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* Corresponding author: S. BakkialakshmiDepartment of Physics,
Annamalai University, Annamalai nagar, Tamilnadu, India-608 002
ISSN: 0976-3031
RESEARCH ARTICLE
EFFECT OF QUENCHING MECHANISM AND TYPE OF QUENCHER
ASSOCIATION
OF STERN VOLMER PLOT IN EGG ALBUMIN
*Bakkialakshmi, S and Barani, V
Department of Physics, Annamalai University, Annamalai nagar,
Tamilnadu, India-608 002
ARTICLE INFO ABSTRACT
Fluorescence quenching techniques have been used extensively in
recent years to examine
reaction rates and the compartmentalization of components in
lipid micelles andmembranes. Steady state fluorescence methods are
frequently employed in such studies
but the interpretation of the resulting Stern Volmer plots is
often hampered byuncertainties regarding the mode of association of
the quencher with the lipid structure andthe nature of the
quenching mechanism. This paper presents a method for
simulatingsteady-state Stern-Volmer plots in two phase systems, and
shows how the forms of suchplots are influenced by the type of
association of the quencher with the membrane and by
the type of quenching mechanism (dynamic and / or static).
Comparisons of simulated plots
with experimental data must take into account the possible
combinations of quencherassociation(s) and quenching mechanism (s).
The quencher used here was Amantadine.
INTRODUCTIONThe quenching of fluorescent molecules has for many
yearsprovided useful information on the nature of
bimolecularinteractions in free solution. In more recent times,
the
experimental techniques and methods of data analysis have
beenextended to two-phase systems to study the structural
anddynamical properties of molecular assemblies such as
detergent
micelles and phospholipid bilayers (1). Analysis of the
time-resolved and steady-state emission yields information on
thepermeability of the lipid-water interface to the quencher,
the
proximity of fluorophore and quencher within the lipid
structureand the dynamical properties of the lipid interior.
In most steady-state fluorescence experiments, data is
initiallypresented in the form of Stern Volmer plots where the
quenching efficiency is related to the total quencher
concentration
(2). In homogeneous solvents of low viscosity, linear Stern
Volmer plots are obtained and the bimolecular rate constant may
be calculated from knowledge of the excited-state lifetime of
thefluorophore. Upward-curving Stern-Volmer plots have also
beenobserved as the viscosity of the solvent increases, and can
be
attributed to a static quenching mechanism (3). Under the
lattercondition,a fluorophore within a spherical volume surrounding
thequencher is quenched instantaneously, while fluorophores
located
outside an active sphere may be quenched by
collisionalinteractions.
These quenching mechanisms (i.e., dymanic and / or static)
havealso been proposed to explain the shapes of Stern-Volmer plots
in
two-phase systems (3-8). However the nature of the distribution
ofquencher between aqueous and lipid compartments has not
beenexamined in detail, and the effects of this distribution on
thecharacteristics of Stern-Volmer plots have not been
explored.
Two distribution processes have been identified: partitioning
andbinding (9). These are separate thermodynamic process and
cannot be interchanged. The former requires that the ratio of
the
concentration of quencher between aqueous and lipid phases
isconstant and independent of the amount of quencher added and
of
the volume fraction of each phase, whereas the latter
impliessaturable binding to a limited number of binding sites
(10).
MATERIALS AND METHODS
Amantadine and Egg Albumin were purchased from sigmaAldrich and
used without further purification. All the experiments
were performed in pH 7.0 at temperature 33oC. Fluorescence
measurements were performed on Shimadzu RFPC
5301spectrofluorimeter equipped with a PC. Absorption
measurements
were performed on Shimadzu 1650 PC spectrophotometer.
RESULTS AND DISCUSSIONBinding of drug with Egg Albumin
Fluorescence measurements give information about the
molecularenvironment in vicinity of the fluorophore molecules.
Therefore,conformational changes of albumin were evaluated by
theintrinsic fluorescence intensity before and after addition of
drug.The binding of small molecule substances to protein, such as
thebinding mechanism, binding constants, binding media, binding
sites and intermolecular distances, can be evaluated by
thefluorescence measurements, for macro molecules. The
ligandquenches the fluorescence emission spectrum of Egg albumin
due
to the changes in environment around tryptophan and / or
tyrosine
caused by interaction of the ligand with albumin. On titration
ofalbumin with the drug solution the fluorescence intensity
decreased due to a variety of molecular interactions, viz.,
excitedstate reactions, molecular rearrangements, energy transfer,
ground
Available Online at http://www.recentscientific.com
International Journal
of Recent Scientific
ResearchInternational Journal of Recent Scientific ResearchVol.
4, Issue, 7, pp.1089 1090, July, 2013
Article History:
Received 11th, June, 2013
Received in revised form 24th, June, 2013
Accepted 18th
, July, 2013Published online 30th July, 2013
Key words:
UV absorption, Fluorescence, Amantadine, Egg
Copy Right, IJRSR, 2013, Academic Journals. All rights
reserved.
7/28/2019 Download 403
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International Journal of Recent Scientific Research, Vol. 4,
Issue, 7, pp. 1089 - 1090, July, 2013
1090
state complex formation and collisional quenching., suchdecrease
in fluorescence intensity in known as quenching .
Fig. 1 Fluoresence quenching Spectra of the Egg Albumin with
different
concentrations of Amantadine mol dm-3 (1) 0, (2) 0.2 (3) 0.4,
(4) 0.6,
(5) 0.8, (6) 1.0, (7) 1.2, (8) 1.4
Fluorescence quenching spectra of Egg Albumin withAmantadine is
given in Fig 1. Table 1, gives the Stern Volmer quenching constant
(KSV) and the regression coefficient
(r). Stern- Volmer Plot is shown in Fig. 2
Fig. 2 Stern-Volmer plot of the Egg Albumin with
different concentrations of Amantadine
UV Vis absorption studies
UV Vis spectroscopy was used to study drug bindinginteractions
[11]. Egg Albumin has a UV Absorption peak at 280nm, at which
particularly three amino acids like tryptophan,
phenylalanine, and tyrosine absorb maximally.
Fig. 3 UV absorption Spectra of the Egg Albumin with
different
concentrations of Amantadine mol dm
-3
(1) 0, (2) 0.2 (3) 0.4, (4) 0.6, (5) 0.8,(6) 1.0, (7) 1.2, (8)
1.4
The formation of drug albumin complexes is evident from UV Vis
adsorption spectral data. Absorbance decreases with the
increase in drug concentration and the shift at 280 nm is
notprominent while no shift was observed from the fluorescence
data. This again confirms the change in polarity around
thetryptophan residue and the change in peptide strand of
albuminand hence the change in hydrophobicity. Fig 3 gives the
UV
absorption spectra of Egg Albumin with Amantadine.
Table 1 Stern-Volmer Quenching Constant (KSV), regression
coefficient (r) quenching rate constant (kq) and standard
deviationQuencher KSV Kq R
S.D
Amantadine 0.5 0.102 0.98 0.24
CONCLUSION
Information about the internal properties of lipid structures
andthe permeability of lipid/water interfaces to quencher
moleculescan be obtained by the fluorescence quenching
technique.
Understanding of the fundamental processes involved, and
theeffects of compartmentalization of these processes, are
requisites
for qualitative and quantitative descriptions. This paper has
shownthe effects of the type of quencher association and
quenchingmechanism on Stern-Volmer plots in compartmentalized
systems,in order to achieve that aim.
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R = 0.984
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
I0/I
[Q] x 10-5M
