Biuret, 1-amino-

Fischer then synthesized a number of di-and tripeptides and showed that their properties were identical to those of the di-and tripeptides which could be obtained after partial protein hydrolysis. A peptide containing 18 amino acid residues was eventually synthesized. Molecules containing peptide bonds were found to give a characteristic pink to purple color in the presence of dilute alkaline copper sulfate. The simplest compound which does this is biuret, formed when urea is heated at 150-160 °C ... 

Many methods are available for measuring TCA-soluble peptides. Possibly the easiest is to measure the absorbance of the solution at 280 nm, as the absorbance at this wavelength is a function of the aromatic amino acid content of the solution. This approach requires a UV spectrophotometer, and the sensitivity of the assay is likely to be lower than that of some of the colorimetric assays. There are also several colorimetric peptide assays that can be applied to this type of peptidase assay, such as the Biuret, Lowry, and Bradford dye-binding methods (for comparison see Piyachomkwan and Penner, 1995). All of these methods measure a relative value rather than an absolute amount of peptide in solution. The results should thus be reported in terms of equivalents, such as BSA equivalents when using a calibration curve prepared using a BSA standard solution. 

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 terminal amino and carboxyl groups react in the same way as the corresponding amino acids (acylation, animation, esterification, etc.). Some of the reactions of the amino groups (with ninhydrin, orthophthaldehyde, fluorescamine, etc.) are used for detection purposes, as will be discussed later. Peptides also react in ways that free amino acids do not, like the classic biuret reaction, which consists of the formation of a colored complex with a transition metal (Cu, Ni, etc.)... 

Alkali metal azides, 1 79 2 139 Alkali metal cyanates, 2 86 Alkali metal pyrosulfites, 2 162 Alkali metal sulfites, 2 162 Alkaline earth azides, 1 79 Allanite, extraction of, 2 44 Allophanyl azide, formation of, from allophanyl hydrazide, 5 51 Allophanyl hydrazide (1-amino-biuret), 5 48 hydrazones of, 5 51 from methyl and ethyl alloph-anates, 5 50 salts of, 5 51... 

Would the amino acid, glycine, give a positive biuret test Explain. 

Bicinchoninic Acid (BCA) Assay. The bicin-choninic acid assay for proteins is based on the same reactions as the Folin-Ciocalteau assay. Proteins are again reacted with alkaline cupric ions to form the biuret complex, and these ions are reduced to cuprous ions by the aromatic amino acids in the proteins. In this case, however, the Cu1+ ions form a complex with bicinchoninic acid (Fig. II-6), which has an intense absorbance maximum at 562 nm. This assay shows the same variation from protein to protein as the Folin-Ciocalteau assay, but is more convenient experimentally and can be made somewhat more sensitive. 

Colour Reactions of Proteins - The colour reactions of proteins are of importance in the qualitative detection and quantitative estimation of proteins and their constituent amino acids. Biuret test is extensively used as a test to detect proteins in biological materials. 

Figure 12.2 LC of carbonylation reaction mixture components on dinitrobenzoylated-amino-propyl-silica (150 x 4.6 mm i.d.) mobile phase n-hexane propan-2-ol (60 40, v/v) flow rate l.Omlmin Peak 1 phenyl ethyl carbamate. Peak 2 N-phenylcarbamylbenzoxazol-2-one. Peak 3 ynj-N-diphenylurea. Peak 4 triphenyl biuret. On silica jy/w-diphenylurea is much more strongly retained than the other three components.

This mode of union is called peptide linkage. The presence of the peptide linkage is thought to account for the development of a violet color when a protein comes in contact with an alkaline solution of cupric ion. The color is given by biuret, formed by urea on heating NH2CONHCONH2. The reaction is known as the biuret reacUon. It is not given by any amino acid. 

Calibration of Total Protein Methods Bovine or human albumin is commonly used to calibrate biuret methods. Albumin has been prepared consistently in high purity, it contains only amino acids, its nitrogen content is a constant fraction of its molecular mass, and the number... 

Fusion of an o-aminonitrile with urea at 175°-200°C gives an annelated 4-aminopyrimidin-2-one, e.g., the quinolino[2,3-d]pyrimidine 123 from 2-amino-3-cyanoquinoline.117 The reaction is thought to pass through a ureide of the type 124. Because urea begins to self-condense to the biuret at... 

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