1,2,4-Fluorodinitrobenzene preparation

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. 

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). 

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. 

When the amount of opium present in a preparation is low, a colorimetric method must be used. The fluorodinitrobenzene method is applicable to most preparations containing 0 1 per cent or more of morphine, but not less since the sample size required becomes too large. Two colorimetric methods are of sufficient importance to discuss here ... 

For application of the fluorodinitrobenzene method to this preparation, transfer 100 ml to a porcelain dish and evaporate to a volume of about 10 ml on a water-bath. Add about 5 g of chromatographic aluminium oxide and continue the evaporation to dryness. Continue by the appropriate method for raw opium. 

The fluorodinitrobenzene assay given above can be applied for morphine. If papaveretum has been prepared from the total alkaloids of opium the full procedure for determination of morphine in raw opium must be used. Since codeine, narcotine and papaverine have been shown to cause no interference in the precipitation of morphine as the dinitrophenylether, however, it is unnecessary to adopt the column procedure where the papaveretum is a mixture of pure alkaloids. 

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