The analysis of amino acids is based on chromato-graphic techniques. Traditional amino acid analyzers involved separation of the amino acid mixture on a column of ion-exchange resin using a series of sodium or lithium citrate buffers of increasing pH. The column effluent was then reacted with ninhy-drin and passed through a spectrophotometer that would detect and quantify a series of peaks. This method is still used, although high-performance liquid chromatography (HPLC) hardware is usually employed. Other postcolumn detection systems can be used, replacing the ninhydrin reagent with orthophthalaldehyde (OPA) or fluorescamine and detecting the product fluorimetrically, thereby increasing the sensitivity.
Amino acids can also be separated by HPLC on a reversed-phase column. The mobile phase is usually based on an aqueous buffer with a gradient of increasing concentration of acetonitrile. In this case, the amino acids are usually converted to a fluorimetrically detectable (or ultraviolet-absorbing) form before being injected onto the column. A wide variety of derivatizing agents can be used for this, including OPA, 1-fluoro-2,4-dinitrobenzene, dansyl chloride, phenylisothiocyanate, and 9-fluorenyl-methyl chloroformate.
It is also possible to measure amino acids using gas-liquid chromatography, but this has never been popular, perhaps because the sample cleanup and deri-vatization steps are more laborious. The amino acids have to be converted to volatile derivatives before analysis, commonly either N-trifluoroacetyl-n-butyl or N-heptafluorobutyl-isobutyl esters. Gas-liquid chromatography is potentially a very sensitive method. It can also be coupled with mass spectro-metry for identification of unknown compounds or for measurement of tracer enrichment when carrying out metabolic studies with stable isotopes.
These analytical methods can be applied equally to the measurement of amino acids in proteins, after hydrolysis, or free amino acids in physiological fluids such as plasma, urine, or tissue extracts. For physiological fluids, the protein must first be removed, and this is usually accomplished by precipitating with an acid such as sulfosalicylic acid or an organic molecule such as acetonitrile. The chromatographic requirements for physiological fluids are more demanding than for protein hydrolysates because there are many more contaminating substances producing extra peaks from which the amino acid peaks must be resolved, so the run time is generally longer.
Proteins have to be hydrolyzed before their amino acid composition can be measured. This is done by heating to 110°C with an excess of 6M HCl, either under nitrogen or in a vacuum. Proteins are usually hydrolyzed for 24 h, but this actually represents a compromise since some amino acids, including valine and isoleucine, may take longer than 24 h to liberate completely, whereas others, including tyro-sine, threonine, and serine, are progressively destroyed. Thus, for complete accuracy a protein should be hydrolysed for different lengths of time (usually between 16 and 72 h) and appropriate extrapolations made to the analytical values for each amino acid.
Acid hydrolysis destroys tryptophan, so a separate alkaline hydrolysis is needed to measure this amino acid. The sulfur-containing amino acids are also partially oxidized during acid hydrolysis, so the protein may be oxidized with performic acid before hydrolysis and the oxidation products of cysteine and methionine measured. Finally, acid hydrolysis converts the amides glutamine and asparagine to their parent dicarboxylic acids, so values are often reported as total [glutamic acid plus glutamine] and [aspartic acid plus asparagine]. If separate values are required for the amides, the protein must be subjected to enzymic hydrolysis.
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