Chapter XXVI

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 (§58-61, page 39).

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.

239. Study of the Composition of Gelatin and of Wool. - Apply the precipitation and color reaction of proteins to a solution of gelatin made by dissolving 1 gram of the substance in 100 cc. of hot water. Cool the solution before it is used. State what conclusion can be drawn from the results as to the presence or absence of certain amino-acids in gelatin.

Apply the color tests to a sample of wool.

240. Separation of Protein by Salting Out (Section 580). - (a) Separation of a globulin from an albumin by means of sodium chloride. - Add to 10 cc. of the egg-white solution finely powdered sodium chloride until the solution is saturated and a slight residue of salt is obtained (3.6 grams for each 10 cc. of solution). The globulin is precipitated. Filter the solution; add to the filtrate 2 to 3 drops of 0.5 per cent acetic acid, and heat to boiling. The albumin which was in solution is coagulated.

(b) Separation of a globulin from an albumin by means of ammonium sulphate. - Prepare a saturated solution of ammonium sulphate by dissolving grams of the salt in 10 cc. of water. Add 10 cc. of this solution to 10 cc. of egg-white solution. In this way the latter solution is one-half saturated with ammonium sulphate, and the globulin is precipitated. Filter, and to one-half of the filtrate add 2 drops of a 0.5 per cent solution of acetic acid and boil. The albumin is coagulated. Saturate the other half with solid ammonium sulphate; the albumin is precipitated. Filter, and test the filtrate as before for protein.

241. Products of the Hydrolysis of Proteins. - (a) Meta-proteins. - In a test-tube place 10 cc. of the egg-white solution and 1 cc. of a 10 per cent solution of sodium hydroxide. In a second tube place 10 cc. of the egg-white solution and 1 cc. of concentrated hydrochloric acid. Place the tubes in a beaker containing about 300 cc. of water at 500, and allow them to stand from 15 to 20 minutes. The temperature should not fall below 40°. The protein is partly converted into alkali-albumin and into acid-albumin.

Boil 2 cc. of the solution in each tube. Is protein precipitated? Add to 2 cc. of the alkali albumin a drop of dilute hydrochloric acid. Is the precipitate soluble in dilute alkali? Add to 2 cc. of the alkali albumin a drop of dilute hydrochloric acid; heat to boiling. Is the precipitate soluble in dilute alkali as before? Explain. Add to 2 cc. of the acid albumin a drop of dilute alkali. Is the precipitate soluble in dilute acetic acid? Add to 2 cc. of the solution of alkali albumin an equal volume of a saturated ammonium sulphate solution. Is the protein precipitated?

(b) Proteoses and peptones. - Stir 2 grams of Witte's peptone with 40 cc. of cold water, and filter from the small insoluble residue.

Add to 2 cc. of the solution 2 to 3 drops of 0.5 per cent solution of acetic acid and boil. Are the proteins coagulated?

242. Separation of Proteoses and Peptones. - Saturate 20 cc. of the solution prepared in experiment 241b above with ammonium sulphate (16 grams). Filter. Save the filtrate which contains the peptones. Place the filter-paper containing the precipitate in a beaker, and heat it with 25 cc. of water. Filter. This filtrate contains the proteoses. Apply to the solution of the peptones and to the solution of the proteoses the precipitation and color reactions given above. Compare the results in the two eases. In making the biuret test when ammonium salts are present, a large excess of alkali must be added; add a strong (1 to 1) solution of sodium hydroxide or about 10 cc. of a 10 per cent solution to 2 cc. of the solution to be tested.

243. The Proteins of Wheat (Section 590). - (a) Protein soluble in water (leucosin). - Shake up about 1 gram of bread flour with 10 cc. of water, filter, and determine whether the filtrate contains protein by applying three or four tests.

(b) Alcohol-soluble protein (gliadin). - Add 25 cc. of water to 75 cc. of alcohol. Pour the mixture onto about 20 grams of bread flour. Stir occasionally during half an hour. Filter, and evaporate the filtrate to dryness. Observe the properties of the dried protein. Is it brittle? Moisten it with water and after a few minutes note its properties. Is it elastic? Prove that it is a protein by applying a number of tests.

(c) Separation of gluten. - Gluten is a mixture of glutenin and gliadin. In a porcelain dish mix 50 grams of bread flour with enough water to make a stiff dough (about 25 cc.). Let the mixture stand for half an hour. Add about 100 cc. of water and work the dough, keeping it in the form of a ball. The starch passes into suspension in the water. Pour off the water, and add fresh water from time to time until all the starch is removed. Note the properties of the gluten.

The two proteins present can be separated by extracting the gliadin with 75 per cent alcohol. This is a tedious process. Grind a small part of the gluten in a mortar repeatedly with 10 cc. portions of 75 per cent alcohol. Finally heat the residue with some of the alcohol on the steambath. When all the gliadin has been removed the protein is no longer elastic. Dissolve the glutenin in dilute sodium hydroxide and apply a few of the protein tests.

Note. - Wheat contains the following proteins: Soluble in water, leucosin, about 0.4 per cent, and a proteose, about 0.3 per cent; soluble in 10 per cent sodium chloride, edestin, about 0.6 per cent; soluble in 75 per cent alcohol, gliadin, about 4.3 per cent; insoluble in neutral solvents, glutenin from 4.0 to 4.5 per cent.

244. Isolation of a Crystalline Protein: Edestin from Hemp Seed. - Grind in a mortar 25 grams of hemp seed; add the seed in small quantities at a time, and see that each seed is crushed. Dissolve 28 grams of sodium chloride in 700 cc. of water (4 per cent solution of sodium chloride) and heat to 60°. Add the hemp seed and stir frequently during 15 minutes. The temperature of the solution should be kept between 58° and 60° during the extraction of the protein. This can be accomplished by placing a very small flame under the beaker. If the temperature rises above 60°, the flame should be removed. Filter into a dry beaker through a large hot water funnel containing water at 60°. Cover the funnel with a watch-glass to prevent loss in heat from the solution. Receive the first part of the filtrate in a small beaker and return it to the funnel. Set the filtrate aside to cool slowly. When the solution is cold, decant off most of the liquid carefully and filter off the solid. Place a little of the suspended solid on a glass slide, cover, and examine under a microscope. Sketch the crystals. If crystals have not separated from the solution before the end of the laboratory period, leave the solution in a cool place. Test the edestin crystals by applying the following reactions: xanthoproteic, biuret, Hopkins-Cole, Millon, sulphur. Shake up some of the protein with 20 cc. of a 10 per cent solution of sodium chloride. Filter through a dry paper into a dry test-tube. If the first part of the filtrate is cloudy return it to the funnel. Pour some of the clear filtrate into water. Determine whether the protein is coagulated by heat, and whether it is salted out by sodium chloride and by ammonium sulphate.

Note. - During the extraction of edestins it is necessary to avoid a high temperature in order to prevent the coagulation of the protein.

245. Isolation of Casein from Milk (Section 596). - To 50 cc. of milk add 150 cc. of water and heat the mixture to 30°. Add dilute acetic acid, drop by drop, as long as a precipitate is formed (about 1 cc. of a 10 per cent solution). Stir vigorously. It is necessary to avoid an excess of acid, since the latter dissolves some of the protein. Filter. During the filtration test small portions of the filtrate for protein by three or four color reactions. Heat 10 cc. of the filtrate to boiling. What protein is precipitated? Filter this off, and test the filtrate for a sugar with Fehling's solution, and for a phosphate with ammonium molybdate.

Wash the precipitate of casein with 50 cc. of water; press out as much water as possible, and then wash the precipitate twice with 20 cc. of alcohol. Squeeze out the alcohol, and press the casein between a number of pieces of filter-paper. Break up the casein and let it dry in the air. Determine whether it is soluble in water. To do this, grind in a mortar a little of the solid with 10 cc. of water, filter, and test the filtrate for a protein with concentrated nitric acid. If the filtrate is not clear, refilter it through two filter-papers. Determine if casein is soluble in sodium carbonate solution. Proceed as before; acidify the filtrate and test it for a protein.

Put into a mortar 10 cc. of water and 3 or 4 drops of a dilute solution of sodium hydroxide. Test the solution with red litmus paper. Next add some casein and grind it thoroughly in the alkaline solution. Test the solution again with red litmus paper. What conclusion can you draw in regard to the chemical nature of casein. Filter the solution through a wet filter-paper, and add to the filtrate a few drops of a solution of calcium chloride. Explain.


Textile Fibers

246. Appearance of Fibers. - Examine under a microscope a fiber of each of the following: silk, wool, cotton, mercerized cotton, and linen.

247. Properties of Wool, Silk, and Cotton. - (a) Effect of burning. - Burn a thread of wool, of silk, and of cotton. Notice the odor produced during the burning. Explain. At the end of a few seconds blow out the flame and observe the appearance of the charred residue.

(b) Action of sodium hydroxide. - For this and the following experiments use pieces of the material about 1 inch square. Place in a test-tube a piece of cotton, a piece of silk, and a piece of wool; add about 10 cc. of a 10 per cent solution of sodium hydroxide. Heat the solution to such a temperature that the tube can just be held in the hand (about 65°); shake, and keep the tube hot by heating it occasionally until the wool dissolves. Heat to boiling for some minutes and note the effect. Explain the cause of the difference in behavior of the animal and vegetable fibers.

(c) Action of concentrated hydrochloric acid. - Shake pieces of cotton, silk, and wool with about 10 cc. of cold concentrated hydrochloric acid. One of the pieces should dissolve.

Place a piece of cotton and a piece of wool in a mixture of 1 cc. of concentrated hydrochloric acid and 5 cc. of water. Press out as much of the solution as possible. Set aside to dry and examine at the next exercise. Try to tear the material.

(d) Examination of wool and of silk for sulphur. - Add a few drops of a solution of lead acetate to about 20 cc. of a 10 per cent solution of sodium hydroxide. Divide the solution into two portions. In one dissolve a piece of wool and in the other a piece of silk.

(e) Action of Millon's reagent (Section 586). - Millon's reagent is prepared by using the substances in the following proportions: One gram of mercury is dissolved in 2 cc. of hot concentrated nitric acid and the resulting solution diluted with 3 cc. of water.

Dilute 2 cc. of the reagent with 10 cc. of water, add to the solution pieces of cotton, silk, and wool, and heat to boiling.

(f) Test of textile fabrics. - Test the samples submitted, making use of the results obtained in (a), (b), (c), (d), and (e) above. The samples may consist of cotton, silk, or wool, or mixtures of any two of the fibers. It is a common practice to make fabrics in which the threads running in one direction are of one material, and those running in another are or a different material. Designs in pure silk are often woven into a fabric made of mercerized cotton. These designs can be readily developed by warming a sample of the material with Millon's reagent.