36
chapter 3
Protein Isolation and Determination of Amino Acid Sequence
n h 2
1
c = o
1
O H
H H
H O
C u 2 + ------> NH :
fcu 2+
:NH
NH
+
1
\
..•••
••.
.
/
C — N
N — C
- О
II
о
/ / , / \
/ \ , \ \
0 H
H H
H ' o
n h 2
Biuret
V iolet c o m p le x
In the
Folin-Ciocalteu reaction,
the protein reacts
with the phosphomolybdotungstic acid reagent to give
a blue color through reaction with tyrosyl residues. The
sensitivity of this method depends on the amino acid com-
position of proteins in each sample.
Protein content, particularly in urine or cerebrospinal
fluid, may also be estimated by methods based on precipi-
tation using sulfosalicylic acid (an anionic protein pre-
cipitant) or heat. The turbidity, which is a measure of
protein concentration, can be quantitated by spectropho-
tometric absorbance methods or light scattering analysis.
Absorbance of a hydrophobic indicator dye that binds to
protein and changes color is also used.
Specific proteins in biological fluids can be estimated
by more discriminating methods, such as electrophoresis,
specific binding techniques, or immunochemical methods.
3.2 Determination of Primary Structure
The determination of the primary structure of a protein
consists of the following steps:
1. Obtain a pure protein.
2. Determine the amino acid composition and molecular
weight of the pure protein. From amino acid
composition and molecular weight data, calculate the
number of residues of each amino acid present per
protein molecule to the nearest whole number.
3. Reduce disulfide bonds to sulfhydryl groups.
4. Determine amino terminal (N-terminal) and carboxy
terminal (C-tcrminal) amino acids. (A unique residue
for each terminus suggests that the native protein
contains only one peptide chain.)
5. Fragment aliquots of the polypeptide by enzymatic or
chemical hydrolysis and separate the peptide mixtures
into individual fragments. (This process will yield
overlapping sets of smaller peptides.)
6
. Sequence each fragment and, by analyzing the
overlapping parts, assemble the sequence of the
original protein. The logic for arranging overlapping
peptides is as follows:
Assume that cleavage of the above polypeptide with a
specific cleaving agent yields four peptides, А, В, C, and
D. These peptides are separated and sequenced but it is yet
not known whether the correct order is ABCD or ACBD.
A second hydrolytic procedure gives rise to three peptides:
E, F, and G.
-------E ---------- — F ------------------ G--------
H3N---------------
j
---------
j—
і
----------
1
--------
і
---------------
COO
’
A-В
B -C
C -D
b rea k
b rea k
b reak
By comparison of the amino acid sequence of fragments
E and G, the terminal fragments A and D can be matched
with their respective adjacent peptides, В and C. Alterna-
tively, the order of the peptides В and C can be deduced
from the overlapping sequence in F.
Purification of a protein is indispensable for establish-
ing its amino acid sequence. Mixtures of proteins will
yield mixtures of peptides and ambiguous amino acid po-
sitions; a unique amino acid sequence can be obtained only
from a pure protein. Protein purification techniques exploit
differences in size, shape, charge, solubility, and specific
binding affinity of the proteins. The optimal combination
of techniques is usually reached by trial and error.
3.3 Separation of Proteins
Proteins are separated on the basis of differences in their
size, shape, charge, solubility, and binding affinity for
other molecules. Most separations begin with proteins in
solution. However, when proteins are an integral part of an
organelle’s structure, it is first necessary to extract them
from membranous elements. Chemicals used to extract
proteins from membranous particles include dissociating
agents (e.g., urea and mercaptoethanol), chelating agents
(e.g., ethylenediaminetetraacetic acid [EDTA]), and or-
ganic detergents (e.g., sodium deoxycholate, sodium lau-
ryl sulfate, and Triton X-100).
Since the total quantity (or activity) of a protein in a
tissue sample is difficult to determine, the original (
1 0 0
%)
quantity of protein at the start of a purification is usually
based on measurements made on an aliquot of the initial
homogenate. Each step in a purification process should
remove extraneous protein and retain most of the protein
of interest. A pure protein preparation is operationally de-
fined as one that maintains a high activity per gram of pro-
tein following several purification steps, i.e., the optimum
