Orange reaction with proteins

II. Xanthoproteic Reaction.—When proteins are treated with strong nitric acid a yellow coloration or yellow solid is formed. It is sometimes necessary to heat the solution to boiling to get a positive test. If an excess of sodium hydroxide is added to the solution, the yellow substance formed by the acid is changed in color to orange. The production of the color in this test is probably associated with the presence of a benzene ring in the compound. When many simple derivatives of benzene are treated with strong nitric acid, the nitro-substitu-tion-products formed have a yellow color. 

Mix 0.5 ml of the peptide solution with 0.5 ml of the carrier protein solution. Chill on ice. Add 0.4 ml of the fois-diazotized tolidine solution. There should be a color change from orange to red almost immediately. Continue the reaction for 2 hours on ice in the dark. 

The shell precursors in the vitelline cells - proteins, phenols and phenol oxidase (EC 1.14.18.1, monophenol o-diphenol oxygen oxidoreductase) - can all be stained specifically with cytochemical reagents although the reactions are not as intense as in trematodes (810). The most useful of these reagents are probably (a) Fast Red Salt B, which stains phenolic materials orange/purple, and (b) catechol, which can be used for detecting the phenol oxidase. Details of these techniques are given by Smyth (789). 

The non-enzymatlc determination of creatinine in urine and serum developed by van Staden [21] is based on Jaffa s reaction [22,23], which involves the formation of a red-orange compound on Interaction of the analyte with picric acid in an alkaline medium. The manifold used Is shown in Fig. 14.5. The sample —deproteinlzed serum to avoid the Interference from proteins or urine diluted to 1 100— Is placed on a sampler from which it Is Injected into the carrier and merged with a basic stream of picric acid. The indicator reaction takes place along a reactor submerged in a bath thermostated at 35 C, after which It reaches the detector flow-cell, where the absorbance Is monitored at 520 nm. The results obtained by this method are consistent with those found by the standard kinetic method [24,25]. The sampling frequency is 120 h 1. 

Xanthoprotein reactions are given by proteins with aromatic side groups On addition of nitric acid, the product turns yellow and, after further addition of ammonia, orange. The Millon reaction is specific for tyrosine On boiling, e.g., egg-white solution with a solution of mercury in nitric-acid containing nitrous acid, a red-brown precipitate is formed. 

They act as proton donors and also have a hygroscopic (desiccating) action due to their avidity for water. The protective keratin layer of the skin is breached and coagulative necrosis occurs in the tissues with thrombosis of vessels. Proteins are precipitated with often liberation of heat (exothermic reaction) which adds to destrnction with formation of a crust stopping penetration of acid into the tissues. According to the acid, the scab on the skin will differ from brownish black scab for snlfnric acid (Fig. 4.55), yellow-brownish discoloration with hydrochloric acid, and an orange yellow dne to nitric acid [20] (Fig. 4.56). Assessment of the lesion depth is often difficult). 

Redox proteins also hold a clue to the importance of metal chemistry to early Ufe. Your oldest electron-moving proteins are not all CHON, but are bedecked with tiny rocks that look like they were ripped from the Earth. The pictures of these proteins in biochemistry textbooks show unusual colors for unusual atoms, orange for iron and yellow for sulfur, arranged in unusual cubes that look hke sharp-edged rocks against the backdrop of the flexible CHON protein structure. In every cell, important redox reactions are catalyzed by tiny rocks. 

Amino acids having specific groupings which may be readily determined have offered a simple approach to protein analysis. Of these tyrosine (and tryptophan) have received the most attention. Little effort has been made to analyze for protein content by determination of the sulfur containing amino acids, cystine and cysteine. Amino acids containing a benzene ring react with hot concentrated nitric acid (xanthoproteic reaction) to form yellow nitro compounds which become orange on neutralization. Millon s reagent and that of Folin, particularly the... 

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