Ultraviolet fluorescence of the aromatic amino acids

Teale, F. W. J.; Weber, Gregorio

doi:10.1042/bj0650476

Cited by 731 publications

(302 citation statements)

References 6 publications

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“…Protein fluorescence quantum yield was evaluated by comparing areas under fluorescence spectra of the protein sample with that of an aqueous tryptophan solutton [20] wtth the same absorbance at the excitation wavelength of 280 nm. The position of the middle of a chord drawn at the 80% level of the maximum Intensity I,,, was taken as the posrtion of the spectrum.…”

Section: Methodsmentioning

confidence: 99%

Thermal transitions in the purple membrane from Halobacterium halobium

Shnyrov

Mateo

1993

FEBS Letters

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The application of a successive annealing procedure to the scanning calorimetric endotherm of the purple membrane from H&bacterium halobium in phosphate buffer. pH 7.5, leads to five thermal transitions beneath the overall endotherm. Circular dichroism and fluorescence experiments have also been carried out with the natrve membrane heated at the same scan rate as in calorimetric runs (1"Clmin) as well as with previously heated membrane samples. These results, together with others from the literature, have been used to suggest a preliminary explanation of the five thermal transitions.

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Section: Methodsmentioning

confidence: 99%

Thermal transitions in the purple membrane from Halobacterium halobium

Shnyrov

Mateo

1993

FEBS Letters

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“…Since q2, the quantum yield of tryptophan in solution, is known to be 0.20 [4] by measuring the other parameters, one can evaluate ql, the quantum yield of the tested compound. The shapes of emission bands of proteins resemble very much that of the emission band of tryptophan and their maxima fall in the range of 320-350 mp [3]. Assuming that in this narrow range the instrumental response remains constant, the correct quantum yield is obtained by multiplying the (11) in 0.1 N NaCl histidine-tryptophan side chain interaction in proteins.…”

Section: Calculation Of Fluorescence Quantum Yieldsmentioning

confidence: 99%

“…Protonatedimidazole and histidine derivatives were also shown to quench the fluorescence of the indole ring [ 1, 21. Since the fluorescence spectra of proteins are due almost exclusively to emission from the indole side chains of the tryptophan residues [3], intramolecular interaction between tryptophan and a protonated histidine side chain should reduce the total quantum yield of the protein molecule. Moreover, since only the ionized form of the imidazole is involved in this type of quenching, the deprotonation of the ionized imidazole should be followed by an increase in the fluorescence intensity.…”

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confidence: 99%

Fluorometric Detection of Histidine‐Tryptophan Complexes in Peptides and Proteins

Shinttzky

Goldman

1967

European Journal of Biochemistry

148

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A study of the fluorescence properties of cyclo(L-histidine-L-tryptophan), cyc$o(L-histidine-Dtryptophan), a,N-acetyl-L-histidine-L-tryptophan methyl ester, a,N-acetyl-L-histidine-L-tryptophan and (L-histidine-L-tryptophan), has revealed an intramolecular interaction between the indole and the protonated imidazole side chains which results in marked quenching of the fluorescence intensity of the indole moiety. All five model compounds were found to possess a characteristic fluorometric titration curve which practically coincides with the potentiometric titration curve of the imidazole side chain of the histidine residue. Theoretical derivations of the pK from both titration curves are given. The extent of quenching by the protonated imidazole moiety, however, is different for each compound and varies from 40 to 8001,.To detect the presence of intramolecular histidine-tryptophan complexes in natural peptides and proteins, fluorometric titrations in aqueous solutions were carried out in the range of pH 4-9. Marked changes in fluorescence intensity with pH in the range of p H 5 to 8.5, suggesting the existence of well defined complexes, were observed with papain, activated papain, chymopapain, ficin and bromelain. Glucagon, pepsinogen, pepsin and goose egg white lysozyme show no change in fluorescence in this pH range. I n systems where a histidine-tryptophan interaction was observed, the pK value of the interacting histidine was evaluated from the fluorometric titration curve.The formation of complexes of the charge-transfer type in aqueous solution between compounds containing an indole ring and compounds containing a protonated imidazole ring has been recently demonstrated [1,2]. Protonatedimidazole and histidine derivatives were also shown to quench the fluorescence of the indole ring [ 1, 21. Since the fluorescence spectra of proteins are due almost exclusively to emission from the indole side chains of the tryptophan residues [3], intramolecular interaction between tryptophan and a protonated histidine side chain should reduce the total quantum yield of the protein molecule. Moreover, since only the ionized form of the imidazole is involved in this type of quenching, the deprotonation of the ionized imidazole should be followed by an increase in the fluorescence intensity. It might therefore be expected that a fluorometric titration (i,e., a determination of the change in fluorescence intensity with pH) of various systems containing interacting histidine and tryptophan side chains, will enable the evaluation of the pK of the imidazole moiety participating in the complex. It should be born in mind, however, that proteins usually contain other titrable quenchers, such as the unionized E-amine of lysine [4,5], the unionized carboxyl group [6,7], and the ionized a-amine [5-71.I n this study, we describe the fluorometric titration of a scries of model compounds containing histidyl and tryptophyl residues as well as of several proteins. From these titration curves, we have derived the extent of quenching and the ba...

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“…Complex with NADPH The fluorescence of most proteins is associated with their ultraviolet absorption with a major contribution of the tryptophan residues [16,17]. When excited at 285 nm the aspartokinase I-dehydrogenase I has a single emission band around 340 nm, entirely attributable to its aromatic residues (Fig.…”

Section: Studies Of the Fluorescence Of The Protein And Of Itsmentioning

confidence: 99%

The Threonine-Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K 12. Binding of Threonine and of Pyridine Nucleotides: Stoichiometry and Optical Effects

Janin

Truffa‐Bachi

et al. 1969

Eur J Biochem

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The aspartokinase I-homoserine dehydrogenase I of Escherichk coli K 12, composed of six subunits of molecular weight 60,000, binds cooperatively six molecules of L-threonine. A maximum of three molecules of NADP+ or NADPH, measured by a number of techniques, is bound in the presence or in the absence of threonine.Characteristic effects of L-threonine on the protein include a perturbation of its absorption and fluorescence spectra and a quenching of the fluorescence of the protein-NADPH complex. These findings are discussed in view of the structure of the protein.Physical and chemical studies of the protein carrying the aspartokinase I and homoserine dehydrogenase I of E . coli K12 [i, 21 suggest that its six constitutive subunits are identical. The two enzymic functions imply binding sites for their respective substrates, for activating cations (K+ and Mg++) and for the allosteric inhibitor, L-threonine. Kinetic data gave an approximation of the affinities for the different ligands ; threonine and the pyridine nucleotides were found to be most amenable to study. The stoichiometry of their binding is presented here and discussed in relation with the subunit structure. Effects of threonine upon the ultraviolet spectrum of the protein and the fluorescence of its complex with NADPH are also demonstrated. MATERIAL AND METHODS Preparation of the ProteinThe pure, electrophoretically homogeneous protein was obtained from extracts of E . wli K12 as described by Truffa-Bachi et al. [l]. The specific activity of homoserine dehydrogenase was a t least 47 units per mg in fresh preparations, and the experiments described here were performed on freshly purified protein unless specified. Enzymic activities were measured and expressed as described in [i]. BuffersBuffer P contained 20 mM potassium phosphate pH 7.2, 2 mM magnesium titriplex and 1 mM dithiothreitol. Buffer A has been described earlier [ l ] and is actually buffer P plus 0.15 M KC1 and 4mM m-threonke. Buffer B is buffer P plus 0.15 M KC1.The protein resuspended from 5001, ammonium sulfate precipitate was equilibrated with buffer by passing it on a small column of Sephadex G 25; exchanging buffers was done the same way. Chemicals and RadiochemicalsThe salts and chemicals used were the same as in [i]. L-Threonine (allo-free) was obtained from Fluka, NADP+ and NADPH from Sigma.~-[~4C]Threonine (uniformly labelled, 81.6 mC/ m o l e ) was obtained from the Commissariat 21 1'Energie Atomique, [carbonyl-14C]nicotinamide dinucleotide phosphate 18.7 mC/mmole, from the Radiochemical Centre (Amersham, England). The purity of the labelled nucleotides was checked by thin layer chromatography. Protein DeterminationProtein was determined by biuret related to dry weight by the following method: about 5 mg of purified protein was dialyzed for 2days against distilled water, and lyophilized. Two fractions were then weighed on a microbalance and subjected to the biuret reaction. The remainder was analyzed for C, H, N, content on a Technicon CHN Analyzer. The measured content in carbon...

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Ultraviolet fluorescence of the aromatic amino acids

Cited by 731 publications

References 6 publications

Thermal transitions in the purple membrane from Halobacterium halobium

Thermal transitions in the purple membrane from Halobacterium halobium

Fluorometric Detection of Histidine‐Tryptophan Complexes in Peptides and Proteins

The Threonine-Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K 12. Binding of Threonine and of Pyridine Nucleotides: Stoichiometry and Optical Effects

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