Amino acid Folins reagent

The older methods for the measurement of protein in natural waters usually depended upon the presence of aromatic amino acids in the protein, and calculated total protein on the basis of an average tyrosine, tryptophan, or phenylalanine content. A method representative of this type was the Folin reagent method published by Debeika et al. [281]. While these methods were useful in fresh water and in some coastal regions, they were not sensitive enough for the lower concentrations to be found in oceanic waters. 

The attempts to use some of the more established reagents for specific analysis of protein e.g. Folin-phenol reagent or the variation of the method by Lowry et al. (1951), whereby total protein content is calculated from the content of aromatic amino acids (tryptophan, tyrosine and phenylalanine), lacks the sensitivity for determinations on seawater samples (Wangersky and Zika, 1978). Furthermore, detailed analysis of the combined amino acid fraction of seawater does not show any aromatic amino acid to be present in significant quantity (Garrasi et al., 1979). 

A number of other reagents have been used to give colored derivatives of amino acids, among these being Folin s reagent, 1,2-naphthoquinon-4-sulfonic acid and o-phthalaldehyde. However, the ninhydrin reaction is so easy to perform and sensitive that other reactions are of very limited importance. Derivatization of amino acids to give colored and/or fluorescent products subsequently subjected to chromatographic procedures can be mentioned in connection with the discussion of color reactions for amino acids. 

Other highly sensitive reagents are 2,4,6-trinitro-benzosulfonic acid, l,2-naphthoquinone-4-sulfonic acid (Folin s reagent), and 4,4 -tetramethyldiaminodi-phenylmethane (TDM). Intensely fluorescing amino acid derivatives are formed by reaction with o-phthal-aldehyde in the presence of reducing agents with pyr-idoxal and zinc ions and with dansyl chloride (5-di-methylaminonaphthalene sulfonyl chloride). 

The major drawback of the Folin-Denis method is that the reagent reacts with other phenolic constituents such as xanthine, proteins and amino acids. However, persimmon flesh usually contains small amounts of such substances, so the values obtained by the method probably reflect the amount of soluble tannins present. 

Amino acids having specific groupings which may be readily determined have offered a simple approach to protein analysis. Of these tyrosine (and tryptophan) have received the most attention. Little effort has been made to analyze for protein content by determination of the sulfur containing amino acids, cystine and cysteine. Amino acids containing a benzene ring react with hot concentrated nitric acid (xanthoproteic reaction) to form yellow nitro compounds which become orange on neutralization. Millon s reagent and that of Folin, particularly the... 

The determination of protein by nitration of its brazene ring containing amino acids (xanthoproteic reaction) has the single advantage of a ampler reagent than methods based on the Folin phenol reagent. The reaction is less sensitive and is somewhat difficult to standardize. Techniques for protein analysis by this means have been described by various authors (107,108,109). 

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. 

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. ... 

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