1,2,4-Fluorodinitrobenzene

Natural fluorine consists entirely of the isotope F and fluorine isotope effects (F KIE) are expected to be small. They were measured for the first time in 1993 [45] in the SNAr reaction of i F/ F fluorodinitrobenzenes with piperidine (Scheme 2). Their value (1.0262 0.0007) [45, 60] suggests a substantial C-F bond breaking in the rate-limiting step of the reaction. 

Various experimental evidence suggests that only 2 or 3 of the 9 tyrosine residues are on the surface of the enzyme (19, 55). Indeed only a part of the tyrosine residues can be easily modified by acetylimidazole at pH 7.5 or by tetranitromethane at pH 8.0 (H. Kasai, K. Takahashi, and T. Ando, unpublished). As enzymes thus modified have catalytic activity, the tyrosine residues that are probably located at the surface of the enzyme do not seem to be essential for activity. Consistent results were also obtained from the modification by fluorodinitrobenzene or by diazo-lH-tetrazole (H. Kasai, K. Takahashi, and T. Ando, unpublished). Especially noteworthy is the derivative, in which one to two tyrosine residues, amino terminal alanine, and one lysine residue were modified with diazo-lH-tetrazole. The derivative was deprived of most of its activity toward RNA but retained about 50% of its activity toward guanosine 2, 3 -cyclic phosphate. This may be explained by some steric hindrance owing to the modification of a tyrosine residue near the active center. 

The original evidence for activation by modification of cysteine residues came from studies with 2,4-fluorodinitrobenzene (15). Incubation of the crystalline enzyme preparations with 4 equivalents of FDNB led to a marked increase in activity in the neutral pH range, together with a small decrease in the activity assayed at alkaline pH (Fig. 4). The modified enzyme showed two broad and nearly equal activity maxima one at pH 7.7 and the other at pH 9.0. When dinitrophenylation was carried out at pH 7.5, this change in catalytic properties was associated with the modification of only 2 of the 20 cysteine residues in the protein (16). These 2 highly reactive cysteine residues were found to be completely protected against the action of FDNB by addition of FDP. 

The third step is to determine the polypeptide chain end groups. If the polypeptide chains are pure, then only one N-terminal and one C-terminal group should be detected. The amino-terminal amino acid can be identified by reaction with fluorodinitrobenzene (FDNB) (fig. 3.18). Subsequent acid hydrolysis releases a colored dinitrophenol (DNP)-labeled amino-terminal amino acid, which can be identified by its characteristic migration rate on thin-layer chromatography or paper electrophoresis. A more sensitive method of end-group determination involves the use of dan-syl chloride (see Methods of Biochemical Analysis 3B). 

When RNase A is treated with iodoacetate (ICH2CO2 ), the two major products obtained are carboxymethylated derivatives of His 12 and His 119. Both of these enzymes are severely inhibited, which suggests that both His 12 and His 119 are important in the active site. The enzyme also is completely inhibited by the reaction of Lys 41 with fluorodinitrobenzene. 

Transport inhibitors, dinitrophenol, DNP, 257 fluorodinitrobenzene, FDNB, 251 gramicidin, 255 N-ethylmaleimide, NEM, 250 ouabain, 259, 260 tetraethyl ammonium, TEA, 253 tetrodotoxin, 254, 256 Trifolium subterranean, migration of, 213, 214 Triple helix formation, 141 Two genes, one polypeptide, 2,59... 

Several early workers reported the availability of lysine in breads (4, J 0, 21 ). Palamidis and Markakis (12) reported that the total lysine content decreased with baking or toasting. Available lysine contents significantly decreased as the heat exposure increased. This correlates highly with PER. However, existing techniques, such as fluorodinitrobenzene (FDNB) (2J ) and trinitrobenzenesulfonic acid (TNBS) 22), for chemically determining available lysine suffer interferences and inaccuracies. 

Fluorodinitrobenzene ( FDNB )-reactive lysine The direct FDNB method of Carpenter ( 8 ) was used with some further ( % J 0 ) modifications. 

FDNB = fluorodinitrobenzene SA = succinic anhydride DAN = dansyl chloride DB = dye-binding with Orange 12 nd = not determined... 

Pentz et al. (P5) estimate taurine with fluorodinitrobenzene in urine passed through Dowex 50 H+ columns, but there are doubts as to whether this procedure is really specific for taurine (B38). Dent et al. have compared results obtained for the estimation of sulfur-containing amino acids in urine of cystinuric patients, by polarographic and microbiological methods (D18, D19). Hier (H12) and Schreier and Pliickthun (S10, Sll) have published data on amino acid excretion as determined microbiologically. Enzymatic methods have been used with success in the case of histidine in urine with specific decarboxylase preparations (S23). 

The N-terminal amino acid of a protein can be determined by reacting the protein with dansyl chloride or fluorodinitrobenzene prior to acid hydrolysis. The amino acid sequence of a protein can be determined by Edman degradation which sequentially removes one residue at a time from the N terminus. This uses phenyl isothiocyanate to label the N-terminal amino acid prior to its release from the protein as a cyclic phenylthiohydantoin amino acid. 

The amino-terminal (N-terminal) residue of a protein can be identified by reacting the protein with a compound that forms a stable covalent link with the free a-amino group, prior to hydrolysis with 6 M HC1. The labeled N-terminal amino acid can then be identified by comparison of its chromatographic properties with standard amino acid derivatives. Commonly used reagents for N-terminal analysis are fluorodinitrobenzene and dansyl chloride. If this technique was applied to the oligopeptide above, the N-terminal residue would be identified as Val, but the remainder of the sequence would still be unknown. Further reaction with dansyl chloride would not reveal the next residue in the sequence since the peptide is totally degraded in the acid hydrolysis step. 

Deacylation of deferriferrichrysin occurs in 20 min. in IN HCi at 100°. Trihydrochloride so obtained may be reacylated and, after removal of O-acetyl in methanolic ammonia and re-insertion of iron, ferrichrysin is obtained. The trihydrochloride hexapeptide may be reduced with platinum oxide and H2. Treatment of the latter compound with 2,4-dinitrofluorobenzene followed by hydrolysis gives N8-DNP ornithine. Hydrolysis of ferrichrysin in 57% HI gives serine, glycine, alanine, ornithine and ammonia in the molar ratios 0.24 1.00 1.50 2.91 0.09. Hydrolysis of deferriferrichrysin in 6N HCI, reduction of the hydrolysate with H2 and platinum oxide and addition of excess fluorodinitrobenzene yields crystalline derivatives of glycine, L-serine and L-ornithine. 

All peptides and proteins, unless they are cyclic, contain the so-called N- and C-terminal residues a free a-amino group at one end and a free a-carboxyl group at the other. The identity of such groups may be determined by various chemical and enzymatic means. One of the first such methodologies utilized fluorodinitrobenzene (FDNB) for N-terminal group analysis ... 

Dinitration to obtain 1,2,4-fluorodinitrobenzene was described by Holleman and Beekman [47] who nitrated p- fluoronitrobenzene. Zahn and A. Wurz [48] prepared the same dinitrocompound by nitrating fluorobenzene. 1,2,4,6-Fluorotrini-trobenzene (picryl fluoride) was prepared by Olah, Pavlath, J. Kuhn and Varsanyi [49] by acting on 1,2,4-fluorodinitrobenzene with a nitrating mixture composed of fuming nitric acid and 60% oleum. The yield was 36% of theoretical. 

Cysteine-149 also reacts selectively with a number of other reagents such as p-fluorodinitrobenzene (63), tetrathionate (64-66), iodosobenzoate... 

The iV-terminal groups in elastin fibers and the two derived proteins were estimated by the fluorodinitrobenzene technique. The elastin of the fibers contained 0.29 moles of iV-terminal residues in 100,000 gm and the a- and /3-proteins contained 24.4 and 32.5 moles of i f-terminals respectively in the same weight of protein. By comparison with the molecular weight of these samples it was deduced that -protein consists of molecules composed on the average of two chains, each containing 27 amino acid residues the a-protein has, as a mean value, 17 such chains containing 35 residues each. 

SYNS 2,4-DiNiTROFLuoROBENZENE 2,4-DNFB l-FLUORO-2,4-DINITROBENZENE 1,2,4-FLUORODINITROBENZENE... 

FLUORO-2,4-DINITROBENZENE see DUW 400 1,2,4-FLUORODINITROBENZENE see DLAX 400 FLUOROETHANE see FIBOOO FLUOROETHANOIC ACID see FICOOO... 

The action of trypsin on proteins and peptides can be limited to the bonds formed by arginine if the e-ammonium groups of lysine are modified. Reaction of proteins with fluorodinitrobenzene (Anfinsen and Redfield, 1956), carbobenzoxychloride (Anfinsen et al., 1956), 0-methylisourea (Weil and Talka, 1957), succinic anhydride (Li and Bertsch, 1960), potassium cyanate (Stark and Smyth, 1963), carbon disulfide (Merigan et al.,... 

Two procedures have been described by Nakane and co-workers. In the first of these free amino groups on the peroxidase are blocked by fluorodinitrobenzene (FDNB) treatment prior to the production of active aldehyde groups by periodate oxidation. A recent modification of this method, described here, omits FDNB blocking and recommends periodate oxidation of the enzyme at low pH prior to coupling with immunoglobulin. ... 

The masking of the reactions of the amino groups of a-amino acids by forming the copper(II) complexes is found to be effective for a large number of other reagents, including fluorodinitrobenzene (59) and acetylation reagents (34f 56),... 

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