Tryptophan nitrous acid

Aromatic amine-containing haptens are converted to diazonium salts with ice-cold nitrous acid. Diazonium salts can then react with a protein at alkaline pH (around 9) through electrophilic attack of the diazonium salt at histidine, tyrosine and(or) tryptophan residues of the carrier protein (Table 7). 

Aromatic amines may be converted to their diazonium salts with nitrous acid. The hapten may then be bound via azo linkages to the tyrosine (shown), histidine, lysine, and possibly arginine and tryptophane residues of the carrier protein by mixing the... 

Much of Landsteiner s pioneer work was carried out with haptens that were aromatic amines. The compounds were converted to diazonium salts with nitrous acid and aUowed to react with proteins at alkaline pH (approximately 9). Reaction occurred primarily with histidine, tyrosine, and tryptophan residues of the protein carrier. For a representative procedure, see Kabat (p. 799 seq.). An interesting application of this procedure was the preparation of a chloramphenicol-protein conjugate which was used to elicit antibodies specific for chloramphenicol. In this case, a prior reduction of the nitro group of chloramphenicol to an amino group was required. As early as 1937, carcinogenic compounds were conjugated to protein carriers by means of their isocyanate derivatives which were prepared from amines. Immune sera were raised, and their properties were studied. - ... 

Pbilpot and Small (8) noted that tryptophan reacts nearly 60 times as fast with nitrous acid as does tsrrosine and that it also becomes yellow but concluded from rate measurements that it was not important in the... 

Although the reaction of nitrous acid with peptides and proteins has been extensively used for analytical purposes (van Slyke estimation), the nature of the organic products has often been overlooked. Recently, Bonnett and Holleyhead 49) reported that treatment of N-acyl-tryptophan derivatives with sodium nitrite under mildly acidic conditions furnishes nitrosamines with the nitroso group located at the indolic nitrogen. 

Conversion of tryptophan or other indole derivatives into a nitro-samine by treatment with nitrous acid followed by diazotization with N-l-naphthylethylene diamine dihydrochloride has been recently studied by Goswami (752). The method seems to be sensitive and to follow Beer s law over of wide range of concentrations of tryptophan. Selective nitrosation of tryptophan is achieved at room temperature in 1 M HCl and diazotization at 10° C or below. Sodium chloride was found to interfere with the assay. The temperature must be controlled with care, since the diazotization reaction is exothermic and diazonium compounds are stable only at low temperatures. This fact would explain the optimum temperature of 10 or 6° C in the diazotization reaction. 

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