The ultraviolet fluorescence of proteins in neutral solution

Teale, F. W. J.

doi:10.1042/bj0760381

Cited by 782 publications

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“…Several years ago, using urea as the denaturing agent, Teale [6] observed that the maxima of the emission spectra of proteins shifted toward that of free tryptophan; this is verified also in the presence of guanidine hydrochloride, the uniformity is even better once the disulfide bridges are reduced by the action of 2-mercaptoethanol. Table 1 gives some data concerning various proteins ; they have been obtained without any correction related to the wavelength dependence of thc particular optical system and photoreceptor.…”

Section: Normalization Of the Fluorescence Parameters Of Proteins In mentioning

confidence: 99%

“…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 : this was shown in the pioneering studies of Weber and his colleagues [5,6] and has been further investigated since. Depending upon the location within the polypeptide structure and the neighboring charges, several classes of residues have been distinguished in native proteins [7-91; each class exhibits a distinct spectral emission maximum, spectral band width, lifetime and quantum yield.…”

mentioning

confidence: 99%

“…The luminescent properties of tyrosine and tryptophan are affected by the microenvironment of the chromophores : this was shown in the pioneering studies of Weber and his colleagues [5,6] and has been further investigated since. Depending upon the location within the polypeptide structure and the neighboring charges, several classes of residues have been distinguished in native proteins [7-91; each class exhibits a distinct spectral emission maximum, spectral band width, lifetime and quantum yield.…”

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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: Normalization Of the Fluorescence Parameters Of Proteins In mentioning

confidence: 99%

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Fluorescence of Proteins in 6‐M Guanidine Hydrochloride

Pajot

1976

European Journal of Biochemistry

207

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“…The fluorescence emission spectrum of cholera toxin (Fig. 3) in 0.02 M Tris-acetate at pH 7 shows a maximum centered at 342 nm which is typical of proteins containing tryptophan (28). Upon addition of GM1 (7 ,uM), the peak emission is shifted to 330 nm.…”

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Cholera toxin interactions with thyrotropin receptors on thyroid plasma membranes.

Mullin¹,

Aloj²,

Fishman³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

115

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Unlabeled cholera toxin inhibits [125I] Cholera toxin can stimulate adenylate cyclase activity in thyroid plasma membranes. Its effect is not additive with that of thyrotropin, and cholera toxin can inhibit thyrotropin stimulation of the adenylate cyclase activity. NAD enhances cholera toxin stimulation of adenylate cyclase activity but has no enhancing effect on the stimulatory activity exhibited by thyrotropin.The GM1 ganglioside [galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)galactosylglucosylceramideI in thyroid plasma membranes can be tritiated by treating membranes with galactose oxidase, followed by reduction with 3-H-labeled sodium borohydride. Cholera toxin at a concentration which yields maximal inhibition of thyrotropin binding to thyroid plasma membrane receptors prevents the labeling of GMI.Fluorescence data indicate that the interaction between cholera toxin and GM1 results in a conformational change in the cholera toxin molecule. Analogous conformational alterations cannot be detected upon exposure of cholera toxin to 5-fold higherIn a previous report (1) we suggested that thyrotropin (TSH) and cholera toxin have an analogous mode of interaction with receptors on thyroid plasma membranes. We hypothesized that in both the case of TSH and cholera toxin, the ,B or B subunit interacts with specific cell surface receptors having gangliosides or ganglioside-like oligosaccharides as part of their core structures. This binding induces a conformational change in the hormone or toxin which results in translocation of an "active" a or A subunit within the cell membrane and adenylate cyclase activation. The evidence which led to this hypothesis can be summarized as follows. Thyrotropin and cholera toxin both bindAbbreviations:

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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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The ultraviolet fluorescence of proteins in neutral solution

Cited by 782 publications

References 9 publications

Fluorescence of Proteins in 6‐M Guanidine Hydrochloride

Fluorescence of Proteins in 6‐M Guanidine Hydrochloride

Cholera toxin interactions with thyrotropin receptors on thyroid plasma membranes.

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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