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207
4.5 RESULTS
PROTEIN DRUG INTERACTION
EFFECT OF ANTI-TUBRCULOSIS (RIFAMPICIN) AND ANTI-
DIABETES (STATINS) ON BUFFALO LIVER CYSTATIN
The binding of proteins to drugs assumes great importance since it influences their
pharmacokinetic and pharmacodynamics properties and may also cause interference
with the binding of other endogenous and/or exogenous ligands as a result of overlap
of binding sites and/or conformational changes. A thorough investigation of drug-
protein interaction generates a curiosity to understand the mechanism of the
pharmacokinetic behaviour of a drug and for the design of analogues with effective
pharmacological properties. Fluorescence quenching is a useful method to study the
reactivity of chemical and biological systems since it allows non-instrusive
measurements of substances in low concentration under physiological conditions
(Nail 2010; Guo 2007). It can reveal information about binding mechanisms to
compounds and provides clues to the nature of the binding phenomenon.
4.5 INTERACTION OF BUFFALO LIVER CYSTATIN WITH
RIFAMPICIN
Rifampicin is a drug used along with isoniazid as an effective and a long haul
treatment of tuberculosis following a 6 month regimen. The side effects resulting
from rifamycin has been attributed as a reason for hepatic cirrhosis and jaundice with
elevated levels of liver enzymes. Rifampicin hasbeen reported as causing hepatitis in
patients beingtreated for tuberculosis. Protein binding of drugs assumes great
importance as most of the drugs bind to proteins for effective delivery into the target
side. However, drugs can bind off target to proteins not desired for drug delivery
leading to the functional inactivation and structural changes in proteins which are vital
for the overall functioning of the cells leading to pathological conditions. Since it
influences their pharmacokinetic and pharmacodynamics properties and may also
208
cause interference with the binding of other endogenous and/or exogenous ligands as
a result of overlap of binding sites and/or conformational changes. A detailed
investigation of drug-protein interaction can give an effective insight for thorough
understanding of the pharmacokinetic behaviour of a drug and for the design of
analogues with effective pharmacological properties. Fluorescence quenching is a
useful method to study the reactivity of chemical and biological systems since it
allows non-instrusive measurements of substances in low concentration under
physiological conditions (Nail 2010; Guo 2007). It can reveal information about
binding mechanisms to compounds and provides clues to the nature of the binding
phenomenon.
The interaction between antitubercluosis drug rifampicin with buffalo liver cystatin
was studied using fluorescence, UV-vis absorption spectroscopy and papain inhibitory
activity of purified protein (BLC). These are powerful tools for the study of the
reactivities of chemical and biological systems since they allow non-intrusive
measurements of substances in low concentrations under physiological
conditions.Buffalo liver cystatin (1µM) was treated with increasing concentrations of
rifampicin (0.1-1 µM) and the data was analyzed spectroscopically by above
mentioned techniques.
4.5.1 Intrinsic fluorescence spectra of statin-cystatin complex
Intrinsic fluorescence measurements were carried out to determine the structural
changes induced by rifampicin to BLC. The excitation wavelength for protein was
taken as 280 nm to assess changes induced in globular conformation of the inhibitor
on interaction with the drug. The emission range was from 300-400 nm. The results
are shown in fig 8.2. Rifampicin caused a decrease in the fluorescence intensity of
BLC with a red shift of around 20 nm. Maximum unfolding was observed at 0.8 µM
concentration of the drug while at 1 µM, liver cystatin was completely denatured.
Increase in fluorescence intensity was also accompanied by a red shift of 5 nm.
Fluorescence measurements of macromolecules like proteins can provide information
about the binding to small molecules especially drugs which is used to calculate
209
binding constants, binding sites and binding mechanism. The fluorescence quenching
data was analysed by the Stern-Volmer equation:
F0/F = 1+Ksv [Q]
Where F0 and F are the steady-state fluorescence in the absence and presence of
quencher (rifampicin), respectively, Ksv is the Stern Volmer quenching constant and
[Q] is the concentration of the drug.
Static quenching involves the formation of a stable complex between the fluor and
quencher. On the other hand, in dynamic quenching the quencher collides with
excited fluor leading to the loss of some energy from excited asa kinetic energy. The
plot of F0 / F vs [Q] exhibited a good linear relationship indicating that the interaction
was purely static in nature (fig 8.3).
4.5.1 Determination of binding constatnt (K) and number of binding
sites (n)
When small molecules bind independently to a set of equivalent sites on a
macromolecule, the equilibrium between free and bound molecules is given by the
equation (Feng et al., 1998; Goa et al., 2004),
Log Fo – F / F = log K + n log [Q]
Where K and n are the binding constant and the number of binding sites, respectively.
Binding constant was found to be 3.31×105 M
-1 and the number of binding sites was
found to be less than 1 (fig 8.4).
210
Figure 8.2: Fluorescence spectra of buffalo liver cystatin in the
presence and absence of rifampicin
Intrinsic Fluorescence emission spectra of rifampicin-cystatin complex
in the presence of different concentrations of rifampicin obtained in
sodium phosphate buffer, pH 7.5. Inhibitor concentration was 1�M.
Concentration of rifampicin was taken in a range of 0.01 to 1�M
respectively. Fluorescence measurements were carried out on a
Shimadzu spectrofluorimeter model RF-450 equipped with a data
recorder 300-400 nm after exciting the protein solution at 280 nm for
total protein fluorescence. This slits were set at 5nm for excitation and
10 nm for emission, the path length of the sample was 1 cm.
211
Figure 8.3: Determination of Stern-Volmer constant
Stern-volmer constant was determined by the equationFo/F=1+Ksv
[Q]where Fo and F are the steady-state fluorescence intensities in the
absence and presence of rifampicin, respectively; Ksv the Stern-
Volmer quenching constant and [Q] is the concentration of rifampicin.
212
Figure 8.4: Binding constant and the number of binding sites
determination by Stern-Volmer plot.
BLC (1µM) was incubated with various concentration of rifampicin
varying from 1 µM to 0.01 µM for 30 min at 298 K and their
fluorescence spectra were recorded between 300-400 nm after exciting
BLC at 280 nm. The fluorescence quenching data was further analysed
by the stern-volmer equation as described in methods. The plot of Fo/F
vs concentration of rifampicin gives binding constant (K) and the
number of binding sites (n) between rifampicin-BLC complex. [Q] is
the concentration of rifampicin.
213
4.5.2 UV-visible absorption studies of rifampcin-BLC complex
Absorption spectral measurements of buffalo liver cystatin in the presence of drug
provided information related to their interaction. Difference spectra of drug protein
complex was measured aganist cystatin alone (fig 8.5). For the difference spectra
obtained at 0.1 µM rifampicin a positive peak at 240 nm was observed. A red shift of
around 20 nm was observed as the concentration of rifampicin increased from
0.01µM to 1 µM with 0.04 µM showing an increase in difference spectra intensity
and a red shift of 15 nm.
4.5.3 Inhibitory activity of cystatin in the presence of rifampicin
The papain inhibitory activity of buffalo liver cystatin (BLC) incubated for 30 min
with increasing concentration of rifampicin is shown in the Table 2.0. At
concentration as low as 0.01 µM, BLC lost its ability to inhibit papain to some extent.
However, complete loss of activity was observed at 1 µM rifampicin. This suggests
that increasing concentration of rifampicin resulted in the functional inactivation of
cystatin.
214
Figure 8.5: UV-vis spectra of buffalo liver cystatin in the presence of
rifampicin
The interaction between rifampicin-BLC was studied by UV-vis
absorption spectral data. BLC concentration was fixed at 1µM while
the rifampicin concentration was 0.01-0.5 µM. Absorption spectra of
native BLC and in presence of rifampicin were recorded in the range of
200-300 nm on a Shimadzu UV- mini vis-spectrophotometer UV-1700
using a cuvette of 1 cm path length.
215
TABLE 2.0: ANTIPROTEOLYTIC ACTIVITY OF BLC IN THE
PRESENCE OF RIFAMPCIN AFTER INCUBATION AT
VARIOUS TIME INTERVALS
Concentration of
rifampcin
30 min of
incubation
2 hrs of
incubation
6 hrs of
incubation
0.01 �M 89.33±1.8 80.12±2.0 72.4±25
0.02 µM 83.4±1.3 75.1±1.7 65.3±2.3
0.04 �M 77.3±1.6 69.4±2.4 53.1±1.9
0.06 �M 62±2.1 54±1.1 41.2±2.1
0.08 �M 55.5±2.5 42.7±1.0 29.5±1.6
0.1 �M 43.1±1.1 24.3±2.2 14.1±2.1
0.5 µM 37±0.6 19±1.5 9.4±1.2
LC (1 µM) was incubated with (0-1 µM) statin for 30 min, 2 and 6 hours respectively
at 37°C.
216
4.5 (B) INTERACTION OF BUFFALO LIVER CYSTATIN WITH
STATIN (ATORVASTATIN)
4.5.4 Fluorescence spectra of statin-cystatin complex
Fluorescence and UV-vis absorption spectroscopy are powerful tools for the study of
the reactivities of chemical and biological systems since they allow non-intrusive
measurements of substances in low concentrations under physiological conditions.
Buffalo liver cystatin (1µM) was treated with increasing concentrations of statin (0.1-
10 µM) and the data was analyzed spectroscopically by above mentioned techniques.
In case of intrinsic fluorescence measurements, the excitation wavelength for protein
was taken as 280 nm to assess changes induced in globular conformation of the
inhibitor on interaction with the drug. The emission range was from 300-400 nm. The
results are shown in fig 8.6. Binding of Atorvastatin with liver cystatin led to an
increase in the fluorescence intensity. Maximum unfolding was observed at 5 µM
concentration of the drug while at 10 µM, liver cystatin was completely denatured.
Increase in fluorescence intensity was also accompanied by a red shift of 5 nm.
4.5.4 Determination of binding constatnt (K) and number of binding
sites (n)
When small molecules bind independently to a set of equivalent sites on a
macromolecule, the equilibrium between free and bound molecules is given by the
equation (Feng et al., 1998; Goa et al., 2004),
Log Fo – F / F = log K + n log [Q]
Where K and n are the binding constant and the number of binding sites, respectively.
The plot of F0 / F vs [Q] exhibited a good linear relationship indicating that the
interaction was purely static in nature (fig 8.7). Binding constant was found to be
2.78×106 M
-1 and the number of binding sites was found to be one (fig 8.8).
217
Figure 8.6: Intrinsic fluorescence studies of buffalo liver cystatin in
the presence and absence of atorvastatin.
Buffalo liver cystatin (1µM) was incubated with various
concentrations of statin (0.1– 10 µM) for 30 min. Fluorescence
measurements were carried out on a Shimadzu spectrofluorimeter
model RF-540 equipped with a data recorder DR- 3 at 25°C. The
fluorescence was recorded in the wavelength region 300-400 nm after
exciting the protein solution at 280 nm for protein fluorescence. The
slits were set at 5 nm for excitation and 10 nm for emission
respectively. The path length of the sample was 1 cm.
218
Figure 8.7: Determination of Stern-Volmer constant
Stern-volmer constant was determined by the equation Fo/F=1+Ksv
[Q]where Fo and F are the steady-state fluorescence intensities in the
absence and presence of Atorvastatin, respectively; Ksv the Stern-
Volmer quenching constant and [Q] is the concentration of
atorvastatin.
219
Figure 8.8: Determination of binding constant and the number of
binding sites by Stern-Volmer plot.
Buffalo liver cystatin BLC, (1µM) was incubated with various
concentration of atorvastatin varying from 0.2 to 10 µM for 30 min at
298 K and their fluorescence spectra were recorded between 300-400
nm after exciting BLC at 280 nm. The fluorescence quenching data
was further analysed by the stern-volmer equation as described in
methods. The plot of Fo/F vs concentration of atorvastatin gives
binding constant (K) and the number of binding sites (n) between
atorvastatin-BLC complex.
220
4.5.5 UV-vis absorption spectra of Atorvastatin-cystatin complex
UV-vis absorption difference spectra was computed at the drug concentrations
varying from 0.1µM-5µM (Fig 8.9). Profound changes were noted only for those
obtained at 1µM, 2µM and 5µM concentrations of statin. A sharp positive peak was
noticeable at 230 nm in the difference spectra at 1µM and 2 µM concentration of
statin with a blue shift. Difference spectra of drug protein complex at 5 µM drug
concentration also showed broad shoulder at 230 nm and a blue shift (15 nm).
4.5.6 Inhibitory activity profile of statin-cystatin complex
Changes in the inhibitory activity of buffalo liver cystatin with increasing
concentration of atorvastatin are shown in table 2.1. The results show that buffalo
liver cystatin lost significant amount of inhibitory activity at 1 µM concentration of
drug.
221
Figure 8.9: UV-vis absorption spectra of Atorvastatin-BLC complex
The interaction between statin-BLC was studied by UV-vis absorption
spectral data. Liver cystatin concentration was fixed at 1 µM while the
statin concentration was in the range of 0.2 µM to 10µM. Absorption
spectra of native BLC and in presence of statin were recorded in the
range of 200-300 nm on a Shimadzu UV-mini vis spectrophotometer
UV-1700 using a cuvette of 1 cm path length.
222
TABLE-2.1: EFFECT OF ATORVASTATIN ON INHIBITORY
ACTIVITY OF BUFFALO LIVER CYSTATIN
Concentration of
atorvastatin
30 mins of incubation 2 hrs of incubation
0.2 �m 93.1±2.3 86.3±1.2
0.4 µM 84.2±1.1 75.3±1.5
0.8 �M 67.3±1.7 57.2±2.0
1 �M 49.8±2.3 36.4±1.0
2 �M 31.2±1.4 19.5±1.2
5 �M 23.1±1.3 11.3±0.5
10 µM 14.3±2.1 7.8±1.5
LC (1 µM) was incubated with (0-1 µM) statin for 30 min and 2 hours at 37°C.
223
4.5 (C): INTERACTION OF PESTICIDE (MALATHION) WITH
BUFFALO LIVER CYSTATIN
Malathion is one of the more frequently used organophosphorothioate (OPT)
insecticides in the world, both in agriculture and in residential settings. Malathion
[O,O-dimethyl-S-(1,2-dicarbethoxyethyl) phosphorodithioate] is one of the most
widely used OP insecticides for agriculture and public health programs (Maroni et al.,
2000). Malathion is soluble in lipids and is stored in liver and other lipophilic tissues
(Garcia-Repetto et al., 1995). Malathion has been found to exhibit rapid but a
symmetrical transmembrane uptake by the liver. Therefore, the liver which is the most
important organ inglucose and lipid homeostasis and production of related enzymes
can be a target for malathion toxicity (Yang et al., 2000).
Fluorescence quenching technique is an important method to study the interactions of
several substances with protein, which can reveal the accessibility of quenchers to
protein fluorophore groups, it helps in understanding the protein binding mechanisms
to these substances andprovide clues to the nature of the binding phenomenon. In the
past years, many researches had been concentrated on the binding of drugs to
albumin. Nowadays, some researches on the binding of organophosphorous pesticides
to BSA or HSA have been carried out (Cortez et al., 2004) to elucidate the mechanism
and toxicity of pesticides. Cystatins help to downgrade or upgrade the activity of vital
cathepsins. The interaction between malathion with buffalo liver cystatin was studied
using fluorescence, UV-vis absorption spectroscopy and papain inhibitory activity of
purified protein (BLC). These are powerful tools for the study of the reactivities of
chemical and biological systems since they allow non-intrusive measurements of
substances in low concentrations under physiological conditions. Buffalo liver
cystatin (1µM) was treated with increasing concentrations of malathion (0.1-1 ppm)
and the data was analyzed spectroscopically by above mentioned techniques.
4.5.7 Intrinsic fluorescence spectra of malathion-cystatin complex
Intrinsic fluorescence measurements were carried out to determine the structural
changes induced by malathion to BLC. The excitation wavelength for protein was
taken as 280 nm to assess changes induced in secondary structure conformation and
224
the internal environment of the inhibitor on interaction with the pesticide. The
emission range was from 300-400 nm. The results are shown in fig 9.0. Malathion
caused a decrease in the fluorescence intensity of BLC with a red shift of around 15
nm. Maximum unfolding was observed at 30 ppm concentration of the drug while at
50 ppm, liver cystatin was completely denatured.
4.5.8 UV-vis absorption spectra of malathion-BLC complex
UV-vis absorption difference spectra was computed at the drug concentrations
varying from 0.1-50ppm (Fig 9.1). There was a consistent change at higher
concentration of malathion. A sharp positive peak was noticeable at 225 nm in the
difference spectra at 30 ppm, 40 ppm and 50 ppm and consequent blue shift (20 nm)
at these very concentrations of malathion.
4.5.9 Inhibitory activity spectra of malathion-cystatin complex
Changes in the inhibitory activity of buffalo liver cystatin with increasing
concentration of malathion are shown in table 2.2. The results show that buffalo liver
cystatin lost significant amount of inhibitory activity at 30 ppm concentration of
malathion while at 50 ppm there was very little or no inhibitory activity.
225
Figure 9.0: Intrinsic fluorescence studies of buffalo liver cystatin in
the presence of malathion.
Buffalo liver cystatin (1µM) was incubated with various
concentrations of statin (0.1–50 ppm) for 30 min. Fluorescence
measurements were carried out on a Shimadzu spectrofluorimeter
model RF-540 equipped with a data recorder DR- 3 at 25°C. The
fluorescence was recorded in the wavelength region 300-400 nm after
exciting the protein solution at 280 nm for protein fluorescence. The
slits were set at 5 nm for excitation and 10 nm for emission
respectively. The path length of the sample was 1 cm
226
Figure 9.1: UV-vis spectra of buffalo liver cystatin in the presence of
malathion
The interaction between malathion-BLC was studied by UV-vis
absorption spectral data. BLC concentration was fixed at 1µM while
the rifampicin concentration was 0.1 ppm to 50 ppm. Absorption
spectra of native BLC and in presence of malathion were recorded in
the range of 200-300 nm on a Shimadzu UV- mini vis-
spectrophotometer UV-1700 using a cuvette of 1 cm path length.
227
TABLE-2.2: INHIBITORY ACTIVITY OF BUFFALO LIVER
CYSTATIN IN PRESENCE OF MALATHION.
Concentration of malathion
(ppm)
% Remaining remaining inhibitory
activity
0.1 85.2±1.7
1 73.5±1.2
5 57.1±2.2
10 43.3±1.4
20 27.2±2.3
30 15.9±2.0
40 8.3±1.1
50 1.4±0.5
BLC (1 µM) was incubated with (0.1-50 ppm) statin for 30 min and 2 hours at 37°C.