2009_Book_FoodChemistry

1.4 Proteins 41
which determines the molecular conformation
(secondary and tertiary structures). Proteins
sometimes occur as molecular aggregates which
are arranged in an orderly geometric fashion
(quaternary structure). The sequences and conformations
of a large number of proteins have
been elucidated and recorded in several data
bases.
(1.82)
Glycoproteins, such as κ-casein (cf. 10.1.2.1.1),
various components of egg white (cf. 11.2.3.1)
and egg yolk (cf. 11.2.4.1.2), collagen from
connective tissue (cf. 12.3.2.3.1) and serum
proteins of some species of fish (cf. 13.1.4.2.4),
contain one or more monosaccharide or oligosaccharide
units bound O-glycosidically to serine,
threonine or δ-hydroxylysine or N-glycosidically
to asparagine (Formula 1.82). In glycoproteins,
the primary structure of the protein is defined
genetically. The carbohydrate components,
however, are enzymatically coupled to the
protein in a co- or post-transcriptional step.
Therefore, the carbohydrate composition of
glycoproteins is inhomogeneous (microheterogeneity).
1.4.1 Amino Acid Sequence
1.4.1.1 Amino Acid Composition, Subunits
Sequence analysis can only be conducted on
a pure protein. First, the amino acid composition
is determined after acidic hydrolysis.
The procedure (separation on a single cationexchange
resin column and color development
with ninhydrin reagent or fluorescamine) has
been standardized and automated (amino acid
analyzers). Figure 1.10 shows a typical amino
acid chromatogram.
As an alternative to these established methods,
the derivatization of amino acids with the subsequent
separation and detection of derivatives
is possible (pre-column derivatization). Various
derivatization reagents can be selected, such as:
• 9-Fluorenylmethylchloroformate
(FMOC, cf. 1.2.4.2.1)
• Phenylisothiocyanate (PITC, cf. 1.2.4.2.3)
• Dimethylaminoazobenzenesulfonylchloride
(DABS-Cl, cf. 1.2.4.2.1)
• Dimethylaminonaphthalenesulfonylchloride
(DANS-Cl, cf. 1.2.4.2.1)
• 7-Fluoro-4-nitrobenzo-2-oxa-1,3-diazole
(NBDF, cf. 1.2.4.2.1)
• 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole
(NBDCl, cf. 1.2.4.2.1)
• o-Phthaldialdehyde (OPA, cf. 1.2.4.2.4)
It is also necessary to know the molecular weight
of the protein. This is determined by gel column
chromatography, ultracentrifugation or SDS-
PAG electrophoresis. Furthermore, it is necessary
to determine whether the protein is a single
molecule or consists of a number of identical or
different polypeptide chains (subunits) associated
through disulfide bonds or noncovalent forces.
Dissociation into subunits can be accomplished
by a change in pH, by chemical modification of
the protein, such as by succinylation, or with denaturing
agents (urea, guanidine hydrochloride,
sodium dodecyl sulfate). Disulfide bonds, which
are also found in proteins which consist of only
one peptide chain, can be cleaved by oxidation of
cystine to cysteic acid or by reduction to cysteine
with subsequent alkylation of the thiol group