100 PHYSIOLOGICAL CHEMISTRY 



biuret coloration. This procedure is not applicable in case the pigment 

 of the original solution is soluble in alcohol. Excess of the copper salt 

 need not be avoided in this test. 



Gies's Biuret Reagent. 1 Gies has devised a reagent for use in the biuret test. 

 This reagent consists of 10 per cent KOH solution, to which 25 c.c. of 3 per cent 

 CuS0 4 solution per liter has been added. This imparts a slight though distinct 

 blue color to the clear liquid. This reagent is of material assistance in performing 

 the biuret test. 



Biuret Paper of Kantor and Gies. According to Kantor and Gies 2 when 

 filter paper is immersed in _ the above reagent and subsequently dried it forms a 

 very satisfactory ''biuret paper" which may be used in a manner analogous to 

 indicator papers. Moist papers may be used in the examination of powders which 

 are neutral or alkaline in reaction. In preparing the "biuret paper," if the filter 

 paper is left for a sufficient length of time in the reagent all traces of the copper 

 sulphate will be removed from the solution. 



5. Ring Biuret Test (Posner). This test is particularly satisfactory for use on 

 dilute protein solutions, and is carried out as follows. To some dilute egg albumin 

 in a test-tube add one-half its volume of potassium hydroxide solution. Now hold 

 the tube in an inclined position and allow some very dilute copper sulphate solution, 

 made as suggested on page 98 to flow down the side, being especially careful to 

 prevent the fluids from mixing. At the juncture of the two solutions the typical 

 end-reaction of the biuret test should appear as a colored zone (see Biuret Test, 

 page 98). 



6. Liebermann's Reaction. Add about 10 drops of concentrated egg albumin 

 solution (or a little dry egg albumin) to about 5 c.c. of concentrated HC1 in a test- 

 tube. Boil the mixture until a pinkish-violet color results. This color was origi- 

 nally supposed to indicate the presence of a carbohydrate group in the protein 

 molecule, the furfural formed through the action of the acid upon the protein react- 

 ing with the hydroxyphenyl group of the protein producing the pinkish-violet color. 

 It is now considered uncertain whether the carbohydrate group enters into the reac- 

 tion. Cole has called attention to the fact that a blue color results if protein mate- 

 rial which has been boiled with alcohol and subsequently washed with ether be used 

 in making the test. He believes the blue color to be due to an interaction between 

 the glyoxylic acid, which was present as an impurity in the ether used in washing 

 the protein, and the tryptophane group of the protein molecule which was split off 

 through the action of the acid. 



7. Acree-Rosenheim Formaldehyde Reaction. Add a few drops of a dilute 

 (i : 5000) solution of formaldehyde to 2-3 c.c. of egg albumin solution in a test-tube. 

 Mix thoroughly and after two to three minutes carefully introduce a little concen- 

 trated sulphuric acid into the tube in such a manner that the two solutions do not 

 mix. A violet zone will be observed at the point of juncture of the two solutions, 

 especially if the mixture is slightly agitated. This color probably results through 

 the union of the protein and the formaldehyde. If the sulphuric acid is added to the 

 protein before the formaldehyde is added the typical end-reaction is not obtained. 

 So far as is known this is a specific test for proteins. The reaction cannot be applied 

 satisfactorily with concentrated formaldehyde. 



1 Gies: Proceedings of Society of Biological Chemists, Journal of Biological Chemistry, 

 7, 60, 1910. 



2 Kantor and Gies: Proc. Soc. Biol. Chem., p. n, 1910. 



