Test, biuret

Ltease test. The enzyme uretwe hydrolyses urea to ammonium carbonate (p. 519). The reaction is sp ific and is frequently used for solu tions of urea to which the biuret test cannot be applied. Add about 5 drops of phenohred to o 2 g. of urea dissolved in 5 ml. of water. To this yellow solution, add 0 2 g. of jack bean meal suspended in 2 ml. of water containing. also 5 drops of phenol-red. The colour changes to red as the solution becomes alkaline. 

Biuret test. Oxamide, having two CONHj groups, will give this test without any preliminary treatment (c/. urea). Shake o-i g. of oxamide with 1 ml, of 10% NaOH solution, add i drop of very dilute CuSO solution and mix well. A rose-pink coloration is produced. 

In alkaline solution biuret, HN(CONH2)2 reacts with copper(II) sulfate to give a characteristic violet colour due to the formation of the complexes [Cu2(/l-OH)2(NHCONHCONH)4] (Fig. 28.6a) and [Cu(NHCONHCONH)2] . This is the basis of the biuret test in which an excess of NaOH solution is added to the unknown material together with a little CUSO4 soln a violet colour indicates the presence of a protein or other compound containing a peptide linkage. 

The biuret test became a standard method by which to detect and estimate protein. 

If you are trying to show only the presence of a protein, a quick test to carry out is known as the Biuret test. A mixture of dilute sodium hydroxide and 1% copper( ii ) sulfate solution is shaken with a sample of the material under test. If a protein is present, a purple colour appears after about three minutes (Figure 15.25). 

Biuret test The test for proteins. A mixture of dilute... 

Biuret test for proteins. Warm the sample in water, add concentrated sodium hydroxide solution and a drop of very dilute copper(u) sulfate solution. 

Notes. (1) Remove about 0.25 ml of the hydrolysate, cool it and basify it with 5 m sodium hydroxide solution. To a portion add a few drops of very dilute copper(u) sulphate solution, and note the absence of any colour change. As a control, prepare a specimen of biuret (NH2-CO-NH-CO-NH2) by heating about lOmg of urea just above its melting point for about 2 minutes. Add a little basified hydrolysate warm to dissolve, cool and add a trace of copper(u) sulphate. A deep pink colour superimposed upon the pale brown colour of the hydrolysate should be observed. If the hydrolysate gives a similar colour originally, it contains peptide material and hydrolysis should be continued until the biuret test is negative. 

The biuret test. This is one of the most general tests for proteins. When a protein reacts with copper(II) sulfate, a positive test is the formation of a copper complex which has a violet color. 

The biuret test. Place 15 drops of each of the following solutions in five clean, labeled test tubes. 

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

Percentage of casein in milk Chemical analysis of proteins Biuret test... 

Biuret Test. Compounds containing two or more peptide bonds (e.g., proteins) take on a characteristic purple color when treated with dilute copper sulfate in alkaline solution. The name of the test comes from the compound biuret, which gives a typically positive reaction. The color is apparently caused by the coordination complex of the copper atom and four nitrogen atoms, two from each of two peptide chains (Fig. II-5). The biuret test is fairly reproducible for any protein, but it requires relatively large amounts of protein (1 to 20 mg) for color formation. Because of its low sensitivity, the biuret assay is no longer widely used. 

Folin-Ciocalteu (Lowry) Assay. The quantitative Folin-Ciocalteu assay (also often called the Lowry assay ) can be applied to dried material as well as to solutions. In addition, the method is sensitive samples containing as little as 5 /Ug of protein can be analyzed readily. The color formed by the Folin-Ciocalteu reagent is thought to be caused by the reaction of protein with the alkaline copper in the reagent (as in the biuret test) and the reduction... 

This purified polysaccharide gave an almost colorless aqueous solution, a negative biuret test, no coloration with iodine and an intense Molisch... 

These results were confirmed in the main by Dorset and Henley. A polysaccharide was obtained from Long s synthetic medium after growth of M. tuberculosis, human strain. This polysaccharide showed a positive biuret test for protein, and underwent hydrolysis only with difficulty. D-Arabinose and D-mannose were identified in the hydrolysate. No glycuronic acid could be detected. On hydrolysis, the D-arabi-nose seemed to be liberated before the other constituents. It was concluded from the biological tests that the active principle of tuberculin concerned in eliciting the skin reaction was a protein. This work was repeated with polysaccharides derived from the culture medium of the bovine and avian strains of organisms, and very similar results were obtained. D-Arabinose and D-mannose were also identified in those polysaccharides. 

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. 

NOTES.—(a) Excess of copper sulphate must be avoided in making the biuret test, since the color of the salt prevents the recognition of the color produced in the reaction. The presence of ammonium salts interferes with the test. In applying the reaction to solutions containing these salts a large excess of sodium hydroxide must be present. Compounds which give the biuret test must contain at least two —CO—NH— groups. The color formed in the reaction varies in shade with the complexity of the molecules. 

In the biuret test, the sample is treated with an alkaline copper sulfate reagent that produces a violet color and requires a peptide with at least two peptide bonds. The violet color is produced through formation of a coordination complex (between peptide nitrogen atoms and cupric ion) that is analogous to the structure of the complex of biuret with cupric ion, as shown below ... 

A practical application of the coordination of A,0-donors to Cu(II) is the biuret test for peptides and proteins. Compounds containing peptide linkages form a violet complex when treated in NaOH solution with a few drops of aqueous CUSO4. The general form of the complex can be represented by that of 21.62, in which the ligand is the doubly deprotonated form of biuret, H2NC(0)NHC(0)NH2. 

These directions of the Codex necessitate a few remarks. First of all, tryptic digestion takes place in neutral medium, and it leads to compounds strongly biuretic. To make the biuret test, take about 2 c.c. of liquid and add about 0.5 c.c. of concentrated NaOH and a few drops of a 2 per cent solution of copper sulphate. A beautiful purple violet coloration is observed. 

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