Folinic acid peptides

Naturally occurring peptides of the pteroylglutamic acid (PGA) family and of folinic acid, such as p-aminobensoylpolyglutamic acid, are very closely related, by structure as well as by origin, and they may be considered as belonging to the same group. We will therefore discuss them together. 

Naturally Occurring Peptides of the Folinic Acid Family... 

In Goiffon s method (G3) peptides are precipitated with phospho-tungstic acid from a trichloroacetic acid filtrate. The precipitate is dissolved and color is developed by a reaction with Folins phenol reagent. This method, however, is not only specific for peptides and a separate assay of uric acid has to be made since this substance also reacts with Folin s reagent. 

In the Biuret reaction, a purple colour develops when the protein is treated with alkaline copper sulphate. This reaction is dependent on peptide bonds and not on the side chains of individual amino-acids present. In the Folin-Ciocalteu reaction, the protein is treated with tungstate and molybdate under alkaline conditions and the formation of a complex such phenylalanine and tyrosine gives rise to a blue colour. Lowry developed one of the most widely used protein assays in which a combination of the above reactions is involved07, l8). 

When it attacks proteins, pepsin liberates tyrosine bound by central bonds at the carboxyl end of the peptide chain, as well as tyrosine at the amino terminal end. At the Rockefeller Institute, M. L. Anson and A. E. Mirsky made liberation of tyrosine the basis of their method for estimating pepsin. They added a pepsin solution to a standard solution of hemoglobin in 0.6 N HCl. Hemoglobin is a substrate easily prepared in large quantities it can be stored without deterioration and it is uniform from one batch to another. The acidity chosen by Anson and Mirsky is well on the acid side of the pH where small variations in pH cause large changes in peptic activity. Anson and Mirsky stopped the reaction with trichloracetic acid and measured the tyrosine in the filtrate by means of the Ciocalteu-Folin phenol reagent. The method was universally adopted. ... 

The final colour in the Lowry method is a result of two reactions. The first is a small contribution from the biuret reaction of protein with copper ions in alkali solution. The second results from peptide-bound copper ions facilitating the reduction of the phos-phomolybdic-tungstic acid (the Folin reagent) which gives rise to a number of reduced species with a characteristic blue colour. The amino acid residues which are involved in the reaction are tryptophan and tyrosine as well as cysteine, cystine and histidine. The amount of colour produced varies slightly with different proteins. In this respect it is a less-reliable assay than the biuret method, but it is more reliable than the absorbance method since A280 may include contribution from other species, and also the absorption of a given residue is dependent on its environment within the protein. 

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