CHAPTER
Protein Isolation and Determination
of Amino Acid Sequence
To understand the structure and function of a protein, one
must know the number and kinds of amino acids present
in the protein and their order (called
sequence
or
primary
structure).
This information is necessary to understand
the effects of mutations, mechanisms of enzyme-catalyzed
reactions, and chemical synthesis of species-specific pep-
tides that may eliminate undesirable hypersensitivity re-
actions. Studies of amino acid sequences in proteins have
aided in the understanding of the evolutionary develop-
ment of living systems. Another application of amino acid
sequence determination is in recombinant DNA techno-
logy. A desired DNA coding for a given polypeptide can
be constructed from knowledge of the precise sequence of
that polypeptide (Chapter 23). Through the use of avail-
able technologies of sequence determination and peptide
synthesis, peptide fragments have been produced that are
identical to part of large proteins present on the surface
of a virus or other pathogen. In the appropriate host, such
peptides elicit antibody production that is effective against
the active pathogen and are potentially useful in the de-
velopment of synthetic vaccines (Chapter 35). Viruses
for which immunogenic peptides are being synthesized
and clinically applied are hepatitis B, influenza, rabies,
mouse leukemia, and hoof-and-mouth disease. Bovine in-
sulin (M.W. 5,700) was the first protein to be completely
sequenced (Sanger, 1955). Sanger was also the first to
deduce the base sequence of a DNA molecule obtained
from phage
(f)X
174 (a bacterial virus; see Chapter 23).
Bovine insulin comprises two peptide chains of 21 and
30 amino acids each, linked by two interchain disulfide
bonds.
In 1960, Hirs, Moore, Stein, and Anfinsen described
the first primary structure of the enzyme
ribonuclease
(M.W. 13,700), which has a single peptide chain of 124
amino acid residues and four intrachain disulfide bonds.
These investigators established many of the techniques
still used in sequence analysis, such as the use of ion
exchange resins for separation of peptides and amino
acids.
3.1
Quantitative Determination of Proteins
UV absorption at 280 nm
is an inaccurate method of
protein determination because proteins have different
amounts of tyrosine and tryptophan residues, and nucleic
acids also absorb at 280 nm.
In the
biuret test,
the sample is treated with an alkaline
copper sulfate reagent that produces a violet color and
requires a peptide with at least two peptide bonds. The vi-
olet color is produced through formation of a coordination
complex (between peptide nitrogen atoms and cupric ion)
that is analogous to the structure of the complex of biuret
with cupric ion, as shown below:
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