcohol effects;
markedly the high potential of some alcohols such as TFE and t-butanol
cannot be explained by the low polarity of the solvent alone.
Kurpin et al studied the alcohol/water mixtures, including the
halogenated alcohols, by small angle X-ray scattering and reported that the
HFIP has a high tendency to form micelle-like assemblies with maximum at
about 30% (v/v). Other alcohol also tends to form micelle-like but much
lesser extent. In aqueous solution of alcohols, alcohols associates so as to
minimize their contact with water, which results in the formation of micellelike assembly with the hydrophobic group inside, although no macroscopic
phase separation takes places. We consider that this characteristic of TFE
and t-butanol may be the reason for their unexpectedly high potential in
stabilizing a- helix and destabilizing the hydrophobic interaction between the
NCS-chr and apoNCS.
The hydrophobic groups of alcohols can associate with each other,
burying their hydrophobic surface. It is same like as in the hydrophobic
groups in the proteins. Although the monomeric unit of alcohols can interact
with neocarzinostain, the interaction was not extensive. Once its form the
micelle-like the interaction becomes marked because micelles provide
extensive sites of interaction. We assumed that the discussed results plays a
critical role in control release of the drug which is the major factor in
improving the efficacy of the drug delivery system.
Apo-neocarzinostatin Exhibits Unusually High Resistance to the Unfolding
by Chemical Denaturants :
Life on earth exhibits an enormous adaptive capacity. During
evolution, organisms achieved viability under extreme conditions either by
“escaping” or “compensating” the stress or by enhancing the stability of the
cellular inventory. In the case of temperature, solvent and pressure, there is
no alternative to mutative adaptation for survival. In the case of proteins,
interaction with the solvent environment is crucial for protein stability,
protein-protein interaction and ligand binding. A fundamental understanding
of protein-solvent interactions is also important for the development of
protein-based biotechnologies and drug delivery processes. Most of the
researchers have used organic co solvents to control the stability and
solubility of proteins. Certain co solvent, such as polyols and free amino
acids are known to stabilize the protein. They are known as osmolytes,
which are produced by most cells in response to environmental stress. Most
organic co solvents, Acetone, DMSO, etc., are known to be destabilize
proteins and inducing denaturation. Some like methanol, ethanol, isoprotanol
and trifluroethanol (TFE) are induce extended protein conformation with
high alpha helix content.
Neocarzinostatin (NCS) is the first identified enediyne chromoprotein;
it consists of a labile chromophore (NCS-Chr), with an unusual bicyclic
dienediyne structure is very potent in causing DNA damage. Although
apoNCS itself is inactive in inducing cleavage of DNA, it plays an important
role in the drug action by protecting and storing the labile chromophore and
also by releasing the chromophore to the target DNA. The apoNCS plays an
important role as a stabilizer by complexing with the labile chromophore to
regulate its availability. However the mechanism of the protein-bound
chromophore and how it selects and interacts with its natural matched
partner is still not clear. Our perivious results say that chromophore release
does not require major conformational change. Which indicate that the
carrier apoNCS should be stable for the extreme conditions for its action.
Most bacteria including Streptpomyces cannot survive at very high
temperature and the chromophore too. Thus it seems not necessary for the
nature to build up a heat stable protein to protect the chromophore. To
protect the chromophore from this extreme environment, apoNCS need to be
more stable in this denaturants and organic solvents. ApoNCS is stable
probably against chemical reduction of disulfide, unfolding by hydrophobic
interactions, etc. Here the Stability refers to the maintenance of a defined
functional state under extreme condition. The problem of protein stability is
closely related to that of protein denaturation, because it is necessary to
destroy the native structure of the protein in order to obtain information
concerning its stability. Thus, many different organic solvents have been
used for studying the denaturation of apo NCS proteins.
In order to understand the stability of apo NCS, we have studied the effects
of several alcohols, other organic solvents and denaturants on the stability of
the apo protein, which was compared to other reported other proteins. By
comparing Cm values of apoNCS with many other reported proteins, we find
that apoNCS exhibits unusually high ability to resist the conformational
changes. The highly rigid property of apoNCS against chemical denaturants
could be a crucial factor provided by nature to protect the very labile and
biological potent chromophore which make more stable environment for the
chromophore and its function.
Conclusions:
The results described in this and previous paper strongly indicates that
release doesn’t require the conformational change of apoNCS in alcohols
and organic solvents. Which also indicate that the destabilization of the
hydrophobic interaction is the major factor in chromophore release
mechanism. Although the low polarity of alcohols is an important factor
determining the alcohol effects, it does not explained the marked effect of
TFE and t-butanol. We consider that the direct interaction of hydrophobic
groups of alcohols with the hydrophobic groups of apoNCS and NCS-Chr is
responsible for the alcohols effect and also apoNCS exhibits unusually high
ability to resist the conformational changes. The highly rigid property of
apoNCS against chemical denaturants could be a crucial factor provided by
nature to protect the very labile and biological potent chromophore which
make more stable environment for the chromophore and its function.
Figure 1
Neocarzinostatin – An Antitumor Antibiotic.
Figure 2
CD spectrum of the apo NCS in variour alcohols A) Methanol
B) Ethanol C) Isopropanol and D) Acetonitrile.
Figure 3
Fluorescence spectrum of the apo NCS in variour alcohols
A) Methanol B) Ethanol C) Isopropanol and D) Acetonitrile.
Figure 4
The rate of release of chromophore from the holoNCS by
different alcohols and organic solvent.
Figure 5
(A) Decreasing the polarity increases the rate of chromophore
release. (B) Vapor pressure also decreases when the rate of
chromophore release increases. Where holo circle – 50% of
solvents; filled circle – 65% of solvents.
Figure 6
A) Increasing the concentration of NaClO4 the rate of NCS-Chr
also increase. For inner figure, increasing the concentration of
Na2SO4 the rate of release doesn’t change much. B) Salts effect
in the presence of 15% ethanol in termes of chromophore
release rate.
Figure 7
The rate of release of chromophore from the holoNCS by
relative dielectric constant of the alcohol/ water mixtures.
Figure 1
Neocarzinostatin – An Antitumor Antibiotic.
3
Methanol
2.5
Ethanol
IPA
2
Acetonitrile
1.5
1
0.5
0
-0.5
0
10
20
30
40
50
60
70
80
% of Alcohol
Figure 4
The rate of release of chromophore from the holoNCS by
different alcohols and organic solvent.
0.040
Rate of release (k sec-1)
Rate of release (k sec-1)
0.04
A
0.03
0.02
0.01
0.00
B
0.035
0.030
0.025
0.020
0.015
0.010
0.005
4
5
6
Synder's polarity index
7
20
40
60
80
100
120
Vapor pressure at 20C
Figure 5
(A) Decreasing the polarity increases the rate of chromophore
release. (B) Vapor pressure also decreases when the rate of
chromophore release increases. Where holo circle – 50% of
solvents; filled circle – 65% of solvents.
0.35
0.025
0.3
A
0.02
0.015
0.25
0.01
0.005
0.2
0
0
0.2
0.15
0.4
0.6
0.8
1
1.2
Na2SO 4 (M)
0.1
0.05
0
-0.05
0
1
2
3
4
5
6
7
8
NaClO4 (M)
Figure 6
A) Increasing the concentration of NaClO4 the rate of NCS-Chr also
increase. For inner figure, increasing the concentration of Na2SO4 the rate of
release doesn’t change much.
0.25
NH4ClO4
B
0.2
0.15
(NH4)2SO4
0.1
0.05
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Salt with 15% Ethanol (M)
Figure 6
B) Salts effect in the presence of 15% ethanol in termes of
chromophore release rate.
3.5
Methanol
Ethanol
IPA
Acetonitrile
3
2.5
2
1.5
1
0.5
0
30
40
50
60
70
80
Dielectric Constant
Figure7
The rate of release of chromophore from the holoNCS by
relative dielectric constant of the alcohol/ water mixtures.
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