Final Thesis PDF - 209 binding constants, binding sites and binding mechanism. The fluorescence quenching

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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

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    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 3.31×10 5 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