Organic Fluorescence Reagents in the Study of Enzymes and Proteins

Kanaoka, Yuichi

doi:10.1002/anie.197701371

Cited by 76 publications

(24 citation statements)

References 167 publications

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“…ANM is an N-maleimide derivative which modifies selectively the thiol groups of proteins [16]. This hydrophobic probe becomes fluorescent when it reacts with the SH groups, forming a stable adduct.…”

Section: Resultsmentioning

confidence: 99%

Identification of a highly reactive sulphydryl group in human placental glutathione transferase by a site‐directed fluorescent reagent

et al. 1990

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A fluorescent maleimide derivative, N-(4-anilino-l-naphthyl) maleimide (ANM), a specific probe for thiol groups, reacted with human placental glutathione transferase (GST, EC 2.5.1.18), causing a complete inactivation of the enzyme in a few minutes. The modified enzyme was denatured, alkylated and digested with (L-l-tosylamide-2-phenylethyl chloromethyl ketone)-trypsin. The tryptic digest was analysed by HPLC and a fluorescent peptide was obtained. The sequence of this peptide allowed us, by a comparison with a well known primary structure, to assign the position 47 to the most reactive cysteine of GST enzyme.

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Section: Resultsmentioning

confidence: 99%

Identification of a highly reactive sulphydryl group in human placental glutathione transferase by a site‐directed fluorescent reagent

et al. 1990

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“…The high reactivity of maleimide double bonds towards thiol groups has been used by Kanaoka and his group (22,23) in the design of labeling agents that become fluorescent only after reaction with the thiol group. Although capable of considerable variation with respect to the fluorescing moiety, a three-ring structure seems to be the minimum required size.…”

Section: Discussionmentioning

confidence: 99%

Bimane fluorescent labels: labeling of normal human red cells under physiological conditions.

Kosower¹,

Kosower²,

Newton³

et al. 1979

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

236

142

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The bimane fluorescent labels, monobromobimane, dibromobimane, and monobromotrimethylammoniobimane, are derivatives of syn-9,10-dioxabimane:1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione. They efficiently label hemoglobin (reactive thiol groups), membrane proteins, and glutathione of normal human red cells under physiological conditions. Monobromobimane and dibromobimane are effective on intact cells while red cell membranes may be impermeable to the positively charged monobromotrimethylammoniobimane, the latter being effective only on lysed cells. These bimane labels provide a class of labeling agents that may have wide applicability in biological materials. syn-9,10-Dioxabimanes, a new class of compounds, are highly fluorescent (1). Exceptions to this generalization are the essentially nonfluorescent bromoderivatives, illustrated by a monobromobimane (mBBr) (I), a dibromobimane (bBBr) (II), and a monobromotrimethylammoniobimane (qBBr) (III). We have now found that these three bromobimanes are highly efficient labeling agents for cells, proteins, and small molecules like glutathione, giving rise to highly fluorescent derivatives. The fluorescent derivatives are very stable in air and under irradiation. The labeling procedures are simple and can be carried out rapidly under physiological conditions. We shall describe in the present article the use of these reagents on normal human red cells. MATERIALS AND METHODSBromobimanes. The synthesis of the bromobimanes is described elsewhere (1). mBBr and bBBr were dissolved in acetonitrile and the 50 mM stock solutions were stored in the dark; the solutions are stable for at least 2 months. The salt, qBBr, was dissolved in aqueous buffer (pH 7.4) shortly before use; the 1-4 mM solutions have a half-life at 220C of approximately 4 hr.Red Cells. Human blood, anticoagulated with heparin, was obtained from normal individuals. The buffy coat was removed after centrifugation; the red cells were washed twice with 135 mM NaCI/10 mM phosphate buffer, pH 7.4, and resuspended in the same buffer to a packed cell volume of 5-10%. Ghosts were obtained from the cells by the procedure of Steck and Kant (2). Hemoglobin solutions were prepared by the centrifugation of lysed red cells at 22,000 X g for 30 min, followed by dialysis of the membrane-free supernatants against 10 mM phosphate buffer, pH 7.4.Reactions of Bromobimanes with Intact Cells, Lysates, and Hemoglobin Solutions. The reagents were added in a ratio of 5-20 ,umol of reagent per ml of packed red cells (1-4 moles of reagent per mole of hemoglobin). Usually 10-20 Aul of the stock solution was used per ml of cell suspension of 5-10% packed cell volume. The reagent solution was placed in a dry test tube and the-sample (cell suspension, hemoglobin solution) was added with rapid mixing. A stock solution (50 mM) of the mBBr reagent could be diluted with buffer to 1-2 mM before it was mixed with a sample. The bBBr compound, which is less soluble in water, was not diluted. qBBr was dissolved in buffer immediately before use...

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“…2 shows the effect of TC on fluorescence spectra of 1. Evidently addition of TC enhances and shifts to shorter wavelengths the fluorescence maximum of 1 as is observed on the inclusion of 1 into the hydrophobic regions of various host molecules (Kanaoka, 1977;Murakami et al, 1990).…”

Section: Resultsmentioning

confidence: 87%

Molecular Recognition by Natural Macrocycles. I. d‐Tubocurarine as a Host Molecule for Organic Anions

1996

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The binding of 8-anilino-1-naphthalenesulfonate and 15 anions of substituted benzoic, aliphatic dicarboxylic, and N-acetyl-alpha-amino acids to a macrocyclic alkaloid d-tubocurarine in aqueous solution has been studied by fluorometry, conductometry, and 1H NMR. The binding constants vary from ca. 50 to 3300 M-1 depending on the guest structure, charge and hydrophobicity. The results of fluorescence and NMR studies show that the host-guest complexation of the anions of aromatic acids involves the formation of a salt bridge between the quaternary nitrogen of the alkaloid and the anionic group of the guest as well as hydrophobic/Van der Waals interactions between the guest and host aromatic moieties. The binding of dianions of aliphatic dicarboxylic acids most probably is purely electrostatic. In general, d-tubocurarine possesses binding ability comparable to that of synthetic cyclophanes. It binds enantiospecifically anions of N-acetyl-alpha-amino acids and discriminates between positional isomers of anions of hydroxy and carboxy substituted benzoic acids.

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Organic Fluorescence Reagents in the Study of Enzymes and Proteins

Cited by 76 publications

References 167 publications

Identification of a highly reactive sulphydryl group in human placental glutathione transferase by a site‐directed fluorescent reagent

Identification of a highly reactive sulphydryl group in human placental glutathione transferase by a site‐directed fluorescent reagent

Bimane fluorescent labels: labeling of normal human red cells under physiological conditions.

Molecular Recognition by Natural Macrocycles. I. d‐Tubocurarine as a Host Molecule for Organic Anions

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