(a) Biuret Reaction.—Solutions of copper sulfate, added to an alkaline solution of a protein, give a bluish-violet color if the substance contains two, or more, —CONH— groups united together through carbon, nitrogen, or sulfur atoms. Inasmuch as most natural proteins contain several such groups, the biuret reaction is a very general test for proteins.
(b) Millon's Reaction.—A solution of mercuric nitrate containing some free nitrous acid (Millon's reagent) produces a precipitate which turns pink or red, whenever it is added to a solution which contains tyrosine, or a tyrosine-containing protein.
(c) Xanthoproteic Acid Reaction.—This is the familiar yellow coloration which is produced whenever nitric acid comes in contact with animal flesh. It is caused by the action of nitric acid on tyrosine. The color is intensified by heating, and is changed to orange-red by the addition of ammonia.
(d) Adamkiewicz's Reaction.—If concentrated sulfuric acid be added to a solution of a protein to which some acetic acid (or better, glyoxylic acid) has previously been added, a violet color is produced. This color will appear as a ring at the juncture of the two liquids, if the sulfuric acid is poured carefully down the sides of the tube, or throughout the mixture if it is shaken up. It depends upon the interaction of the glyoxylic acid (which is generally present as an impurity in acetic acid) upon the tryptophane group, and is therefore given by all proteins which contain tryptophane.
(e) Molisch's reaction for furfural will be shown by those proteins which contain a carbohydrate group. In applying this test, the solution to be tested is first treated with a few drops of an alcoholic solution of α-naphthol, and then concentrated sulfuric acid is poured carefully down the sides of the test-tube. If carbohydrates are present, either free or as a part of a protein molecule, a red-violet ring forms at the juncture of the two liquids.
(f) Sulfur Test.—If a drop of a solution of lead acetate be added to a solution containing a protein, followed by sufficient sodium hydroxide solution to dissolve the precipitate which forms, and the mixture is heated to boiling, a black or brown coloration will be produced if the protein contains cystine, the sulfur-containing amino-acid.
FOOTNOTES:
[5] Since the proteins are essentially colloidal in nature, many of the terms used in the discussions of their properties, and these properties themselves, will be better understood after the chapter dealing with the colloidal condition of matter has been studied. A more logical arrangement so far as the systematic study of these properties is concerned would be to take up chapter XV before undertaking the study of the proteins (this order is actually followed in some texts on Physiological Chemistry). But from the standpoint of the consideration of the various groups of organic components of plants, it seems a better arrangement to consider these groups in sequence, and then to discuss the various physical-chemical phenomena which govern their activity. However, it is recommended that the student refer at once to [Chapter XV] for an explanation of any terms used here, which may not be familiar to him; and that after the study of [Chapter XV], he return to this chapter dealing with the proteins for an illustrative study of the applications of the principles presented there.
THE CLASSIFICATION OF THE PROTEINS
Formerly, the classification of proteins was based almost wholly upon their solubility and coagulation reactions. More recently, since their products of hydrolysis have been extensively studied, their classification has been modified, in attempts to make it correspond as closely as possible to their chemical constitution and physical properties. As knowledge of these matters progresses, the schemes of classification change. On that account, no one definite scheme is universally used. For example, the English system varies considerably from the one commonly used by American biochemists, which is the one presented below.