Amino acid derivatives, post

Hydrophilic hormones and other water-soluble signaling substances have a variety of biosynthetic pathways. Amino acid derivatives arise in special metabolic pathways (see p. 352) or through post-translational modification (see p. 374). Proteohormones, like all proteins, result from translation in the ribosome (see p. 250). Small peptide hormones and neuropeptides, most of which only consist of 3-30 amino acids, are released from precursor proteins by proteolytic degradation. 

Notice the R group on each amino acid. The R group is called the side chain of the amino acid. Nearly all organisms use the same 20 a-amino acids to synthesize proteins. Many amino acids and amino acid derivatives, such as hydroxyprotine and cystine, can be created by post-translational modifications after the polypeptide is formed. Ten amino acids are essential. ("Essential" means that they cannot be synthesized by the body, so they must be ingested. Some books list 8 or 9 amino acids as essential. The discrepancy involves whether or not to list as essential those amino acids that are derivatives of other essential amino acids.) Each amino acid differs only in its R group. The R groups have different chemical properties. These proper-... 

The small peptides in cheese can be fractionated by various forms of chromatography, e.g., gel permeation, ion-exchange, and especially RP-HPLC. Using these techniques, more than 200 peptides have been demonstrated in Cheddar cheese, many of which have been isolated and identified (see Section IVE). Free amino acids are usually quantified by ion-exchange HPLC with post-column derivitization using ninhydrin or by separation of fluorescent amino acid derivatives by RP-HPLC. 

Pre-column derivatization and subsequent chromatography of the amino acid derivatives potentially provide the highest sensitivity available for amino acid analysis (<10 15 molar). The Important advantage in this approach is that trace amino acid contaminants in the column eluents do not interfere as in the post-column systems. Only amino acids in the sample that have been derivatized are detected, and consequently the baseline is not influenced by possible contaminants contained in the eluents. 

Table III. Amino Acid Derivatives Formed Post- or Cotranslationally... 

Table 2. Some amino acid derivatives formed by post-translational modification in polypeptides. For references see H.B. Vickery [11]...

The preparation of derivatives for TLC has been reviewed by Gasparic (1961) and Edwards (1980). Derivatization has been used in the TLC of amino acids where certain amino acids are easily converted into the so-called DANS forms (Jimethyl amino naphthalene suUbnyl) (Pataki and Niederweiser, 1967). These authors have studied a variety of amino acid derivatives on silica gel. The use of both pre- and post-chromatographic derivatization in TLC applications in food analysis has been discussed (Shalaby, 1996). For a recent update on the use of derivatization in TLC, see Cserhati and Forgacs (1996). 

The formation of an aldehyde group on a macromolecule can produce an extremely useful derivative for subsequent modification or conjugation reactions. In their native state, proteins, peptides, nucleic acids, and oligonucleotides contain no naturally occurring aldehyde residues. There are no aldehydes on amino acid side chains, none introduced by post-translational modifications, and no formyl groups on any of the bases or sugars of DNA and RNA. To create reactive aldehydes at specific locations within these molecules opens the possibility of directing modification reactions toward discrete sites within the macromolecule. 

A wide body of data now supports the idea that infectious prions consist principally or entirely of an abnormal iso form of PrP. Disease associated PrP, designated PrPSc, is derived from PrPc by a post-translational mechanism and neither amino acid sequencing nor systematic study of known covalent post-translational modifications have shown any consistent differences between PrPc and PrIJSt [5,20]. 

The pyridoxal amino acid analog (Pal) was stereoselectively synthesized from a readily available pyridoxol derivative and the residue was incorporated into peptides at the alcohol oxidation state in protected form. Oxidation of the 4 -alcohol group to the desired aldehyde was achieved post-synthetically on free. 

The thyroid hormone thyroxine (tetraiodo-thyronine, T4) and its active form triiodothyronine (T3) are derived from the amino acid tyrosine. The iodine atoms at positions 3 and 5 of the two phenol rings are characteristic of them. Post-translational synthesis of thyroxine takes place in the thyroid gland from tyrosine residues of the protein thyro-globulin, from which it is proteolytically cleaved before being released, iodothyronines are the only organic molecules in the animal organism that contain iodine. They increase the basal metabolic rate, partly by regulating mitochondrial ATP synthesis, in addition, they promote embryonic development. 

The post-column derivatization of amino acids by the ninhydrin technique is a well known method for routine analysis of amino acids [7-9]. The amino acids are usually separated by ion-exchange chromatography and then converted into UV-absorbing derivatives for quantitation. The ninhydrin reaction is often used for TLC detection of amino acids and proteins. 

It is obvious that it is prudent to check the correctness of the amino acid sequence derived from the base sequence of the gene not only at the NHg and COOH termini, which is the common practice, but throughout the entire protein. This would help to uncover any significant errors as well as address the possibility of post-translational modifications. 

As with MID on MS, the more wavelengths that are simultaneously monitored, the greater is the likelihood of valid identification. Another analytical technique is the formation of derivatives which are fluorescent or absorb UV radiation at unique wavelengths. The compound of interest may be derivatized and injected onto the HPLC system the column separates the reactants and then passes them through the detector. The compound may also be derivatized "post column" as done by amino acid analyzers. The de-rivatizing reactant is metered to mix with the column effluent and is then sent to the detector. Ideally, only the derivatized products should be detectable. 

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