Reaction of Folin's Reagent with Proteins and Biuret Compounds in Presence of Cupric Ion

In an earlier paper 1 the writer noted that traces of copper sulfate increased the blue color obtained by treating such proteins as pepsin or gelatin with Folin's phenol reagent. It has now been found that a number of organic substances reduce the Folin's phenol reagent only if a small amount of cupric ion is present. These same substances also show a positive biuret reaction. The copper-phenol values in Table I represent the amount of color per milligram of substance produced as a result of adding m/1300 copper sulfate to the solution under examination with the phenol reagent. One obtains this value from the difference in color values produced with and without copper ion. For many substances no color is produced in the absence of copper but most proteins contain tyrosine and tryptophane and must, therefore, be analyzed with and without cupric ion. No attempt has been made to determine whether each of the substances in Table I produces a blue color exactly in proportion to its concentration. With leucyl-glycyl-glycine the proportionality is quite good while with pure biuret it is not as good. The order of magnitude of the values in Table I is of more significance than the absolute values. The solutions have been read against a tyrosine standard and the values calculated as the number of milligrams of tyrosine that will give the same amount of color under identical conditions. No amino acids show any comparable effect of the presence of cupric ion when tested with the phenol reagent. With certain substances, particularly biuret, the presence of some non-chromogenic substances has fairly pronounced effect on the amount of blue color that develops in the presence of cupric ion. Thus the presence of glycine or glycyl-glycine increases the copper-phenol color of biuret. Urea gives a blue color with the phenol reagent in the presence of copper ion and will, therefore, interfere if present in a solution being analyzed. In view of the fact that the products of hydrolysis have little if any influence on the color of leucyl-glycyl-glycine, it seems likely that one could follow quantitatively the extent of enzymatic hydrolysis of this and similar peptides by means of this color test.