Correction of fluorescence spectra and measurement of fluorescence quantum efficiency

Parker, C. A.; Rees, W. T.

doi:10.1039/an9608500587

Cited by 1,325 publications

(585 citation statements)

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“…It is necessary to justify the fact of simply measuring the fluorescence intensity at the emission maximum instead of measuring the area under the emission spectrum. As Parker and Rees [60] pointed out, it is possible to do so once it has been established that the peak height was proportional to the area; this is expected only if the environment of the chromophores are identical. This was checked with eight different proteins dissolved in 7 M guanidine hydrochloride at 15, 20 and 25 OC.…”

Section: Correlation Between the Actual Number O J Tryptophanyl Residmentioning

confidence: 99%

Fluorescence of Proteins in 6‐M Guanidine Hydrochloride

Pajot

1976

European Journal of Biochemistry

207

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To determine the tryptophan content in proteins, an analytical ultraviolet fluorescence method is proposed based on making uniform the environment of aromatic chromophores in 6 -7 M guanidine hydrochloride.The fluorescence intensity scale is calibrated using standard solutions of free tryptophan. A correlation coefficient between the fluorescence of protein tryptophanyl residues and of free tryptophan was estimated in testing 17 well characterized proteins.This method is particularly suited to proteins carrying groups absorbing in the 290 -370-nm region, such as flavin, heme and pyridoxal phosphate and in the presence of substances such as 2-mercaptoethanol which prohibit the use of the spectroscopic or magnetic circular dichroism methods. It is less time-consuming than techniques requiring hydrolysis or chemical reactions.The determination of the tryptophan content in proteins has been a problem for a long time. In the usual procedures for amino acid analysis tryptophan is extensively destroyed during the acid hydrolysis ; therefore several independent methods have been proposed. The simplest are spectrophotometric techniques based on the 'normalization' of the absorption parameters of tyrosine and tryptophan residues in denatured unhydrolyzed proteins. The older technique of Coodwin and Morton [l] consists in measuring at two wavelengths the absorbance of the protein dissolved in 0.1 N sodium hydroxide, but the measurements are often impaired by changes in the turbidity of the solution. Since, several improvements and modifications have been proposed, for instance, the addition of urea [2] or the denaturation in guanidine hydrochloride.The latter procedure was developed by Edelhoch [3] who showed that, in the presence of guanidine hydrochloride, the absorption spectra of tyrosyl and tryptophanyl residues are effectively normalized and then could be used for quantitative determination of these residues. Nevertheless, the presence of absorbing species such as heme, flavin or added reagents limits somewhat the general applicability of this technique.In this laboratory, F. Labeyrie used the fluorescence of tryptophanyl residues of denatured proteins (6 M guanidine hydrochloride) to determine the tryptophan concentration of hemoprotein subunits. It was assumed that the fluorescence spectra of the tryptophanyl residues of the fully denatured protein is normalized, as is its absorption spectra, and that the specific fluorescence of the free tryptophan and the tryptophanyl residues, under these conditions, are identical. The method was applied to flavocytochrome h,, as was briefly reported elsewhere [4], and should be very convenient for other proteins possessing groups absorbing in the ultraviolet range.The scope of this communication is to investigate in more detail the general applicability of this technique and to establish optimal conditions for a proper determination of tryptophan content in proteins.The luminescent properties of tyrosine and tryptophan are affected by the microenvironment of the chromophores ...

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Section: Correlation Between the Actual Number O J Tryptophanyl Residmentioning

confidence: 99%

Fluorescence of Proteins in 6‐M Guanidine Hydrochloride

Pajot

1976

European Journal of Biochemistry

207

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“…A quantum yield of 0.14 for ISA-modified enzyme was obtained by using the comparative method of Parker and Reese [26]; the fluorescence and absorbance of ISA-modified enzyme is related to a solution of quinine sulfate in 0.05 M H2S04 at 25°C by using a quantum yield of 0.70 for quinine sulfate [27].…”

Section: Fluorescence Experimentsmentioning

confidence: 99%

Distance between the substrate and regulatory reduced coenzyme binding sites of bovine liver glutamate dehydrogenase by resonance energy transfer

Lark

Colman

1990

European Journal of Biochemistry

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Bovine liver glutamate dehydrogenase is known to bind reduced coenzyme at two sites/subunit, one catalytic and one regulatory; ADP competes for the latter site. The enzyme is here shown to be catalytically active with the thionicotinamide analogue of NADPH ([SINADPH) [7], and calf brain tubulin [8]. Energy transfer between two chromophores is possible as long as the emission spectrum of the energy donor overlaps the absorption spectrum of the energy acceptor. When combined with the spectral properties of the donor and acceptor, the amount of quenching of the donor's fluorescence by the acceptor leads to information about the distance between the chromophores.The activity of bovine liver glutamate dehydrogenase is affected by purine nucleotides. Either NADPH or NADH is capable of binding to the catalytic site [9] and high concentrations of these coenzymes inhibit the enzyme by binding to a distinct reduced coenzyme regulatory site [lo]. ADP, which activates the enzyme, has two binding sites per subunit [Ill.

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“…The emission quantum yield, q, of retinol bound to the 'human protein' and to the 'chicken protein' was determined by the method of Parker and Rees [13]. A quinine sulfate solution in 0.1 N H2S04 ( q = 0.55) of a known absorbance served as a reference.…”

Section: Fluorescence Measumnentsmentioning

confidence: 99%

Binding Affinities of Retinol and Related Compounds to Retinol Binding Proteins

et al. 1976

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Fluorimetric titrations were used to determine apparent dissociation constants of the all-trans isomers of retinol, retinoic acid, retinyl acetate and retinyl palmitate to human-retinol binding protein and chicken-retinol binding protein. Enhancement of the fluorescence of retinol and retinyl acetate when bound to the protein was utilized to establish the binding affinity of these compounds. With retinoic acid which is essentially a non-fluorescent compound, quenching of protein fluorescence due to energy transfer to the bound ligand from tryptophanyl residues served to determine the binding affinity. The various ligands display 1 : 1 molecular complexes with both types of retinol binding proteins. Retinol, retinoic acid and retinyl acetate were found to have similar binding affinities to both species of carrier proteins: For retinol, Kd = 1.9 x lop7 M with humanretinol binding protein and Kh = 1.

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Correction of fluorescence spectra and measurement of fluorescence quantum efficiency

Cited by 1,325 publications

References 0 publications

Fluorescence of Proteins in 6‐M Guanidine Hydrochloride

Fluorescence of Proteins in 6‐M Guanidine Hydrochloride

Distance between the substrate and regulatory reduced coenzyme binding sites of bovine liver glutamate dehydrogenase by resonance energy transfer

Binding Affinities of Retinol and Related Compounds to Retinol Binding Proteins

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