Proteins fluorescence spectroscopy

Measuring Protein Sta.bihty, Protein stabihty is usually measured quantitatively as the difference in free energy between the folded and unfolded states of the protein. These states are most commonly measured using spectroscopic techniques, such as circular dichroic spectroscopy, fluorescence (generally tryptophan fluorescence) spectroscopy, nmr spectroscopy, and absorbance spectroscopy (10). For most monomeric proteins, the two-state model of protein folding can be invoked. This model states that under equihbrium conditions, the vast majority of the protein molecules in a solution exist in either the folded (native) or unfolded (denatured) state. Any kinetic intermediates that might exist on the pathway between folded and unfolded states do not accumulate to any significant extent under equihbrium conditions (39). In other words, under any set of solution conditions, at equihbrium the entire population of protein molecules can be accounted for by the mole fraction of denatured protein, and the mole fraction of native protein,, ie. 

The protein-containing colloidal solutions of water-in-organic solvents are optically transparent. Hence, absorption spectroscopy, circular dichroism spectroscopy and fluorescence spectroscopy are found to be convenient for studying biocatalysis [53]. The reversed micelles are interesting models for studying bioconversion, since the majority of the enzymes in vivo act inside or on the surface of biological membranes. 

Lehrer, S. S. (1997). Intramolecular pyrene excimer fluorescence A probe of proximity and protein conformational change. Fluorescence Spectroscopy 278, 286-295. 

Enzyme structure may be studied by fluorescence spectroscopy [238-244]. Excitation in the 280-310 nm absorption bands of proteins, usually results in fluorescence from tryptophan (Trp) residues in the 310-390 nm region. The fluorescence from the Trp residues is a convenient marker for protein denaturation and large decreases or red-shifts in fluorescence are observed when proteins are denatured. These changes are most often due to the exposure of the Trp residues that are buried in the protein and may be due to the changes in the proximities of specific residues that may act as fluorescence quenchers. Fluorescence emission characterization of the immobilized... 

Protein peroxidation Modified tyrosines GC/MS, HPLC, immunoassays Protein carbonyls Atomic absorption spectroscopy, fluorescence spectroscopy, HPLC... 

Mayer, L.M., Schick, L.L., Loder, T.C. 1999. Dissolved protein fluorescence in two main estuaries. Marine Chemistry, 64, 171-179. Miano, T.M., Senesi N., 1992. Synchronous excitation fluorescence spectroscopy applied to soil humic substances chemistry. Sci Total Environ, 117/118, 41-51. 

Such ambiguity and also the low structural resolution of the method require that the spectroscopic properties of protein fluorophores and their reactions in electronic excited states be thoroughly studied and characterized in simple model systems. Furthermore, the reliability of the results should increase with the inclusion of this additional information into the analysis and with the comparison of the complementary data. Recently, there has been a tendency not only to study certain fluorescence parameters and to establish their correlation with protein dynamics but also to analyze them jointly, to treat the spectroscopic data multiparametrically, and to construct self-consistent models of the dynamic process which take into account these data as a whole. Fluorescence spectroscopy gives a researcher ample opportunities to combine different parameters determined experimentally and to study their interrelationships (Figure 2.1). This opportunity should be exploited to the fullest. 

In using the method of the red-edge shift in UV fluorescence spectroscopy, we should take into account the possibility of emission not only of tryptophan but also of tyrosine residues. In many tryptophan-containing proteins, tyrosine fluorescence is not observed. However, it is considerable in serum albumin, and the decrease in its intensity is responsible for the long-wavelength shift of the spectra recorded at Aex < 290 nm. At Aex > 292 nm, the tyrosine component should be completely absent. 

Thus, at present, fluorescence spectroscopy is capable of providing direct information on molecular dynamics on the nanosecond time scale and can estimate the results of dynamics occurring beyond this range. The present-day multiparametric fluorescence experiment gives new opportunities for interpretation of these data and construction of improved dynamic models. A further development of the theory which would provide an improved description of the dynamics in quantitative terms with allowance for the structural inhomogeneity of protein molecules and the hierarchy of their internal motions is required. 

It should be noted that the dynamics studied by fluorescence methods is the dynamics of relaxation and fluctuations of the electric field. Dipole-orientational processes may be directly related to biological functions of proteins, in particular, charge transfer in biocatalysis and ionic transport. One may postulate that, irrespective of the origin of the charge balance disturbance, the protein molecule responds to these changes in the same way, in accordance with its dynamic properties. If the dynamics of dipolar and charged groups in proteins does play an important role in protein functions, then fluorescence spectroscopy will afford ample opportunities for its direct study. 

It is now clear that in the absence of molecular oxygen most proteins phosphoresce in aqueous solutions at ambient temperature.(10) In this chapter we discuss the use of phosphorescence of tryptophan to study proteins, with emphasis on measurements at room temperature. Comparisons between phosphorescence and the more commonly used fluorescence spectroscopy are made. Comprehensive reviews of protein luminescence have been written by Longworth.(n 12 1 A discussion on the use of phosphorescence at room temperature for the study of biological materials was given by Horie and Vanderkooi.(13)... 

D. Horsley, J. Herron, V. Hlady, and J. D. Andrade, Human and hen lysozyme adsorption A comparative study using total internal reflection fluorescence spectroscopy and molecular graphics, in Proteins at Interfaces Physicochemical and Biochemical Studies (J. L. Brash and T. A. Horbett, eds.), ACS Symposium Series No. 343, pp. 290-305, American Chemical Society, Washington, D.C. (1987). 

Fluorescence spectroscopy and its applications to the physical and life sciences have evolved rapidly during the past decade. The increased interest in fluorescence appears to be due to advances in time resolution, methods of data analysis and improved instrumentation. With these advances, it is now practical to perform time-resolved measurements with enough resolution to compare the results with the structural and dynamic features of macromolecules, to probe the structures of proteins, membranes, and nucleic acids, and to acquire two-dimensional microscopic images of chemical or protein distributions in cell cultures. Advances in laser and detector technology have also resulted in renewed interest in fluorescence for clinical and analytical chemistry. 

The metaiioporphyrins form a diverse class of molecules exhibiting complex and varied photochemistries. Until recently time-resolved absorption and fluorescence spectroscopies were the only methods used to study metailoporphyrln excited state relaxation in a submicrosecond regime. In this paper we present the first picosecond time-resolved resonance Raman spectra of excited state metaiioporphyrins outside of a protein matrix. The inherent molecular specificity of resonance Raman scattering provides for a direct probe of bond strengths, geometries, and ligation states of photoexcited metaiioporphyrins. 

Similarly, this amphiphilic polymer micelle was also used to dismpt the complex between cytochrome c (Cc) and cytochrome c peroxidase (CcP Sandanaraj, Bayraktar et al. 2007). In this case, we found that the polymer modulates the redox properties of the protein upon binding. The polymer binding exposes the heme cofactor of the protein, which is buried in the protein and alters the coordination environment of the metal. The exposure of heme was confirmed by UV-vis, CD spectroscopy, fluorescence spectroscopy, and electrochemical kinetic smdies. The rate constant of electron transfer (fc°) increased by 3 orders of magnimde for the protein-polymer complex compared to protein alone. To establish that the polymer micelle is capable of disrupting the Cc-CcP complex, the polymer micelle was added to the preformed Cc-CcP complex. The observed for this complex was the same as that of the Cc-polymer complex, which confirms that the polymer micelle is indeed capable of disrupting the Cc-CcP complex. 

The possibility to carry out conformational studies of peptides at low concentrations and in the presence of complex biological systems represents a major advantage of fluorescence spectroscopy over other techniques. Fluorescence quantum yield or lifetime determinations, anisotropy measurements and singlet-singlet resonance energy transfer experiments can be used to study the interaction of peptides with lipid micelles, membranes, proteins, or receptors. These fluorescence techniques can be used to determine binding parameters and to elucidate conformational aspects of the interaction of the peptide with a particular macro-molecular system. The limited scope of this chapter does not permit a comprehensive review of the numerous studies of this kind that have been carried and only a few general aspects are briefly discussed here. Fluorescence studies of peptide interactions with macromolecular systems published prior to 1984 have been reviewed. 

Another technique that often utilises the UV spectral range is Fluorescence Spectroscopy, ft also relies on a UV excitation, and subsequent emission perpendicular to the incident beam (see Figure 7.9). The emission can either take place with the same frequency (resonance fluorescence) or at a lower frequency (stimulated fluorescence). The latter phenomenon is rooted in the ability of the UV excited state to interact with the local enviromnent, typically through the excitation of vibrational states of the surrounding part of the protein molecule or of the solvent molecules. 

Fluorescence spectroscopy Binding of substrates, association reactions between species, denaturation of proteins and other macromolecules ... 

Nevin A, Cather S, Anglos D, Fotakis C (2006) Analysis of protein-based binding media found in paintings using laser induced fluorescence spectroscopy. Anal Chim Acta 573-574 341-346. 

---