In all, about twenty amino acids have been obtained from the various proteins by hydrolysis, and a single protein may yield all of them, and usually does yield the greater number of them. Some of these twenty-odd amino acids contain more than one amino group, some more than one carboxyl group, some contain sulphur, and there are various other complications.

The proportions of the various amino acids yielded by the various proteins differ widely. For instance, comparing the following proteins - casein from milk, gelatin prepared from the tendons of beef, gliadin from wheat, and zein from cornwe find that no glycine is yielded by casein, gliadin, or zein, while the amount yielded by gelatin amounts to one sixth of the weight of the gelatin. Again, while gelatin yields only 0.6 per cent of alanine, casein yields 1.5, gliadin 2, and zein nearly 10 per cent of this amino acid. Once more, tryptophane, one of the very complex amino acids, is not found among the hydrolysis products of gelatin or of zein, but gliadin yields I per cent and casein 1 per cent of this substance.

In addition to amino acids, ammonia is formed in the hydrolysis of proteins, and from certain classes of proteins— the so-called "conjugated" proteins still other products are obtained. Thus, hemoglobin, the red coloring matter of the blood, yields a pigment containing iron; casein (the curd of milk) and vitellin of egg yolk yield phosphorus compounds; the mucins yield carbohydrates; and nucleins yield nucleic acid, a complex organic acid containing phosphorus. The amino acids and the other groups which enter into the molecules of the more complex proteins are often referred to figuratively as the "building stones" of the protein molecules. "Native" proteins (i.e. such as exist in plant and animal tissues and fluids) may be divided into two classes: 1. Simple proteins, which yield no hydrolysis products other than amino acids and ammonia.

2. Conjugated proteins, which yield other hydrolysis products in addition to amino acids and ammonia.

Just as in the hydrolysis of starch, intermediate products -soluble starch, dextrins, maltose-between the starch and its ultimate product, glucose, were obtained, so also in the hydrolytic cleavage of proteins, intermediate products are obtained. Some of these products have still the essential characteristics of proteins. When egg-white is heated above 73° C. (160° F.), for example, it hardens or "coagulates." It is thus converted from a substance soluble in water into one which is insoluble but is still essentially a protein. It is believed that this change is due to slight hydrolysis or hydration of the protein molecule. Similarly, the soluble caseinogen of milk is readily converted into the insoluble protein, casein, by the action of the ferment rennin; and the soluble fibrinogen of the blood clots into fibrin when the blood is exposed to air. Such slightly altered proteins are called "derived proteins or protein derivatives. Another class of derived proteins are obtained by further hydrolysis of such primary derivatives as coagulated egg albumin, fibrin, and casein. Such hydrolysis can be accomplished by the action of digestive ferments (such as the pepsin found in the stomach and the trypsin found in the small intestines) and the products are substances soluble in water and not coagulated by heat. The substances so produced are called proteoses and peptones. Proteoses are more complex and less soluble in salt solutions than peptones, but no very sharp line of distinction can be drawn between the two classes of compounds. The term peptone was formerly applied to both, and commercial peptone" consists largely of proteoses.

Protein Tests

The following are some of the most general tests for proteins:

1. Decomposition Test. - Proteins when subjected to dry heat (e.g. in a test tube) give off vapors having an alkaline

reaction and a characteristic, disagreeable, "empyreumatic odor - that of burning meat, feathers, leather, hair, wool, etc. When the protein is mixed with lime and heated, the odor of ammonia is distinctly noticeable.

Experiment 104.

Materials:

Egg albumin, dry.

Blood albumin, dry.
Gelatin.

Casein.

Quicklime.

Turmeric paper.

Red litmus paper.

Heat a little of each of the proteins in a dry test tube. Note the odor. Add quicklime and heat again. Note odor and hold turmeric paper and red litmus paper at the mouth of the test tube. Also expose turmeric paper and red litmus paper to ammonia gas by holding them to the open mouth of the ammonium hydroxide reagent bottle.

2. Xanthoproteic Test (Greek, xanthos = yellow). — Proteins heated with concentrated nitric acid impart a yellow color to the acid, owing to the formation of xanthoproteic acid. If the acid be then neutralized with ammonia, the color deepens.

Experiment 105.

Materials:

The same proteins as for Experiment 104.
Sugar.

Gently heat a little of each of the proteins with concentrated nitric acid. Note the coloration. Cool the tube, and then neutralize the acid with ammonium hydroxide. Heat a very little sugar with nitric acid in the same manner as the proteins. Is the acid colored by the sugar? Cool and neutralize with ammonia. In making the xanthoproteic tests on the proteins notice the difference in behavior between gelatin and the other proteins. Chemically pure gelatin gives no xanthoproteic test. This test is due to the action of nitric acid upon the amino acids of a certain

class, none of which is yielded by the hydrolysis of pure gelatin. Commercial gelatin, however, contains small quantities of other proteins.

3. Millon's Test. Proteins boiled with Millon's reagent (prepared by dissolving mercury in its own weight of concentrated nitric acid, diluting with twice the volume of water, and allowing to settle) yield a red precipitate which collects at the surface of the liquid.

Experiment 106.

Materials:

The same proteins as for Experiment 104.

Boil small portions of the proteins in test tubes with Millon's reagent. Note the difference in behavior between gelatin and the others. Pure gelatin gives no Millon's test, this reaction being due to an amino acid of the same class as those to which the xanthoproteic test is due.

4. Biuret Test. — Proteins dissolved in strong alkali and treated with minute quantities of copper sulphate give a violet to blue coloration. The test takes its name from the substance with which it was first obtained. Biuret, NH2. CO. NH. CO. NH2, although neither a protein, a peptide, nor an amino acid, gives a coloration like that given by the proteins.

Experiment 107.

Materials:

The proteins used in Experiment 104.
Peptone.

Dissolve a little of each in a 50 per cent solution of potassium hydroxide. To a test-tubeful of water add a drop or two of copper sulphate solution. Add a few drops of this diluted copper sulphate solution to the alkaline solutions of the proteins. Note any differences observed.

CHAPTER XXXV

THE PROTEINS. II

We have seen that the "building stones which go to make up the molecules of proteins are numerous and varied. We have also seen that a single pair of these amino acids can be joined together into two different products, e.g. glycine and alanine into either glycyl alanine or alanyl glycine. Remembering, now, that the protein molecules are built up, not of two, but of a large number of building stones, we can realize that a great many different kinds of protein molecules might be constructed, not only by varying the selection of the building stones, but also by varying their arrangement.

Whether this be the explanation or not, it is certainly true that the proteins exhibit a remarkable diversity of physical properties, particularly as regards their solubilities. It is customary to attempt to classify the proteins according to their solubilities in a number of solvents.

The albumins dissolve in water and are coagulated by heat. Egg albumin (ovalbumin), milk albumin (lactalbumin), and blood albumin (seralbumin) are typical examples.

The term albumins has been much used as the general class name for proteins. The definition here given is that now recognized by the leading American and British societies of physiology and biological chemistry. Other terms formerly used as synonyms for the modern term proteins are proteids and albuminoids. The societies referred to have agreed to drop the word proteid altogether. The English societies also drop albuminoid, while the American societies use it to designate a special class of proteins. (See below.)