Transcript
Page 1: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

eexxááââÄÄààáá@@HH

\\ÇÇààxxÜÜttvvàà||ÉÉÇÇ ÉÉyy ÑÑââÜÜ||yy||xxww UUââyyyyttÄÄÉÉ ÄÄ||ääxxÜÜ vvççááààttàà||ÇÇ ãã||àà{{ wwÜÜââzzáá

ttÇÇww ÑÑxxááàà||vv||wwxxMM \\ààáá uu||ÇÇww||ÇÇzz ttÇÇww ááààÜÜââvvààââÜÜttÄÄ ááààââww||xxáá

Page 2: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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

Page 3: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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

Page 4: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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).

Page 5: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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.

Page 6: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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.

Page 7: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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.

Page 8: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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.

Page 9: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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.

Page 10: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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.

Page 11: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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).

Page 12: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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.

Page 13: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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.

Page 14: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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.

Page 15: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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.

Page 16: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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.

Page 17: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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.

Page 18: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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

Page 19: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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.

Page 20: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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

Page 21: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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.

Page 22: Final Thesis PDF - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/21073/18... · 209 binding constants, binding sites and binding mechanism. The fluorescence quenching data

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.


Recommended