Bratton-Marshall assay

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... 

However, more-rigorous treatment (5% acetic acid, 100°C, 17 hours) opened the imidazole ring and produced /V -cyclohexyl-a-formylaminoacetamidine (57), characterized as the crystalline picrate. Amidine 57 produced no dye in the Bratton-Marshall assay. The same behavior can be expected from AIR (46), although the product of hydrolytic ring-opening was not actually isolated. On the other hand, it was observed that a solution of AIRs (0.2 mM in 0.01-M ammonium hydroxide) prepared by biosynthesis, when stored at 4°C, did not change appreciably within a day. A decrease in the concentration of AIRs of about 30% occurred within a month. 

An important step forward in drug detection analysis utilizes techniques as the Bratton-Marshall approach in the sulfadimidine assay. This drug is converted to an azo dye in the Bratton-Marshall assay. However, the acetyl metabolite, if it is to be assayed, requires hydrolysis back to sulfadimidine and application of a different assay calculation. Unfortunately, the necessary hydrolysis conditions cause some decomposition of the unmetabolized drug, which leads to analytical errors. These errors, however, can be overcome with liquid chromatography approaches. 

Fig. 24. Activation of tryptophan pyrrolase by RNAase T,. Drosophila melanogaster were homogenized in 4 vol of 0.05 M potassium phosphate (pH 7.5) per g of frozen adult flies and centrifuged at 15,000 g for 30 min. The tryptophan pyrrolase was assayed by using a modified Bratton-Marshal assay to detect kynurenine production that results from adding 0.1 ml of homogenate to 1 ml of assay mixture consisting of 0.005 M L-tryptophan, 0.001 M 2-mercaptoethanol, 0.1 M potassium phosphate (pH 7.5) and oxygen at a concentration that results from equilibration with air. The reaction occurred at 37 °C and was stopped with 0.33 ml of 20% trichloroacetic acid at the times indicated. RNAase T, was either present at 2.5 U/ml...

Kajganovic (90) have used Bratton-Marshall reagent for quantitation after separation and elution of sulfonamide mixtures containing sulfacetamide. The solvent system chloroform-methanol-25% ammonia solution (90 15 2.4) gives good separation. The results show a relative standard deviation of about 4%. Sarsunova et al. (86) have used chloroform-ethanol (100 8) as solvent system for the separation of sulfacetamide, sulfathiazole and sulfadimidine in tablets. The sulfonamides are assayed by extraction with acetone-ethanol, separation on alumina, and determination at 270 and 290 nm. The relative standard deviations of 2.5%, 4.1% and 3.2% have been obtained for the three components with six replicates. 

Examples of the chemical reaction method are the assay of sulfonamide by the Bratton-Marshall procedure. The drug in solution is reacted with sodium nitrite to form an unstable diazonium intermediate. This unstable salt couples with a reagent yielding a reaction product with an intense color. The dye-salt extraction method may also be exemplified by the assay of atropine. Atropine in solution reacts with bromthymol blue at an appropriate pH. After shaking, the color complex formed is extracted from the aqueous layer into the organic layer and measured quantitatively. 

Averell and Norris (3) have developed an analytical method adapted to the determination of parathion in spray or dust residues, which is sensitive to about 20 micrograms. It is based upon the reduction of parathion with zinc to the amino compound, diazotiza-tion, and coupling with Bratton and Marshall s amine, which gives an intense magenta color with an absorption peak at 555 millimicrons. Bowen and Edwards (6) have used the polarograph to assay technical grades of parathion and its formulations. 

Two of these systems were studied as models—the acetylation of choline in brain to give acetyl choline (Hebb, Nachmansohn), and of sulfanilamide (the active component in prontosil, Chapter 3) in liver (Lipmann). Sulfanilamide is rapidly inactivated by acetylation on the p-amino group and then excreted. Sulfanilamide is easily diazotized the diazonium salt formed can be coupled with N-( 1 -naphthyl)ethylenedi-amine dihydrochloride to give a pink derivative (Bratton and Marshall, 1939). This formed the basis for an elegant colorimetric assay. Only the free p-amino group reacts, so that as acetylation proceeded color formation diminished. 

Pyrrolidone carboxylyl peptidase activity has been followed using pyrrolidone carboxylyl L-alanine (or other pyrrolidone carboxylyl-amino acids) as substrate by following the amount of amino acid released as determined by the ninhydrin method (137). In another assay procedure the release of -naphthyl amine from L-pyrrolidone carboxylic-/ -naphthylamide (135) is determined by the diazotization procedure of Bratton and Marshall (138) as modified by Goldberg and Rutenberg (139). 

Chemical Methods. Assay of these peptides by chemical methods is based upon the following reactions. Pteroylglutamic acid is reduced by metallic zinc in acid solution, and the liberated arylamine is diazotized and coupled with Bratton and Marshall reactant (90). The coupled compound is colored and therefore determined by photometry (305). p-Aminobenzoylpolyglutamic acid is also determined by the same method. 

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