Dinitrophenyl -amino acids quantitation

In a few cases, alkaline hydrolysis has proved applicable to special problems. Tryptophan is not destroyed in alkali, and analysis of alkaline hydrolyzates forms the basis of one method for quantitative determination of this amino acid (e.g., Dreze, 1960). Despite the fact that tryptophan-containing peptides should be more stable in alkali than acid, partial alkaline hydrolysis has not been employed for identification of this type of peptide. Amino acids often can be regenerated by alkaline hydrolysis from derivatives obtained by the amino-terminal end-group methods. Dinitrophenyl amino acids and phenylthiohydantoin (Fraenkel-Conrat et al., 1955) as well as hydantoin (Stark and Smyth, 1963) derivatives of amino acids can be treated in this manner. 

The dinitrophenyl group has been used to protect the imidazole — NH group in histidines (45% yield)" by reaction with 2,4-dinitrofluorobenzene and potassium carbonate. Imidazole —NH groups, but not a-amino acid groups, are quantitatively regenerated by reaction with 2-mercaptoethanol (22°, pH 8, 1 h)." The 2,4-... 

The sequencing methods and determination of C-terminal and N-terminal amino acids are now widely used in biochemical research. The identification and quantitation of the characteristic degradation products can be accomplished by the gas-phase analytical methods. Thus, GC of both dinitrophenyl and various hydantoin amino acid derivatives has now been widely documented. Separation of thiohydantoins [244,245], phenylthiohydantoins [490,491] and methylthiohydantoins [492] generally requires additional silylation for the sake of volatility. Furthermore, acyl derivatives of similar substances have also been reported [493,494]. The most obvious advantage of GC for determination of the Edman degradation products is sensitivity which is particularly important in the sequence analysis of only minute amounts of proteins and peptide hormones. 

Kerr and Godin [90], using the dinitrophenylation method of Sanger [91], have identified valine, threonine, glycine, alanine, serine, glutamic acid, and aspartic acid as N-terminal amino acids in human hair. Quantitative data... 

In order to be able to detect and determine quantitatively amino acids in biological material (serum, urine, sperm, lymph, milk, tissue) other components of the body fluids and organ extracts, such as proteins, peptides, lipids, carbohydrates, salts and urea, must be removed (see p. 736). If the amino acids are to be separated as their dinitrophenyl derivatives, these may be efficiently separated from the seriously inter-... 

Studies of the structure of papain were initiated by Thompson (165), who determined the amino or N-terminal amino acid sequence and the free amino groups. Crystalline papain and mercuripapain were allowed to react with l-fluoro-2,4-dinitrobenzene (FDNB) by the procedures of Sanger (136). The dinitrophenyl (DNP) proteins were subsequently hydrolyzed in 6 iV HCl and the DNP-amino acids isolated by partition chromatography of the ether extracts of the hydrolyzates. Quantitative estimates of the DNP-amino acids were made spectrophotometrically. The results are given in Table V. 

The chemical structures of these peptides were established in the usual way by quantitative amino-acid analysis and sequence analysis by a combination of chemical and enzymic methods, including dinitrophenylation, dansylation, Edman degradation, hydrazinolysis and digestion with leucine aminopeptidase and carboxypeptidases A and B. The Neutral Protease peptides of the salmine and iridine components or derivatives thus identified are listed in Table VIII-6 with their recovery values. Differences in the amino-acid sequences and amounts of the peptides obtained from iridine I aroused the suspicion that two molecular species (a and b) were present in the iridine I component. This result, as already described, was supported by observations on the thermolysin peptides of iridine I. 

---