42 1 Amino Acids, Peptides, Proteins
Fig. 1.10.Amino acid chromatogram. Separation of a mixture of amino acids (10 nmol/amino acid) by an amino
acid analyzer. Applied is a single ion exchange column: Durrum DC-4A, 295×4 mm buffers P 1 /P 2 /P 3 :0.2N
Na-citrate pH 3. 20 / 0 .2 N Na-citrate pH 4. 25 / 1 .2 N Na-citrate and NaCl of pH 6.45. Temperatures T 1 /T 2 /T 3 :
48 / 56 / 80 ◦C. Flow rate: 25 ml/h; absorbance reading after color development with ninhydrin at 570/440 nm:
—/––––
(cf. 1.2.4.3.5) to prevent reoxidation. Separation
of subunits is achieved by chromatographic or
electrophoretic methods.
1.4.1.2 Terminal Groups........................................
N-terminal amino acids can be determined
by treating a protein with l-fluoro-2,4-
dinitrobenzene (Sanger’sreagent; cf. 1.2.4.2.2) or
5-dimethylaminonaphthalene-1-sulfonyl chlor-
ide (dansyl chloride; cf. 1.2.4.2.1). Another pos-
sibility is the reaction with cyanate, followed by
elimination of the N-terminal amino acid in the
form of hydantoin, and separation and recovery
of the amino acid by cleavage of the hydantoin
(cf. 1.2.4.2.3). The N-terminal amino acid (and
the amino acid sequence close to the N-terminal)
is accessible by hydrolysis with aminopeptidase,
in which case it should be remembered that
the hydrolysis rate is dependent on amino acid
side chains and that proline residues are not
cleaved. A special procedure is required when
the N-terminal residue is acylated (N-formyl- or
N-acetyl amino acids, or pyroglutamic acid).
Determination of C-terminal amino acids is
possible via the hydrazinolysis procedure
recommended byAkabori:(1.83)The C-terminal amino acid is then separated from
the amino acid hydrazides, e. g., by a cation ex-
change resin, and identified. It is possible to mark
the C-terminal amino acid through selective titra-
tion via oxazolinone: