Analisis de aa

The Ninhydrin Reaction
Amino acids can be readily detected and quantified by reaction with ninhydrin.
As shown in Figure 4.10, ninhydrin, or triketohydrindene hydrate, is a
strong oxidizing agent and causes the oxidative deamination of the _-amino
function. The products of the reaction are the resulting aldehyde, ammonia,
carbon dioxide, and hydrindantin, a reduced derivative of ninhydrin.The
ammonia produced in this way can react with the hydrindantin and another
molecule of ninhydrin to yield a purple product (Ruhemann’s Purple) that
can be quantified spectrophotometrically at 570 nm. The appearance of CO2
can also be monitored. Indeed, CO2 evolution is diagnostic of the presence of
an _-amino acid. _-Imino acids, such as proline and hydroxyproline, give bright
yellowninhydrin products with absorption maxima at 440 nm, allowing these
to be distinguished from the _-amino acids. Because amino acids are one of
the components of human skin secretions, the ninhydrin reaction was once
used extensively by law enforcement and forensic personnel for fingerprint
detection. (Fingerprints as old as 15 years can be successfully identified using
the ninhydrin reaction.) Moresensitive fluorescent reagents are now used routinely
for this purpose.
Specific Reactions of Amino Acid Side Chains
A number of reactions of amino acids have become important in recent years
because they are essential to the degradation, sequencing, and chemical synthesis
of peptides and proteins. These reactions are discussed in Chapter 5.
In recent years, biochemists have developed anarsenal of reactions that
are relatively specific to the side chains of particular amino acids. These reactions
can be used to identify functional amino acids at the active sites of enzymes
or to label proteins with appropriate reagents for further study. Cysteine
residues in proteins, for example, react with one another to form disulfide
species and also react with a number of reagents,including maleimides (typically
N-ethylmaleimide), as shown in Figure 4.11.
4.6 ● Separation and Analysis of Amino Acid Mixtures
Chromatographic Methods
The purification and analysis of individual amino acids from complex mixtures
was once a very difficult process. Today, however, the biochemist has a wide
variety of methods available for the separation and analysis of amino acids, or
for thatmatter, any of the other biological molecules and macromolecules we
encounter. All of these methods take advantage of the relative differences in
the physical and chemical characteristics of amino acids, particularly ionization
behavior and solubility characteristics. The methods important for amino
acids include separations based on partition properties (the tendency to associate
with onesolvent or phase over another) and separations based on electrical
charge. In all of the partition methods discussed here, the molecules of
interest are allowed (or forced) to flow through a medium consisting of two
phases—solid–liquid, liquid–liquid, or gas–liquid. In all of these methods, the
molecules must show a preference for associating with one or the other phase.
In this manner, themolecules partition, or distribute themselves, between the
two phases in a manner based on their particular properties. The ratio of the
concentrations of the amino acid (or other species) in the two phases is designated
the partition coefficient.
In 1903, a separation technique based on repeated partitioning between
phases was developed by Mikhail Tswett for the separation of plant pigments(carotenes and chlorophylls). Tswett, a Russian botanist, poured solutions of
the pigments through columns of finely divided alumina and other solid media,
allowing the pigments to partition between the liquid solvent and the solid support.
Owing to the colorful nature of the pigments thus separated, Tswett called
his technique chromatography. This term is now applied to a wide variety of...