**8. Acknowledgment**

The author is grateful to Prof. Victor Fadeev for providing the ability to work in his group and for great help in mastering the fluorescence spectroscopy methods. The author also thanks Evgeny Vrzheshch for the samples of the red FPs and for valuable discussions.

### **9. References**

Agranovich, V.M. & Galanin, M.D. (1982). *Electronic Excitation Energy Transfer in Condensed Matter*, Elsevier Science Ltd, ISBN 978-0444863355, North-Holland

Banishev, A.A.; Vrzhechsh, E.P. & Shirshin, E.A. (2009). Application of laser fluorimetry for determining the influence of a single amino-acid substitution on the individual

because of non-polarity of substituted residues. Therefore, there is no defined correlation between the chromophore geometry (consequently, the volume of the substituted amino

At the present time the red FPs whose molecules are monomers are of particular interest (Piatkevich et al., 2010) as fluorescent markers. Attempts to find new variants of red FPs in order to improve their properties (higher brightness, photo and pH stability, etc.) are performed. However, the interrelation between optical or photophysical parameters and structural properties of FPs, which is necessary for development of these studies, is rather unclear. A method for prediction of properties of FPs based on their structure is still not developed. This problem might be solved by analysis of properties of mutant proteins with point mutations. Therefore, the results obtained in this Section can be used to tackle the general problem of the development of an algorithm, which could provide the prediction of the spectral properties of FPs based on their structures. The data will also be useful for

In current work the approach based on the simultaneous use of nonlinear laser fluorimetry, spectrophotometry and conventional fluorimetry methods has been applied for investigation of the photophysical properties of the protein molecules of different complexity. The full set of photophysical parameters of the fluorophores (tryptophan residues) of human and bovine serum albumins has been determined. The photophysical processes in the spectral forms of the red FP mRFP1 under UV (266 nm) and visible (532 nm) irradiation are described quantitatively. The individual photophysical parameters of the new mutants of the mRFP1 protein (a single substitution at the 66 amino acid position) were determined. It was shown that the individual extinction coefficient of the red chromopore of the proteins correlate positively with the volume of the substituted amino acid residue at position 66 (for polar substitution). A similar correlation has been described for the position of the maximum of the absorption, fluorescence excitation and emission spectra: the position of the maximum moves to the red with increasing the volume of the residue. In addition, the partial concentration of the fluorescent spectral form in the resultant solution of each FP

The author is grateful to Prof. Victor Fadeev for providing the ability to work in his group and for great help in mastering the fluorescence spectroscopy methods. The author also thanks Evgeny Vrzheshch for the samples of the red FPs and for valuable discussions.

Agranovich, V.M. & Galanin, M.D. (1982). *Electronic Excitation Energy Transfer in Condensed* 

Banishev, A.A.; Vrzhechsh, E.P. & Shirshin, E.A. (2009). Application of laser fluorimetry for

determining the influence of a single amino-acid substitution on the individual

*Matter*, Elsevier Science Ltd, ISBN 978-0444863355, North-Holland

acid residue) and the optical properties of the proteins.

revealing promising positions for directed mutagenesis.

**7. Conclusion** 

variant has been determined.

**8. Acknowledgment** 

**9. References** 

photophysical parameters of a fluorescent form of a fluorescent protein mRFP1. *IEEE J. Quantum Electron.*, Vol. 39, No. 3, pp. 273-278, ISSN 10637818


**12** 

**Current Achievement and Future Potential** 

*Jordan University of Science and Technology, Department of Chemistry, Jordan* 

Spectrofluorometric methods of analysis are the most commonly analytical techniques and continue to enjoy wide popularity. The wide availability of the instrumentation, the simplicity of procedure, sensitivity, selectivity, precision, accuracy, and speed of the technique still make the spectrofluorometric methods attractive. These features make fluorescence spectroscopy an attractive technique as compared to other forms of optical spectroscopy or other analytical techniques such as chromatography and electrophoresis. Fluorescence spectroscopy has been used widely as a tool for quantitative analysis, characterization, and quality control in the pharmaceutical, environmental, agricultural,

The emission of light from an excited electronic state of a molecular species is called luminescence. The discovery and characterization of luminescence begun from the 15th century. In 1506 Nicolas Monardes was the first to describe the bluish opalescence of the water infusion from the wood of a small Mexican tree. In 1612 Galileo described the emission of light (phosphorescence) from the famous Bolognian stone, which discovered by Vincenzo Casciarolo, a Bolognian shoemaker. Galileo wrote: "It must be explained how it happens that light is conceived into the stone, and is given back after some time, as is childbirth". Even though, some of the first scientific reports of luminescence appeared in the middle of the 18th century. In 1845 Sir J.F.W. Herschel reports on an experiment he did twenty years earlier. Herschel made the first observation of fluorescence from quinine sulfate (quinine: (*R*)-(6-methoxyquinolin-4-yl)-((2*S*, 4S, 8R)- 8-vinylquinuclidin-2-yl)methanol, C20H24N2O2, quinine absorbs in the UV region), he observed that an otherwise colorless solution of quinine in water emitted a blue color under certain circumstances. Herschel concludes that a species in the solution, "exert its peculiar power on the incident light" and disperses the blue light. The experiment can be repeated simply by observing glass of tonic

water exposed to sunlight. Often a blue glow is visible at the surface (Rendell, 1987).

The phenomenon of fluorescence was known by the middle of the nineteenth century. British scientist Sir George G. Stokes first made the observation that the mineral fluorspar exhibits fluorescence when illuminated with ultraviolet light, and he coined the word "fluorescence". In 1852, Sir G.G. Stokes studied the same compound (quinine) that has been used by Herschel and found that the fluorescing (*emitted*) light has longer wavelengths than the excitation (*absorbed*) light, a phenomenon that has become to be known as the Stokes

**1. Introduction** 

nanotechnology and biomedical fields.

**of Fluorescence Spectroscopy** 

*United Arab Emirates University, Department of Chemistry, UAE* 

Nathir A. F. Al-Rawashdeh

