Diazotized arsanilic acid

Studies in solution indicate that the conformations of tyrosine-248 in the dissolved and crystalline states may be quite different. This conclusion is based on the work of Johansen and Vallee. They have examined the absorption and circular dichroic spectral properties of a carboxy-peptidase A derivative in which tyrosyl-248 is selectively labeled with the conformational probe, diazotized arsanilic acid 62, 63),... 

Zinc arsanilazocarboxypeptidase, the product of coupling crystalline carboxypeptidase with diazotized arsanilic acid, contains one arsanilazo-tyrosyl residue per molecule. No other residues are modified. Approximately 95% of the azotyrosine can be accounted for by the isolation of a peptide containing the label on tyrosine-248 (54). 

Dissolve and diazotize arsanilic acid (Sigma Chemical Co.) as in Table 12.4. 

Protein Antigens.—The immunizing antigens used for inoculations were made from diazotized arsanilic acid and sheep serum by the method described by Landsteiner and van der Scheer. The ratio of arsenic to protein in these antigens ranged from 2 to 3%. 

Test antigens were similarly made from piuified ovalbumin by treatment with diazotized arsanilic acid, diazo-... 

Diazotized arsanilic acid reacts with tyrosine and with histidine, lysine, tryptophan and arginine ... 

Recently Spande and Glenner (376) reinvestigated the reaction of 3-substituted indole derivatives with diazonium salts. In earlier work, it was proposed that the reaction of diazotized sulfanilic acid with tryptophan takes place at the 3-position. On the other hand, reaction of skatole with diazotized arsanilic acid was assumed to give a 2-substituted derivative, solely by analogy to the pyrroles (259, 395). 

A. Preparation of immunogens for the production of anti-hapten antibodies Arsanilic acid and A- 4-aminobenzoyl)-L-glutamic acid are diazotized and coupled to KLH (0.05 mmol/100 mg) and to BSA (0.1 mmol/100 mg), respectively, according to the method given in Table 12.4. These preparations are used for the production of antibodies which can be purified by the methods given in Section 7. 

The antigen for the feed was prepared by conjugating arsanilic acid to bovine serum albumin (Sigma, RIA grade) by the diazotization technique (14). As this material is deep red, flow distribution and the progress of column saturation can be followed visually. Breakthrough curves were detected with an Altex 153 spectrophotometer at 335 nm. 

The Bart reaction is successful with a wide variety of aromatic and heterocyclic amines. A variation in which an aromatic amine, in the presence of arsenic trichloride, is diazotized in an organic solvent (the Scheller reaction) has also found wide application. Both arsonic and arsinic acids can be prepared by the Scheller reaction which often gives better yields than the Bart reaction with electron-attracting substituents on the aromatic ring. For the commercial preparation of 4-aminophenylarsonic acid [98-50-0] (arsanilic acid), C HgAsNO, and 4-hydroxyphenylarsonic acid [98-14-6], C FTAsO the Bnchamp... 

Arsanilic acid was diazotized in hydrochloric acid solution by the addition of sodium nitrite solution at 0 to the starch-iodide end-point. The diazotizations of p (p-aminophenylazo)-phenylarsonic acid and of >-amino-benzanilide- -arsonic acid were similarly carried out at 10 ° with end-point the disappearance of the slightly soluble amine hydrochlorides. 

Carboxypeptidase.— The resonance Raman technique has been used to study the microenvironment of azotyrosyl-248 in carboxypeptidase A (CPA) which has been modified with diazotized /j-arsanilic acid the co-ordination number of the zinc is thought to be five (17). Steady-state kinetic studies on both CPA and carboxypeptidase B (CPB) have revealed significant differences between the activities of the enzymes in solution and their activities in the crystalline state, thus reinforcing the significance of the spectral results (reported in Vol. 5, p. 287) with solid and aqueous arsanolazot)nosyl-248 CPA. 

It has been found by experiment that substances of the type RX, containing two different haptenic groups, do not form precipitates with either anti-R serum or anti-X serum Alone, but do form precipitates with a mixture of the two specific antisera. This provides proof of the effective bivalence of the dihaptenic precipitating antigen, and thus furnishes further evidence for e framework theory of antigen-antibody precipitation. In these experiments tiie anti-R serum and anti-X serum were made by injecting rabbits with sheep serum coupled with diazotized p-arsanilic add and diazotized p-aminobenzoic acid, respectively, and the RX substances used were l-amino-2-/>-(p-azophenylazo)-phenylarsonic acid-3,6-disul-fonic add 7-p-(p-azophenylazo)-benzoic acid-8-hy-droxynaphthalene and l,8-dihydroxy-2-/>-azo-phenylarsonic acid-3,6-disulfonic acid-7-p-(p-azo-phenylazo)-benzoic acid-naphthalene. 

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