Labeling with diazotized

Papain, Bandeiraea simplicifolia lectin, and lima bean lectin have been labeled with diazotized [ I]aniline according to the procedure described... 

For quantitative work, it is necessary to estimate the concentration of 5-amino-l-(P-D-ribofuranosyl)imidazole in aqueous solution. It seems that the only available method is the Bratton-Marshall assay, which was originally developed for the estimation of arylamines in biological fluids. The principle of the method is the spectrometric estimation of a salmon-pink colored dyestuff obtained by diazotation in situ, followed by coupling with /V-( 1 -naphthyl)ethyl-enediamine.65 The only remaining problem then is to know the molar extinction of this dye because pure samples of AIRs are not available. A value of 16800 at 520 nM was obtained for the dyes prepared from a model compound, 5-amino-l-cyclohexylimidazole-4-carboxylic acid (54), which is crystalline. A comparable molar extinction can be expected for the dye prepared from imidazole 55, if the carboxyl group does not exert too much influence on the chromophore. Actually, its influence is perceptible even with the naked eye, the dyestuff prepared from 53 having a somewhat different, wine-red color, with max>520 nM. The molar extinction for 55 is 17400 at 500 nM. When the decarboxylation of 54 was conducted under mild acidic conditions (pH 4.8, 50°C, 1 hour), estimation of 5-aminoimidazole 55 by the Bratton-Marshall method led to the conclusion that the reaction was almost quantitative.66 Similar conditions for the final decarboxylation were adopted in the preparation of samples of AIRs labeled with stable isotopes.58... 

In 1982, the first enzyme immunoassay of clenbuterol was described (134). It was used to determine clenbuterol levels in plasma of human patients treated by oral route with this drug. It was a highly sensitive double-antibody and heterologous immunoassay based on a competition for binding to a clenbuterol-specific antibody between a diazotized clenbuterol analogue labeled with -galactosidase and unlabeled standard or sample clenbuterol. The antibody-bound enzyme hapten was separated from free hapten by anti-rabbit IgG immobilized to a polystyrene ball. The assay could detect levels as low as 0.5 pg clenbuterol per tube. 

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). 

Various preparations of antisera to atropine have been reported. A racemic hemisuccinate ester was prepared and conjugated to bovine serum albumin by the carbodiimide technique. Antisera formed to the original immunogen selectively bound the R isomer (34), but a later antiserum prepared by this approach was reported to bind both R and S forms with "equal efficiency" (35). R, S-atropine was treated with diazotized p-amino-benzoic acid, and the resulting compound (which was not further characterized) was used for conjugation to bovine serum albumin by means of a carbodiimide-mediated reaction. Antisera resulting from use of this material were quite selective for the R isomer, with a cross-reaction of only about 2% for the S isomer (36). Virtanen et al. followed this procedure with S-atropine. Their antiserum bound equally to S- and R,S-atropine, as measured by displacement of tritium-labeled R,S-atropine (37). In another study (31), both racemic atropine and the S isomer were coupled to human serum albumin by the technique of Wurtzburger et al. (36), Antisera were obtained that were selective for both the R and S isomers (33). 

The potential use of diazotized [ I]di-iodosulphanilic acid as a label for cell-surface membranes has been investigated. Colorimetric determination of the compound is possible as it reacts at neutral pH with naphth-l-ol to yield a coloured complex (X ax 430 nm). Results obtained surest that when used under appropriate conditions di-iodo-sulphanilic acid can serve as a highly selective membrane label with minimal incorporation into intracellular soluble protein. 

Brief reaction of 3-aminodibenzofuran with chlorine in carbon tetrachloride at room temperature gave the 4-chloro product. After 5 min, a 1 0.4 ratio of 3-amino-4-chloro and 3-amino-1,2,4-trichloro derivatives had formed. Subsequent diazotization and reaction with Cu(I)37 Cl gave products with a labeled chlorine in the 3-position (90SC2501). 2-Methoxydibenzofuran was brominated to give the 3-bromo (33%) and a little of the 1-bromo derivative [39JA1365 84AHC(35)2],... 

Amino-3-hydroxy[3-13C]naphthalene, 88, has been obtained77 in seven steps starting with 2-(l,3-dioxolan-2-yl)-benzyl chloride and Na13CN (equation 40). The diazotation of the labelled amino compounds 88 followed by deprotonation provided [9a-13C]naphthol [2,3-d]-l,2,3-oxadiazole, 89 (equation 41). 

Labelling of proteins with 211 At via diazotized 1,4-diaminobenzene and para-aminobenzoic acid... 

Boyd and coworkers583 also labelled lima-bean lectin with 131I (indirectly, by coupling of [131I]-p-iodoaniline to protein by diazotization)584 and visible dyes,584,585 and quantitated the binding to type A erythrocytes. 

N , Reactions with. The course of the reaction between diazomethane and hydrazoic acid was studied using N -labeled reactants. It was also found that labeled methyl azide could be reliably analyzed by decompn with HI, and that unambiguously N -labeled niethyl azide could be made by treating methylurea with HN 02 and decompg the product with KOH to CHjNN (Ref 2). Another study noted that in the course of the diazotization of benzhydrazide and 2,4-dinitrophenylhydrazine with HN 02, only azides of the structure R—N=N=N were formed (Ref 1)... 

In the other method, I-labeled aniline was prepared by the chlora-mine-T method, diazotized, and allowed to react with the protein under basic conditions (Fig. 1). Presumably it couples to the phenol moiety of tyrosine residues. If this is the case, the product is analogous to that obtained by direct iodination by the chloramine-T or lactoperoxidase procedures. Papain and certain plant lectins in which cysteine residues are part of the active site were labeled without loss of activity. When variations of the chloramine-T or lactoperoxidase methods were used, 64-83% of the activity was lost. 

In the labeling of intact cells or subcellular fractions, it is imp>ortant to have the cell suspension washed thoroughly and free from extraneous proteins or protein-containing macromolecules that could couple with the diazotized ISA. 

Procedure. An aliquot of the neutralized diazotized [ I]ISA is added to a washed, buffered suspension of cells (pH 7.5) at 5°. The reaction goes to completion within 15-30 min depending on the cells being labeled. The reaction is stopped by washing the cell suspension 3 or 4 times with a lOx volume of 0.15 M NaCl containing % bovine serum albumin or serum-containing tissue culture medium (e.g., RPMI 1640 with 10% fetal calf serum). 

In a more detailed examination of the mechanism, Clusius and Schwarzenbach found that the same mixture of isotopic isomers arises both from diazotization of phenylhydrazine with labelled nitrous acid and from dehydration of specifically labelled a-nitroso-phenylhydrazine (276). It was concluded that a-nitrosophenylhydra-... 

A more selective modification, with concomitant loss of activity, has been achieved using diazotized [ °S]sulfanilic acid (130,194). The free thiol groups were protected by mercuric chloride before the tyrosine modification was performed. It was found that an incorporation of 1.5 radioactive sulfur atoms per subunit inhibited the activity. The labeled peptide was isolated and partially sequenced. By comparison with the dogfish LDH sequence the labeled tyrosine corresponds to residue 220. However, this tyrosine is nowhere near the active center of the subunit. It is most improbable that it can be associated with catalysis. 

C-labelled Direct Blue 6 (17) and Acid Red 114 (18), bis-azo biphenyl dyes, possible human carcinogens2 3b, needed in order to study their distribution and biological transformations in animals, have been prepared24 from uniformly ring-labelled 14C-benzidine and 14C-3,3 -dimethylbenzidine according to equations 7 and 8. 17 has been prepared by reacting the diazotized benzidine with two equivalents of H acid under basic conditions to insure the occurrence of the attack of the electrophilic species at the ortho... 

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