Fluorodinitrobenzene method

The N-terminal amino acids are generally determined by the fluorodinitrobenzene method (DNP method, Sanger s reagent) ... 

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. 

Fluorodinitrobenzene method(Fig. 4). This method very slightly overestimates available lysine, because F-DNB reacts a little with the e-NH linkage of blocked lysine". This slight imperfection of the F-DNB method for early Maillard damage does not affect the general validity and usefulness of this method for detecting heat damage to food. 

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). 

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

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). 

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. ... 

For the fluorodinitrobenzene assay apply the method for raw opium using a weight of powdered tablets equivalent to 0 2 g of papaveretum. 

It is of equal importance to ascertain the sequence of amino acids in naturally occurring peptides as in peptides found in protein hydrolyzates (cf. Chapt. IV-2). The methods can be indicated only briefly. Terminal amino groups are allowed to react with fluorodinitrobenzene. Hydrolysis frees the characteristically yellow dinitrophenylamino compounds (Sanger). 

---