III. COUPLING OF ACETYLATED BOVIXE SERUM ALBUMIN WITH ARSANILIC ACID AND p-AMINOBENZOIC ACID*

In the first report (1) of this series, we described a spectroscopic method for the determination and differentiation of the azo dyes resulting from coupling of arsanilic acid and related compounds with tyrosine and histidine. The bisazo compound of tyrosine and the bisazoamino compound of lysine were also characterized. In the second paper (2) we showed that the spectroscopic method can be applied to the products obtained by coupling diazotized arsanilic acid with a few representative proteins. Landsteiner and other early investigators of azoproteins were interested in the immunological properties of these compounds. They tacitly assumed that the “haptene” was attached to aromatic residues, without further studying quantitative relationships, the influence of pH, and other factors. That residues other than aromatic amino acids are involved in the coupling reaction was observed by Friedheim: who had noticed that some of the coupled azo residues are cleaved off by dilute acid. Gelewitz et al. (3) ascribed the excess of arsenic over the number of azo groups, which are titratable with titanous chloride, to the formation of bisazoamino compounds from aliphatic w-amino groups, a reaction which had been reported by Busch et al. (4). A comparison of the figures for the coupling products of native bovine serum albumin, acetylated to varying degree, showed an increase in the percentage of haptene coupled with tyrosyl and histidyl radicals. For example, with equivalent amounts of haptene (see under “Experimental Procedure”) at pH 9, the percentages were 46 for nonacetylated bovine serum albumin (Table III (a)), and 71 for 87% acetylated bovine serum albumin (Table I). We have now duplicated this finding in the case of p-aminobenzoic acid; other aspects of N-acetylation will be discussed below. The complete N-acetylation of bovine serum albumin is accompanied by acetylation of the phenolic hydroxyl groups of the tyrosyl residues. Subsequent treatment with alkali permits partial or complete hydrolysis of the acetoxy groups. The acetylation of phenolic hydroxyl blocks the coupling reaction, but it will be shown that in the case of p-aminobenzoic acid coupling this effect is specifically absent. We shall also show that part of the diazotized haptene is incorporated in the protein without coupling.