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A method for quantitation of amino-terminal residues in proteins is presented. The method is a modification of a double isotope-labelling technique, using 3H-labelled dansyl chloride and 14C-labelled amino acids as internal standards. The method is demonstrated on human fibrinogen, horse myoglobin and on mouse myoloma IgA. A linear relationship between the ratio 3H/14C in the separated amino-terminal amino acid of the protein and the amount of protein added in the labelling mixture was obtained with standard deviations of +/- 7.4% +/- 3.4% and +/- 10.3%, respectively. An application of the method is demonstrated by measuring the increase in amino-terminal glycine in fibrinogen following the proteolytic action of thrombin. The method seems to be useful when 0.1 nmol or more of protein is used.  相似文献   

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A sensitive method is described for the detection of amino-terminal tryptophan in peptides and proteins as the dansyl derivative. The use of the method is illustrated with a tetrapeptide and with the enzyme phospholipase C from Bacillus cereus. The method may also be applicable when internal tryptophanyl residues are encountered during dansyl-Edman manual sequencing of peptides and proteins.  相似文献   

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This report describes a simple procedure for the dansylation of membrane proteins on a thin layer of solid dansyl chloride.  相似文献   

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F-actin has been specifically labeled with a fluorescent probe, dansyl aziridine, at cysteine-373 of the protein. The fluorescence property of the conjugated probe serves as a spectroscopic indicator of several processes in which actin participates. The sulfhydryl modification does not impair the G-F transformation of actin, nor does it affect the complex formation of actin and myosin or the dissociation of the complex by ATP as judged by viscosity measurements. However, both labeled actin and actin modified by N-ethylmaleimide, which also reacts at cysteine-373, stimulate the Mg2+-ATPase of myosin only about 75% as well as unmodified actin. The probe attached to actin exhibits a 65-nm blue shift of its emission maximum from 560 to 495 nm and a sixfold fluorescence enhancement indicating that it is located in a hydrophobic environment. The excitation spectrum of labeled actin indicates that a tryptophan and a tyrosine residue are close to the probe and transfer excitation energy to the dansyl fluorophore. Upon depolymerization of F-actin, the fluorescence intensity of labeled actin increases about 20%. The fluorescence of labeled actin is also enhanced by the addition of EDTA, ATP, and pyrophosphate, but Mg2+ antagonizes this effect reversibly. However, in the presence of 10 mm orthophosphate buffer (pH 7.4) these effects disappear. When labeled F-actin binds with myosin subfragment-1 (SF-1) or heavy meromyosin (HMM), the fluorescence of the actin adduct is enhanced. The fluorescence properties of labeled acto-SF-1 and acto-HMM become insensitive to EDTA and polyphosphates even in the absence of orthophosphate. These results suggest that the two-stranded helical structure of the F-actin filament is stabilized by the presence of phosphate and/or the binding of the myosin “head”.  相似文献   

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Two dansyl derivatives: 1-(5-dimethylaminonaphthalene) sulfonyl (4-amino)-benzyl amine and 1-(5-dimethylaminonaphthalene) sulfonyl beta(4-aminophenyl) ethylamine, have been recently synthesized. Reaction of these compounds with nitrous acid lead to the corresponding dansyl-bearing diazonium salts. The latter derivatives can couple, under mild basic conditions, to the imidazole moiety of histidine, the phenolic ring of tyrosine and to the epsilon-amino function of lysine. The applicability of the two reagents was tested in the modification of several peptides, including [D-Phe6]LHRH, [D-Gln6]LHRH, Leu-enkephalin and Tyr-tuftsin, and proteins such as calmodulin, bovine serum albumin and nerve growth factor.  相似文献   

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W T Hsieh  K S Matthews 《Biochemistry》1985,24(12):3043-3049
Chemical modification using 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride) has been used to explore the importance of lysine residues involved in the binding activities of the lactose repressor and to introduce a fluorescent probe into the protein. Dansyl chloride modification of lac repressor resulted in loss of operator DNA binding at low molar ratios of reagent/monomer. Loss of nonspecific DNA binding was observed only at higher molar ratios, while isopropyl beta-D-thiogalactoside binding was not affected at any of the reagent levels studied. Lysine residues were the only modified amino acids detected. Protection of lysines-33 and -37 from modification by the presence of nonspecific DNA correlated with maintenance of operator DNA binding activity, and reaction of lysine-37 paralleled operator binding activity loss. Energy transfer between dansyl incorporated in the core region of the repressor protein and tryptophan-201 was observed, with an approximate distance of 23 A calculated between these two moieties.  相似文献   

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Dehydroalanine residues in peptides and proteins react with 4-pyridoethanethiol in alkaline solution to form 4-pyridoethyl cysteine residues, which after acid hydrolysis, give the corresponding amino acid. An experimental protocol has been developed, and with simple dehydroalanine derivatives, conversions of 96–99% are achieved. Tests with peptides and proteins show that serine and cysteine/cystine residues do not interfere with this analytical procedure. A sample of wool keratin contained 57 µmol dehydroalanine/g.  相似文献   

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