PROTEINS

236. Detection of Nitrogen, Sulphur, and Phosphorus in Proteins. - The methods described below illustrate those commonly used in the analysis of proteins.

(a) Analysis for nitrogen by means of soda-lime. - Hood. - Heat about 0.1 gram of casein with five times its weight of soda-lime in a dry test-tube over a small flame. Observe the odor of the gases which escape, and test them with moist litmus paper. (b) Analysis for phosphorus and sulphur by fusion with potassium nitrate. - Hood. - Fuse together cautiously in a small porcelain crucible about 0.1 gram of casein, 0.2 gram of potassium nitrate, and 0.1 gram of anhydrous sodium carbonate. When the mixture is colorless, let it cool; then add 10 cc. of water and heat to boiling. The fusion has converted the phosphorus into sodium phosphate. The acid radical is tested for in the usual way. Acidify about 2 cc. of the solution with nitric acid, and add a solution of ammonium molybdate. If no precipitate forms, warm gently (about 60) and set aside. A yellow precipitate indicates the presence of phosphorus. Acidify about 2 cc. of the solution from the fusion mixture, which contains a sulphate if sulphur is present, with hydrochloric acid, and add a solution of barium chloride. Heat the solution to boiling and set aside. Examine the tube for a white precipitate. (c) Analysis for sulphur by decomposition with sodium hydroxide. - Boil about 0.1 gram of casein with 5 cc. of a 10 per cent solution of sodium hydroxide to which a drop of lead acetate solution has been added. If a black precipitate is not evident, filter the solution through a paper and observe if a precipitate is left on the paper. When proteins containing sulphur are decomposed by sodium hydroxide, sodium sulphide is formed; the latter is converted into lead sulphide by lead salts. (d) Analysis for nitrogen, phosphorus, and sulphur by fusion with sodium. - Analyze a sample of dried egg-white for nitrogen, phosphorus, and sulphur by the method commonly used in the qualitative analysis of organic compounds. 237. Precipitation Reactions of Proteins (Section 586). - The protein solution to be used in the following experiments is prepared as follows: Beat egg-white for a short time to break the membranes, and squeeze it through cotton cloth. Mix with ten times its volume of water and filter through paper. This gives a solution which contains about 1 per cent of protein. (a) Strong mineral acids: Heller's test. - Put 2 cc. of egg-white in a test-tube; incline the tube, and pour cautiously down the side concentrated nitric acid in such a way that the acid sinks to the bottom of the tube and two layers are formed. Shake the tube. Is the precipitate dissolved? Dilute 2 cc. of the egg-white solution with 100 cc. of water, and test 2 cc. of this solution as before with nitric acid. Determine whether the protein is precipitated from the 1 per cent solution by dilute hydrochloric acid and by dilute acetic acid, and if it is soluble in an excess of the acid. (b) Salts of the heavy metals. - To 2 cc. of egg-white solution add 1 drop of a solution of mercuric chloride. For what purpose can this reaction be used? Test in the same way the effect on the protein of a solution of lead acetate, of silver nitrate, and of copper sulphate. To the tube containing the precipitate with copper sulphate add 3 to 4 drops of a solution of sodium hydroxide, dilute, and note the color.

(c) Alkaloidal reagents in acid solutions. Hydroferrocyanic acid. - Add to 2 cc. of egg-white solution 5 drops of glacial acetic acid and 1 drop of a solution of potassium ferrocyanide. Tannic acid. - Add, drop by drop, to 2 cc. of egg-white solution a solution of tannic acid. Picric acid. - Repeat with a solution of picric acid. Potassium mercuric iodide. - Prepare the reagent by adding to a few drops of a solution of mercuric chloride a solution of potassium iodide until the precipitate first formed is dissolved. Add to 2 cc. of egg-white solution a drop of dilute hydrochloric acid and a drop of the reagent. Bromine. - Add bromine-water to 2 cc. of the egg-white solution until a permanent yellow color is formed. (d) Coagulation by alcohol. - Add to 5 cc. of the egg-white solution twice its volume of alcohol. Set aside until the next exercise. Filter and test the solubility of the precipitate in water. To do this, wash the precipitate with water, and test the solution for protein. (e) Heat coagulation. - Heat 2 cc. of the egg-white solution to boiling. To 2 cc. of the egg-white solution add 1 or 2 drops of a 0.5 per cent solution of acetic acid, and heat to boiling. Compare the results in the two cases (see note below). Determine the temperature of coagulation of egg-white as follows: Add to 5 cc. of the protein solution 5 drops of a 0.5 per cent solution of acetic acid. The mixture should be faintly acid to delicate litmus paper. Put a thermometer in the solution, and place the test-tube in cold water in a beaker; heat the water and determine the temperature at which the solution clouds and when the precipitate separates in flocks. Stir from time to time with the thermometer. Notes. - (a) Heller's reaction is a very delicate test for most proteins; peptones are not precipitated by nitric acid. (c) A reaction with potassium ferrocyanide takes place only in acid solution; neutral salts interfere to some extent with the reaction. Peptones are not precipitated by hydroferrocyanic acid. (d) Alcohol precipitates proteins unchanged, but on standing the latter are converted into a form which is insoluble in water. (e) Egg-white is faintly alkaline. Complete precipitation takes place only in faintly acid solution. The temperature at which coagulation takes place depends to a large extent on the amount of acid and of salts present. 238. Color Reactions of Proteins (Section 586). - (a) Biuret reaction. - Add to 2 cc. of egg-white solution 2 cc. of a 10 per cent solution of sodium hydroxide and 2 drops of a 1 per cent solution of copper sulphate. (b) Xanthoproteic reaction. - To 2 cc; of the egg-white solution add 5 drops of concentrated nitric acid and heat to boiling. Note the change in color. Cool the solution and make it alkaline with ammonia or sodium hydroxide. (c) Millon's reaction. - To 2 cc. of the egg-white solution add 5 drops of Millon's reagent. Is a precipitate formed? Heat to boiling. (d) Molisch reaction. - Add to 2 cc. of egg-white solution 3 drops of a solution of a-naphthol in chloroform; shake and pour cautiously down the side of the test-tube concentrated sulphuric acid so that two layers are formed. If no color is produced, put the tube aside and examine in a few minutes.

(e) Sulphur reaction. - Add 1 drop of a solution of lead acetate to 2 cc. of the egg-white solution and then a dilute solution of sodium hydroxide until the precipitate is dissolved. Heat to boiling. (f) Hopkins-Cole reaction. - Add to 2 cc. of the egg-white solution 5 drops of a solution of glyoxylic acid (see Appendix). Pour down the side of the tube concentrated sulphuric acid so that two layers are formed. If no color develops examine the tube in a few minutes. 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. (b) The color is produced as the result of the formation of nitro-derivatives of the compounds which contain a benzene ring, for example, tyrosine. (c) The color produced in Millon's test is given by derivatives of benzene in which a hydrogen in the ring has been replaced by a hydroxyl group. The reaction serves as a test for the presence of tyrosine. (d) If the Molisch test is positive, a carbohydrate is present in the protein molecule. (e) If sulphur is present a black precipitate or brown coloration is produced. If the result is in doubt the solution should be filtered and the paper examined. The precipitate, which is lead sulphide, is formed as the result of the decomposition of the cystine by the alkali. (f) The color produced is due to the formation of a compound from the glyoxylic acid in the reagent and the tryptophane in the protein. A similar color is produced when sulphuric acid is added to a protein solution in the presence of a trace of formaldehyde. The reaction is used as a test for formaldehyde in milk.