Secondary amino acids, proline hydroxyproline

The advantages of this method are a short reaction time and the nonfluorescence of the OPA reagent. Therefore, excess reagent must not be removed before the chromatography stage. Using this method, it is possible to measure tryptophan, but not secondary amino acids such as proline or hydroxyproline. Cysteine and cystine can be measured, but because of the low fluorescence of their derivatives, they must be detected using an UV system, or alternatively oxidized to cysteic acid before reaction. 

Ornithine (a) and hydroxyproline (d) are both a-amino acids, because an amino group is attached to the same carbon atom as that which carries a carboxyl. Although ornithine is not used in protein synthesis, it is an intermediate in the urea cycle (Chap. 15). /3-Alanine (b) and y-aminobutyrate (c) have their amino group attached to a carbon atom different from that bearing the carboxyl, and are a /3- and a y-amino acid, respectively. Strictly speaking proline is a secondary amino acid, because the amino nitrogen is covalently connected to the side chain. It is sometimes referred to as an imino acid. 

Secondary amino acids, imino acids , such as proline and hydroxyproline, do not possess an a-amino group, and react with ninhydrin to form a yellow product which is usually detected at 440 nm. Therefore, amino acid analyzers are equipped with a photometer capable of measuring at two different wavelengths (570 nm and 440 nm). The sensitivity of this detection method is about 200 pmol. 

The OPA reagent was first reported in 1971 by Roth as a postcolimm fluorogenic reagent for amines [5] and has been widely used for the sensitive determination of primary amino compounds. However, the fluorescent derivatives are not sufficiently stable, and it is sometimes difficult to obtain reproducible results using the postcolumn derivatization system. A precolumn derivatization technique has also been developed using OPA in the presence of alkylthiol compounds such as 2-mercaptoethanol. OPA rapidly reacts with primary amino compounds within 2 min at room temperature, and the derivatives can be separated by reversed-phase liquid chromatography [20]. Fluorescence detection of the derivatives is performed at 440 nm (emission wavelength) with excitation at 330 nm. Because OPA does not react with secondary amino compounds, proline and hydroxyproline can not be determined by this method. Replacement of 2-mercaptoethanol with other thiols, such as 2-ethanethiol [21] and 3-mercaptopropionic acid [22], produced more stable fluorescent derivatives. 

The same is true for hydroxyproline, which occurs only in structural proteins. Collagen is rich in both proline and hydroxyproline. The hydroxy group actually would justify the inclusion of this amino acid in Group II. These secondary amino acids are still called imino acids after some archaic chemical nomenclature. Compounds containing the group... 

Primary amino acids will react with o-phthalaldehyde in the presence of the strongly reducing 2-mercaptoethanol (pH 9-11) to yield a fluorescent product (emission maximum, 455 nm excitation maximum, 340 nm). Peptides are less reactive than a-amino acids and secondary amines do not react at all. As a result, proline and hydroxyproline must first be treated with a suitable oxidizing agent such as chloramine T (sodium A-chloro-p-toluene-sulphonamide) or sodium hypochlorite, to convert them into compounds which will react. Similarly cystine and cysteine should also be first oxidized to cysteic acid. 

All primary amines react with fluorescamine under alkaline conditions (pH 9-11) to form a fluorescent product (Figure 10.12) (excitation maximum, 390 nm emission maximum, 475 nm). The fluorescence is unstable in aqueous solution and the reagent must be prepared in acetone. The secondary amines, proline and hydroxyproline, do not react unless they are first converted to primary amines, which can be done using A-chlorosuccinimide. Although the reagent is of interest because of its fast reaction rate with amino acids at room temperature, it does not offer any greater sensitivity than the ninhydrin reaction. 

Some special features of proteins are elaborated by secondary transformations that are not part of the translation process. The A-formylmethionine initiator may be hydrolysed to methionine, or, as we have already indicated, the methionine unit may be removed altogether. Other post-translational changes to individual amino acids may be seen, e.g. the hydroxylation of proline to hydroxyproline (see Section 13.1) or the generation of disulfide bridges between cysteine residues (see Section 13.3). 

Proline and hydroxyproline differ from the other amino acids in that they contain a secondary amino group in a pyrrolidine ring therefore, they do not produce aminoketones and Strecker aldehydes in the reaction with dicarbonyls, ffowever, nitrogen heterocyclics are produced, including 1-pyrroline, pyrrolidine, 2-acetyl-1-pyrroline and 2-acetyltetrahydropyridine (Scheme 12.5) [19]. 

Reinecke salt is of value as a precipitant for primary and secondary amines, proline and hydroxyproline, and certain amino acids.1... 

When a Boc amino acid is being coupled to a N-tenninal proline, hydroxyproline, or other secondary amino group, the Kaiser test is negative or gives an inconclusive brownish color. Such N-terminal residues give a good blue color with isatin.P ... 

Only proline (or hydroxyproline), being a secondary amine, will form Amadori compounds with a glycosylamine as an intermediate. Other amino acids contain primary amino groups, although secondary amines are present in some of them (tryptophan, histidine). 

Only certain a-amino acids are present in any protein structure, and they have the general formula R-CH(NH2)-COOH, with the exception of proline and hydroxyproline that have secondary amino groups. With few exceptions, amino acids have chiral molecules with L (S) configuration at the chiral a carbon ... 

The nitrosation of -amino acids is especially interesting because of the biological importance of N-nitrosoamines (see Sect. 4.2). The nitrosation mechanism was investigated first with amino acids containing a secondary amino group, namely proline (4.1, X = H), 4-hydroxyproline (4.1, X = OH), and sarcosine (4.2), but also with cysteine (4.3). 

---