Dinitrophenyl method

End group analysis, determination of N-terminal (— dinitrophenyl method, —> dansyl method, Edman degradation) and C-terminal C-terminal end group analysis, Akabori method) amino acid residues in the primary structure analysis of peptides and proteins. 

Kerr and Godin [90], using the dinitrophenylation method of Sanger [91], have identified valine, threonine, glycine, alanine, serine, glutamic acid, and aspartic acid as N-terminal amino acids in human hair. Quantitative data... 

Lessen rearrangement and identification of the amino acid involved in intermediate formation. The dinitrophenylation method and Lessen rearrangement procedure followed the description of Gross and Morrell (22). The solution of hydroxamate modified was adjusted to pH 8 by the use of sodium bicarbonate and incubated with an equal amount of 1% 2,4-dinitrofluorobenzene in ethanol for 30 min at room temperature. After the reaction, the excess of 2,4-dinitrofluorobenzene was extracted with ether several times, until no yellow color was observed. The protein was dissolved in 0.1 N NaOH and the resultant solution was immersed in boiling water for 10 min. After acidification, the product was lyophilized, then hydrolyzed in a sealed tube containing 6 N HCl for 24 hr at 110 (23). The amino acid analysis was performed on a 2 x 53-cm column of PA-15 in a Beckman amino acid analyzer Model 121, eluting with 0.35 N sodium citrate, pH 3.8, at 30o. The peak of 2,3-diaminopropionic acid appeared at 231 min, and 2,4-diaminobutyric acid was eluted at 240 min. 

New Methods for the Synthesis of 2-Amino-2-deoxyglucosides Utilizing N-2, 4-Dinitrophenyl (DNP) Derivatives. P. F. Lloyd and M. Stacey, Chem. Ind. (London), (1955) 917. 

GPT activity was determined by the colorimetric method with 2,4-dinitrophenyl-hydrazine (refs. 12,13). Results were calculated on the basis of the calibration prepared from pyruvate, made for each series of determinations. The amount of pyruvate (pmol/cm ) formed during 1 h incubation at 37 °C was assumed as the activity unit. 

A simple and rapid method of separating optical isomers of amino acids on a reversed-phase plate, without using impregnated plates or a chiral mobile phase, was described by Nagata et al. [27]. Amino acids were derivatized with /-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA or Marfey s reagent). Each FDAA amino acid can be separated from the others by two-dimensional elution. Separation of L- and D-serine was achieved with 30% of acetonitrile solvent. The enantiomers of threonine, proline, and alanine were separated with 35% of acetonitrile solvent and those of methionine, valine, phenylalanine, and leucine with 40% of acetonitrile solvent. The spots were scraped off the plate after the... 

Unfortunately, there are no universal methods to detect all types of protein oxidation, because the products formed can be so diverse in nature. However, some forms of protein oxidation can be assayed using chemical modification (Davies et al., 1999 Shacter, 2000). In particular, the formation of carbonyl groups on proteins can be targeted using the reagent 2,4-dinitrophenyl-hydrazine (DNPH). This compound reacts with aldehydes to form 2,4-dinitrophenylhydrazone derivatives, which create chromogenic modifications that can be detected at high sensitivity in microplate assays or Western blot analysis (Buss et al., 1997 Winterbourn et al., 1999). 

The most commonplace substrates in energy-transfer analytical CL methods are aryl oxalates such as to(2,4,6-trichlorophenyl) oxalate (TCPO) and z s(2,4-dinitrophenyl) oxalate (DNPO), which are oxidized with hydrogen peroxide [7, 8], In this process, which is known as the peroxyoxalate-CL (PO-CL) reaction, the fluorophore analyte is a native or derivatized fluorescent organic substance such as a polynuclear aromatic hydrocarbon, dansylamino acid, carboxylic acid, phenothiazine, or catecholamines, for example. The mechanism of the reaction between aryl oxalates and hydrogen peroxide is believed to generate dioxetane-l,2-dione, which may itself decompose to yield an excited-state species. Its interaction with a suitable fluorophore results in energy transfer to the fluorophore, and the subsequent emission can be exploited to develop analytical CL-based determinations. 

In earlier studies the in vitro transition metal-catalyzed oxidation of proteins and the interaction of proteins with free radicals have been studied. In 1983, Levine [1] showed that the oxidative inactivation of enzymes and the oxidative modification of proteins resulted in the formation of protein carbonyl derivatives. These derivatives easily react with dinitrophenyl-hydrazine (DNPH) to form protein hydrazones, which were used for the detection of protein carbonyl content. Using this method and spin-trapping with PBN, it has been demonstrated [2,3] that protein oxidation and inactivation of glutamine synthetase (a key enzyme in the regulation of amino acid metabolism and the brain L-glutamate and y-aminobutyric acid levels) were sharply enhanced during ischemia- and reperfusion-induced injury in gerbil brain. 

The best previous method of preparation, the chlorinolysis of bis- (2,4-dinitrophenyl) disulfide by sulfuryl chloride in the presence of pyridine,11 requires much more time and effort with results that are uncertain, even for experienced operators. 

Die Methode ist an dem Protein Ribonuklease ausprobiert worden Sokolovsky nnd Patchornik (P5)). Es hat sich hicrbci als notwendig erwiesen, nach der Einfiilirung des Dinitrophenyl-Restes eine Acetylie-ning der Aminogruppcn dcs Proteins vorzunehmen, um Reaktionen des Dehydroalanins mit Aminogruppcn zu vermeiden. 

Die Methode von Sanger. 2,4-Dinitrofluorbcnzol reagicrt bei pjj 8-9 mit Aminogruppen quantitativ unter Bildung von N-Dinitrophenyl-Aininosauren (DNP-Aminosauren). Diese sind gelb gefarbt und meist sehr saurestabil. 

A new method of prepn of 2,4-dinitrophenyl deci vs oi ami no alcohols, among them amino-ethanols)... 

Solid-phase techniques can be applied to the N-activated esters.16 In the activated ester method, 4-nitrophenyl, 12 21-27 2,4-dinitrophenyl, 614 28 2,4,5-trichlorophenyl, 29 and penta-fluorophenyl 8 esters can be used, the amide bonds being formed with aminolytic release of the corresponding phenol (Scheme 4). For example, the use of pentafluorophenyl esters in the synthesis of pure azapeptides (named azatides for the first time) proved to be advantageous. 8 ... 

The high reactivity of di- and trinitrophenyl fluorides towards nucleophiles has been used for the arylation of various N-nucleophiles. A method was developed for the determination of N-terminal amino acids in peptides. Thus, nucleophilic attack of the amino acid nitrogen at Sanger s reagent (2,4-dinitrophenyl fluoride, 4), hydrolysis and subsequent analysis of the N-(2,4-dinitrophenyl)amino acid allows determination of the amino acid.162,163 Although this method has been replaced by more efficient procedures, it marked a milestone in the elucidation of peptide structures (Nobel Prize 1958). A variety of N-nuclcophilcs (no amino acids) which have been used in the nucleophilic substitution of 2,4-dinitrophcnyl fluoride is listed. 

N,N B is(dinitrophenyl) urea T etranitro-sym -diphenyl-urea or Tetranitro-carbanilide, (O2N)2C6H3.NH.CO.NH.C6H3(N02)2 mw 392.24, N 24.43%. Two isomers are described in the literature N,N -B is(2, 4-dinitropbenyl) -urea, yel ndls(from coned HN03), mp begins to dec ca 150°, melts ca 218 with decomposi-tion(Ref 1) and N,N -Bis(3,5-dinitropbenyl) urea, yel ndls(frorn alc+w), mp 265° (Ref 2). Other props and methods of prepn are given in the Refs. Expl props of these tetranitro... 

These amino-acid derivatives can be separated from the ordinary amino acids resulting from hydrolysis of the peptide because the low basicity of the 2,4-dinitrophenyl-substituted nitrogen (Section 23-7C) greatly reduces the solubility of the compound in acid solution and alters its chromatographic behavior. The main disadvantage to the method is that the entire peptide must be destroyed in order to identify the one A-terminal acid. 

---