Intrinsic fluorescence spectroscopy

Molecular weight of heavy and light chains Peptide mapping Amino acid analysis Intrinsic fluorescence spectroscopy Thermal denaturation monitored by fluorescence Fourier transfrome infrared spectroscopy Binding (e.g., ELISA, BiaCore, etc) Potency (e.g., cell based, ELISA)... 

Muller MG, Georgakoudi I, Zhang QG, Wu J, Feld MS. Intrinsic fluorescence spectroscopy in turbid media disentangling effects of scattering and absorption. Applied Optics 2001, 40, 4633 1646. 

T. Le Bihan and C. Gicquaud, Kinetic study of the thermal denaturation of G actin using differential scanning calorimetry and intrinsic fluorescence spectroscopy, Biochem. Biophys. Res. Commun. 194, 1065-1073 (1993). 

Structural versus functional information from intrinsic fluorescence spectroscopy... 

Ultimately though, whilst intrinsic fluorescence spectroscopy of biological macromolecules appears to be rather limited, the technique of fluorescence spectroscopy becomes considerably... 

I. Georgakoudi, M.S. Feld, M.G. Muller, Intrinsic fluorescence spectroscopy of biological tissue. In M.-A. Mycek, B.W. Pogue (eds.), Handbook of Biomedical Fluorescence. Marcel Dekker Inc. New York, Basel, 109-142... 

The major reasons for using intrinsic fluorescence and phosphorescence to study conformation are that these spectroscopies are extremely sensitive, they provide many specific parameters to correlate with physical structure, and they cover a wide time range, from picoseconds to seconds, which allows the study of a variety of different processes. The time scale of tyrosine fluorescence extends from picoseconds to a few nanoseconds, which is a good time window to obtain information about rotational diffusion, intermolecular association reactions, and conformational relaxation in the presence and absence of cofactors and substrates. Moreover, the time dependence of the fluorescence intensity and anisotropy decay can be used to test predictions from molecular dynamics.(167) In using tyrosine to study the dynamics of protein structure, it is particularly important that we begin to understand the basis for the anisotropy decay of tyrosine in terms of the potential motions of the phenol ring.(221) For example, the frequency of flips about the C -C bond of tyrosine appears to cover a time range from milliseconds to nanoseconds.(222)... 

As shown above, the intrinsic fluorescence spectra of proteins as well as coenzyme groups and probes shift within very wide ranges depending on their environment. Since the main contribution to spectral shifts is from relaxational properties of the environment, the analysis of relaxation is the necessary first step in establishing correlations of protein structure with fluorescence spectra. Furthermore, the study of relaxation dynamics is a very important approach to the analysis of the fluctuation rates of the electrostatic field in proteins, which is of importance for the understanding of biocatalytic processes and charge transport. Here we will discuss briefly the most illustrative results obtained by the methods of molecular relaxation spectroscopy. 

Fluorescence spectroscopy S Conformational change with ligand binding induces change in fluorescence properties of intrinsic or extrinsic fluorophore... 

In fluorescence spectroscopy, however, diffuse reflectance correction of spectral distortions in biological media has been studied extensively. Analytical models based on photon migration theory,44 diffusion theory46,60,61 as well as empirical models,62 have been reported to obtain intrinsic fluorescence. In the following, we will review a particular correction method based on photon migration theory for fluorescence spectroscopy and introduce its Raman counterpart. 

The same general principle that applies for intrinsic fluorescence should hold true for Raman spectroscopy as well. Unlike in fluorescence spectroscopy, spectral distortion owing to prominent absorbers is less of an issue in the NIR wavelength range. However, for quantitative analysis the turbidity-induced sampling volume variations become very significant and usually dominate over spectral distortions. 

Gardner CM, Jacques SL, Welch AJ. Fluorescence spectroscopy of tissue recovery of intrinsic fluorescence from measured fluorescence. Applied Optics 1996, 35, 1780-1792. 

A peculiar case is certainly that of the green fluorescent protein (GFP). Functional GFP variants have been identified by visual inspection of colonies based on the intrinsic fluorescence of GFP under UV illumination [24, 32 - 34]. To increase accuracy of signal quantification as well as throughput, digital image spectroscopy has been applied in some cases [32],... 

Among the other spectroscopies, either in the direct or reflection mode, fluorescence spectroscopy may be mentioned. A promising variant is Total Internal Reflection Fluorescence spectroscopy (TIRF). The decay rate of an excited fluorescing probe is usually Interpreted in terms of the local fluidity and polarity. The technique has been used to estimate the extent of ordering inside adsorbed surfactant layers, but this is not an absolute method because a fluorescent probe has to be inserted, and such probes themselves affect the local fluidity. More rigorous are fluorescence experiments with molecules that possess such a probe as an intrinsic part of their structure, such as tryptophans in... 

Neyroz P, Zambelli B, Ciurli S. Intrinsically disordered structure of Bacillus pasteurii UreG as revealed by steady-state and time-resolved fluorescence spectroscopy. Biochemistry 2006 45 8918-8930. 

Eftink MR. Intrinsic fluorescence of proteins. In Topics in fluorescence spectroscopy. Lakowicz JR, ed. 2000. Kluwer Academic/Plenum Pubhshers, New York. pp. 1-15. 

Adsorption of Fluorescent Components of Sea Water. Further investigation concerning the nature of the film substance has also been undertaken using fiuorescence spectroscopy. This technique is quite sensitive and is applicable to aqueous solutions as well as to adsorbed films in contact with aqueous media (29). One may observe the intrinsic fluorescence of a material of interest and, in addition, reagents and pro-... 

A.A. Heikal, S.T. Hess, G.S. Baird, R.Y. Tsien, W.W. Webb, Molecular spectroscopy and dynamics of intrinsically fluorescent proteins Coral red (dsRed) and yellow (Citrine). PNAS 97(22), 11996-12001 (2000)... 

Brennan, J. D., Using intrinsic fluorescence to investigate proteins entrapped in sol-gel derived materials. Applied Spectroscopy 1999, 53(3), 106A-121A... 

The effect of thermal curing on the morpholgy and conformation of PMDA-ODA and BTDA-ODA polyimides is investigated using fluorescence spectroscopy. These polyimides are found to exhibit intrinsic fluorescence. 

---