Tyrosine biuret reaction

In the Biuret reaction, a purple colour develops when the protein is treated with alkaline copper sulphate. This reaction is dependent on peptide bonds and not on the side chains of individual amino-acids present. In the Folin-Ciocalteu reaction, the protein is treated with tungstate and molybdate under alkaline conditions and the formation of a complex such phenylalanine and tyrosine gives rise to a blue colour. Lowry developed one of the most widely used protein assays in which a combination of the above reactions is involved07, l8). 

The Folin-Ciocalteau Assay of Protein Concentration. The Folin-Ciocalteau assay is one of the most sensitive and most commonly used assays to determine protein concentration (sensitive to about 10 /rg/m I protein). This procedure employs two color-forming reactions to assay protein concentration photometrically. In the first reaction (a biuret reaction), compounds with two or more peptide bonds form a dark blue-purple color in the presence of alkaline copper salts. In the second reaction, tryptophan and tyrosine side chains react with the Folin solution to produce cuprous ions. This reaction is most efficient under basic condi-... 

The latter group (M13, M14) separated two types of KIK factor from cancerous gastric juice. The first was a mucopolysaccharide and the second a mucoprotein. Others thought it to be a mucopolypeptide. A similar factor from cancerous ascitic fluid (19) contained 50-60% hexoses, 1.5% tyrosine, and 10% protein by biuret reaction. It formed a single peak at pH range 1.5-10.0 on paper electrophoresis, and at pH 8.6 moved with the mobility of serum y-globulin. 

The color produced in the Lowry method results from the biuret reaction plus the reduction of the phosphomolybdate-phosphotungstate reagent (Folin CiOcalteu phenol reagent) by tyrosine residues. The Lowry method is suitable for solutions containing 20 to 400 /rg protein/ml. 

The final colour in the Lowry method is a result of two reactions. The first is a small contribution from the biuret reaction of protein with copper ions in alkali solution. The second results from peptide-bound copper ions facilitating the reduction of the phos-phomolybdic-tungstic acid (the Folin reagent) which gives rise to a number of reduced species with a characteristic blue colour. The amino acid residues which are involved in the reaction are tryptophan and tyrosine as well as cysteine, cystine and histidine. The amount of colour produced varies slightly with different proteins. In this respect it is a less-reliable assay than the biuret method, but it is more reliable than the absorbance method since A280 may include contribution from other species, and also the absorption of a given residue is dependent on its environment within the protein. 

In addition to all the other groups noted by them, Blum and Strauss (282) also reported a disappearance of unoxidized sulfur, and stated that the iodide ion formed during iodination was over four times that expected from a straight substitution reaction. They suggested that cystine sulfur had been oxidized. They also found that the biuret value was lower in the iodinated protein. Sometimes considerably more iodine is bound to proteins than can be accounted for on the baas of thmr tyrosine and histidine content (286, 287). 

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