On the Nature of the Reaction between Diazo- Tized Sulfanilic Acid and Proteins

It has been generally assumed since the work of Pauly (6) and of Inouye (5) that the reactivity of proteins with diazo compounds is due to the imidazole group of hiitidine and the phenyl group of tyrosine. There are, however, several indications that these are not the only groups concerned in the reaction. Thus, Hooker and Boyd (4) found that casein coupled with diazoarsanilic acid contained approximately 78 atoms of As in each molecule of protein. Similarly, the experiment cited below shows that each molecule of serum protein can apparently inactivate on the order of several hundred molecules of diazotized sulfanilic acid. If we take 100,000 as an “average” molecular weight for the serum proteins, and take 6 and 3 per cent for the approximate tyrosine and histiclme contents, respectively (Cohn (3) p. 870), it follows that each molecule of serum protein contains on the order of twenty histidine and thirty-five tyrosine groups. Even the assumption that every imidazole and phenol group incorporated in the protein molecule remains free and reactive would not account for the observed reactivity of the protein molecule with diazo compounds. Preparation of Diazotized Sulfanilic Acid-For 200 cc. of a ~:/7 solution, 5 gm. of sulfanilic acid were treated with 1.75 gm. of anhydrous Na&03 dissolved in 75 cc. of water and with 2.0 gm. of NaNOz dissolved in 10 cc. of water. The solution was cooled in ice water and diazotized at 3-5”, a mechanical stirrer being used, by the slow addition of 35 cc. of N HCl from a separatory funnel, the tip of which was immersed in the liquid. The pale yellow, somewhat acid diazonium solution was brought to pH 7.2 to 7.6 with dilute NaOH. Since the solutions coupled