1. Materials

An egg, fibrin, gelatin, acid albumin, commercial peptone (mixed albumoses, proteoses, and peptones), Grubler's pure peptone.

(1) To Prepare Dilute Egg Albumin

Make an opening in end of the shell of an egg; drain off the white of the egg, catching it upon a coarse linen cloth - a towel serves the purpose well; press the albumin through the meshes of the linen into a beaker; add 400 or 500 c.c. of distilled H20; transfer the mixture to a 1-liter cylinder, and shake vigorously; after a short time filter through pure absorbent cotton or strain through fine linen.

(2) To Prepare Acid Albumin

To 100 cc. of dilute egg albumin add an equal quantity of 0.2 per cent hydrochloric acid; place the mixture in the incubator for two or three hours. Though the change begins at once, it will probably not be complete before the time suggested. If one wishes to isolate the acid albumin from the mixture, he has only to neutralize carefully with sodic hy-droxid, precipitating the acid albumin, and to wash the precipitate with distilled water. For the purpose for which it is to be used in the following demonstation, it may be left in the acid solution, which represents 0.1 per cent HC1. Label: Acid Albumin Solution in 0.1 per cent HCl.

(3) Make an aqueous solution of the commercial "peptone," and, though the peptone is present in small proportions, label: Proteoses.

(4) Make an aqueous solution of a few grams of Grubler's pure peptone, and label: Peptone.

(5) Dissolve a few grams of gelatin in distilled water.

2. Experiments And Observations. - (1) The Heat Test

Pour into test-tubes a few cubic centimeters of each of the following protein solutions and subject each in turn to a temperature of 63° C. (145° F.), and, finally, to a temperature of 100° C. (212° F.), by dipping the tubes into water baths of the temperatures named;

(a) Dilute egg albumin.

(b) Acid albumin in acid solution.

(c) Gelatin in aqueous solution.

(d) Proteoses.

(e) Peptone.

Record results in a table and formulate conclusions.

(2) Subject the same series of proteins to the Cold Nitric Acid Test by pouring, first, 1 c.c. or 2 c.c. of strong nitric acid into a test-tube; then, with a fine-pointed pipette, carefully floating the protein liquid upon it. In the case of the dilute egg albumin, a characteristic white ring forms between the acid and the albumin. Note in each case whether or not a typical ring is formed.

Tabulate results and formulate conclusions in a concise statement.

(3) The Xanthoproteic Test

Use the tubes and materials already prepared in the cold nitric-acid test. Shake the tubes to mix the acid with the protein. In some cases a coagulum will be formed, and this coagulum turns yellow on boiling, if the tube is held in a Bunsen flame. After the coagulum has been boiled in the acid, cool under the hydrant or in a jar of cold water, and add strong ammonia to alkaline reaction. The light-yellow coagulum which forms in the case of the egg albumin turns to an orange-color. This test is usually given as a universal protein test. Tabulate results on the above suggested series (a) to (e), noting any variations of the reaction with different proteins. Note variations in the reaction with different strengths of solution of the same proteid.

(4) The Biuret Test

To a suspected liquid add an excess of sodic hydrate; shake well, and to the mixture add one or two drops of a very dilute solution of cupric sulphate. A violet color appears, which, on heating, becomes deeper in shade.

Tabulate results on the protein series (a) to (e).

(5) Subject each of the series of proteins (a) to (e) to each of the following reagents, tabulating results:

I. Absolute alcohol.

II. Mercuric chlorid, saturated solution.

III. Tannic acid, saturated solution.

IV. Silver nitrate, 10 per cent solution.

On which of the protein solutions would one get a precipitate with silver nitrate independent of the presence of protein?

(6) The Difusibility Of Proteins

Fill dialyzers with proteins above studied. On the following day test the diffusates for protein.

Experiments And Observations

(I) What reagent may best be used to determine whether or not any of the egg albumin has diffused through the animal membrane?

(II) How may one determine whether or not any of the salts of the egg albumin have diffused through the membrane?

(III) In the case of the solution of acid albumin is there any contraindication against silver nitrate as a reagent to determine whether protein has diffused?

(IV) What tests would be most reliable in these cases to detect the presence of protein in the diffusate?

(V) Would a trace of protein in the diffusate necessarily demonstrate the diffusibility of these proteins through the walls of the alimentary tract? If not, why not?

(VI) What tests may be used to determine the presence of gelatin in the diffusate? Is gelatin diffusible?

(a) If peptone is diffusible, the diffusate will certainly contain peptone. Do peptone and proteoses respond alike to all the general tests for proteoses?

(b) How may peptone be separated from the proteoses? What single reagent is indicated in the case?

(VII) Demonstrate the diffusibility of peptone.