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International Journal of Recent Scientific Research Vol. 4, Issue, 7, pp.1089 1090, July, 2013 ISSN: 0976-3031

International Journal of Recent Scientific Research

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
Article History:
Received 11th, June, 2013 Received in revised form 24th, June, 2013 Accepted 18th, July, 2013 Published online 30th July, 2013

ABSTRACT
Fluorescence quenching techniques have been used extensively in recent years to examine reaction rates and the compartmentalization of components in lipid micelles and membranes. Steady state fluorescence methods are frequently employed in such studies but the interpretation of the resulting Stern Volmer plots is often hampered by uncertainties regarding the mode of association of the quencher with the lipid structure and the nature of the quenching mechanism. This paper presents a method for simulating steady-state Stern-Volmer plots in two phase systems, and shows how the forms of such plots 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 quencher association(s) and quenching mechanism (s). The quencher used here was Amantadine. Copy Right, IJRSR, 2013, Academic Journals. All rights reserved.

Key words: UV absorption, Fluorescence, Amantadine, Egg albumin.

INTRODUCTION
The quenching of fluorescent molecules has for many years provided useful information on the nature of bimolecular interactions in free solution. In more recent times, the experimental techniques and methods of data analysis have been extended to two-phase systems to study the structural and dynamical properties of molecular assemblies such as detergent micelles and phospholipid bilayers (1). Analysis of the timeresolved and steady-state emission yields information on the permeability of the lipid-water interface to the quencher, the proximity of fluorophore and quencher within the lipid structure and the dynamical properties of the lipid interior. In most steady-state fluorescence experiments, data is initially presented 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 the fluorophore. Upward-curving Stern-Volmer plots have also been observed as the viscosity of the solvent increases, and can be attributed to a static quenching mechanism (3). Under the latter condition,a fluorophore within a spherical volume surrounding the quencher is quenched instantaneously, while fluorophores located outside an active sphere may be quenched by collisional interactions. These quenching mechanisms (i.e., dymanic and / or static) have also been proposed to explain the shapes of Stern-Volmer plots in two-phase systems (3-8). However the nature of the distribution of quencher between aqueous and lipid compartments has not been examined in detail, and the effects of this distribution on the characteristics of Stern-Volmer plots have not been explored.

Two distribution processes have been identified: partitioning and binding (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 is constant and independent of the amount of quencher added and of the volume fraction of each phase, whereas the latter implies saturable binding to a limited number of binding sites (10).

MATERIALS AND METHODS

Amantadine and Egg Albumin were purchased from sigma Aldrich and used without further purification. All the experiments were performed in pH 7.0 at temperature 33oC. Fluorescence measurements were performed on Shimadzu RFPC 5301 spectrofluorimeter equipped with a PC. Absorption measurements were performed on Shimadzu 1650 PC spectrophotometer.

RESULTS AND DISCUSSION

Binding of drug with Egg Albumin Fluorescence measurements give information about the molecular environment in vicinity of the fluorophore molecules. Therefore, conformational changes of albumin were evaluated by the intrinsic fluorescence intensity before and after addition of drug. The binding of small molecule substances to protein, such as the binding mechanism, binding constants, binding media, binding sites and intermolecular distances, can be evaluated by the fluorescence measurements, for macro molecules. The ligand quenches 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 of albumin with the drug solution the fluorescence intensity decreased due to a variety of molecular interactions, viz., excited state reactions, molecular rearrangements, energy transfer, ground

* Corresponding author: S. Bakkialakshmi Department of Physics, Annamalai University, Annamalai nagar, Tamilnadu, India-608 002

International Journal of Recent Scientific Research, Vol. 4, Issue, 7, pp. 1089 - 1090, July, 2013 state complex formation and collisional quenching., such decrease in fluorescence intensity in known as quenching . increase in drug concentration and the shift at 280 nm is not prominent while no shift was observed from the fluorescence data. This again confirms the change in polarity around the tryptophan residue and the change in peptide strand of albumin and 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 deviation
Quencher Amantadine KSV 0.5 Kq 0.102 R2 0.98 S.D 0.24

CONCLUSION
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 with Amantadine 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
2 1.8 1.6 1.4 1.2
I0/I

Information about the internal properties of lipid structures and the permeability of lipid/water interfaces to quencher molecules can be obtained by the fluorescence quenching technique. Understanding of the fundamental processes involved, and the effects of compartmentalization of these processes, are requisites for qualitative and quantitative descriptions. This paper has shown the effects of the type of quencher association and quenching mechanism on Stern-Volmer plots in compartmentalized systems, in order to achieve that aim.

R = 0.984

References
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1 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 0.8 1 [Q] x 10-5M 1.2 1.4 1.6

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 binding interactions [11]. Egg Albumin has a UV Absorption peak at 280 nm, 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

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