Biuret reagent

It is recommended that the protein be precipitated and extracted by Bloor s reagent before adding the biuret reagent ... 

K7. Kingsley, G. 11., Procedure for serum protein determinations with a triphosphate biuret reagent. Stand. Methods Clin. Chem. 7, 199-207 (1972). 

When substances containing two or more peptide bonds react with the biuret reagent, alkaline copper sulfate, a purple complex is formed. The colored product is the result of coordination of peptide nitrogen atoms with... 

Add 1.5 mL of biuret reagent to each tube and mix well by inverting several times while holding a piece of hydrocarbon foil over the opening. 

Using a multichannel pipettor, add 250 pi biuret reagent to each well. Mix well on a plate shaker for 30 sec. 

The biuret total protein reagent is considerably less sensitive to total protein than the other three protein assay reagents discussed in this unit. This limits the applications in which the Biuret reagent can be used. Since the primary use of the Biuret reagent has been for serum total protein in the clinical laboratory, there is little published information about its compatibility with substances and reagents common to nonclinical samples. 

Gornall, A.G., Baardawill, C.J., and David, M.M. 1949. Determination of serum proteins by means of the biuret reagent. J. Biol. Chem. 177 751-766. 

Accurate total protein quantitation of fatty tissues was achieved by adding 0.1 ml 10% sodium deoxy-cholate, pH 8.0 and 2.9 ml biuret reagent to each protein pellet following an acetone/ether wash step. After sonication, each sample was heatedfor 30 sec in a boiling water bath to develop full color. 

Dextran at high concentrations causes a slight overestimation of the total protein concentration with the biuret reagent. 

A modified biuret reagent was formulated (sodium tartrate replaces sodium potassium tartrate, the sodium hydroxide concentration is reduced, and potassium iodide was deleted). When the modified biuret reagent was mixed with samples containing 2% detergent (SDS or sodium cholate or Triton X-I00), it resulted in less protein-to-protein variation among six proteins. 

Used sodium potassium tartrate as a stabilizer and added potassium iodide to prevent autoreduction of the biuret reagent however, this reagent was found to be unstable after long storage. 

After comparing spectra it was determined that the species which tends to decrease in absorbance during the reaction was the cupric tartrate complex found in the biuret reagent. Its absorbance is significant even down to wavelengths of slightly below 530 nm. The complications caused by the absorbance of the cupric complex at 550 nm appear to be a substantial cause of the unusual rate curve observed in the early part of the reaction. 

Mix the contents thoroughly and transfer them to a plastic bottle. 2-6. The biuret reagent just prepared should be a deep royal blue. This solution may be stored indefinitely. If a black precipitate is observed in the storage container, however, discard the solution and prepare a fresh solution. 

Add 4.0 ml biuret reagent to each tube and vortex the mixture for a few seconds to effect thorough mixing of the solutions. 

Figure 2-14. Absorbances observed with increasing amounts of protein assayed by means of biuret reagent.

Biuret Reaction - A compound, which is having more than one peptide bond when treated with Biuret reagent, produces a violet colour. This is due to the formation of coordination complex between four nitrogen atoms of two polypeptide chains and one copper atom. 

A protein solution (0.3 ml) was diluted with 0.9 ml of water. To 0.5 ml of this diluted solution, 4.5 ml of biuret reagent were added and the color was allowed to develop. The absorbance of the mixture at 540 nm was 0.18 in a 1 cm diameter test tube. A standard solution (0.5 ml, containing 4 mg of protein/ml) plus 4.5 ml of biuret reagent gave an absorbance of 0.12 in the same-size test tube. Calculate the protein concentration in the undiluted unknown solution. 

That is, 1 mg of protein (in a standard sample size of 0.5 ml) plus 4.5 ml of biuret reagent will yield an absorbance of 0.06 at 540 nm in the particular test tube used. 

The one-piece alkaline copper biuret reagent was introduced by Kingsley (K21) and enables the reaction to be carried out without the precipitation of cupric hydroxide. At least 12 improved reagents of this type but of different composition have been proposed. 

The earlier alkaline copper tartrate reagents required the addition of potassium iodide to prevent their slow autoreduction. However, if the constituents are of analytical quality, tartrate-biuret reagents are stable indefinitely, and potassium iodide can safely be omitted. 

The use of Benedict s qualitative reagent and NaOH solution has been suggested for protein estimation (G6, H9, H28). This practice is not recommended as opalescence may occur during an estimation, although it has been claimed that this opalescence can be removed by ether extraction (H9). Another biuret reagent contains a high concentration of ammonium hydroxide (L9), but is inconvenient to work with and has lower sensitivity to protein. 

K17. Kibrick, G. R., On the determination of protein in serum and in fractions obtained from serum with a biuret reagent prepared with sodium hydroxide. J. Lab. Clin. Med. 34, 1171-1174 (1949). 

W9. Watson, D., and Farrance, I., Biuret reagents for determining protein in plasma. Proc. Australian Assoc. Clin. Biochem. 1, 87-88 (1964). 

Protein, total s Spectrophotometry Biuret reagent (alkaline copper tartrate) forms complex with proteins measure absorbance at 550 inn after 30 min... 

C. E. Shideler, B. W. Renoe, J. Crump, M. R. Wills, J. Savory, and K. K. Stewart, Automated Multiple Flow-Injection Analysis in Clinical Chemistry Determination of Total Protein with Biuret Reagent. Clin, Chem, 26 (1980) 1454. 

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