Chapter 3 Flashcards
(48 cards)
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Substances having this dual (acid-base) nature are amphoteric and are o
led ampholytes
(from “amphoteric electrolytes”). A simple monoamino monocarboxylic α-amino acid, such as
alanine, is a diprotic acid when fully protonated; it has two groups, the —COOH group and the
—NH
+
3 group, that can yield protons
good pics on pg 373
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The 20 amino acids commonly found as residues in proteins contain an \and thus amino acids can exist in at least two
stereoisomeric forms.
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Only the L stereoisomers of amino acids, with a configuration related to the absolute
configuration of the reference molecule L-glyceraldehyde, are found in proteins.
Amino acids can be classified into five types on the basis of the polarity and charge (at pH 7) of
their R groups.
Other, less common amino acids also occur, either as constituents of proteins (usually through
modification of common amino acid residues a
α-carboxyl group, an αamino group, and a distinctive R group substituted on the α-carbon atom.
The α-carbon atom of
all amino acids except glycine is asymmetric,
\and thus amino acids can exist in at least two
stereoisomeric forms.
Only the L stereoisomers of amino acids, with a configuration related to the absolute
configuration of the reference molecule L-glyceraldehyde, are found in
proteins
Amino acids can be classified into five types on the basis of
the polarity and charge (at pH 7) of
their R groups.
Other, less common amino acids also occur, either as
s constituents of proteins (usually through
modification of common amino acid residues a
Amino acids vary in their acid-base properties and have characteristic
titration curves.
Monoamino monocarboxylic amino acids (with nonionizable R groups) are diprotic acids
(+H3NCH(R)COOH) at low pH and exist in several different ionic forms as the pH is increase
Amino acids with ionizable R groups have additional
ionic species, depending on the pH of the
medium and the pKa of the R group
Ionizable R groups in a peptide (Table 3-1) also
contribute to
the overall acid-base properties of the molecule
Many small peptides exert their effects at
very low
concentrations. For example, a number of vertebrate hormones
(Chapter 23) are small peptides. These include oxytocin (nine
amino acid residues), which is secreted by the posterior pituitary
gland and stimulates uterine contractions, and thyrotropinreleasing factor (three residues), which is formed in the
hypothalamus and stimulates the release of another hormone,
thyrotropin, from the anterior pituitary gland. Some extremely
toxic mushroom poisons, such as amanitin, are also small
peptides, as are many antibiotics.
Amino acids can be
joined covalently through peptide bonds to
form peptides and proteins. Cells generally contain thousands of
different proteins, each with a different biological activity.
The ionization behavior of peptides reflects their ionizable side
chains as well as
the terminal α-amino and α-carboxyl groups.
Proteins can be very long polypeptide chains of
100 to several
thousand amino acid residues. However, some naturally
occurring peptides have only a few amino acid residues. Some
proteins are composed of several noncovalently associated
polypeptide chains, called subunits.
Simple proteins yield only amino acids on
hydrolysis;
conjugated proteins contain in addition some other component,
such as a metal or organic prosthetic group.
Ion-exchange chromatography
exploits differences in the sign
and magnitude of the net electric charge of proteins at a given pH
Affinity chromatography
is based on binding affinity (Fig. 3-17c).
The beads in the column have a covalently attached chemical
group called a ligand — a group or molecule that binds to a
macromolecule such as a protein.
Protein purification protocols o
fuse additional amino acids or peptides (tags) to the target
protein. Affinity chromatography can be used to bind this tag,
achieving a large increase in purity in a single step (see Fig. 9-11).
In many cases, the tag can be subsequently removed, fully
restoring the function of the native protein.
electrophoresis
Protein purification is usually complemented by electrophoresis,
an analytical process that allows researchers to visualize and
characterize proteins as they are purified.
The electrophoretic method commonly employed for estimation
of purity and molecular weight makes use of the detergent
sodium dodecyl sulfate (SDS) (“dodecyl” denoting a 12-carbon
chain).
Isoelectric focusing
is a procedure used to determine the
isoelectric point (pI) of a protein (Fig. 3-20).
A pH gradient is
established by
y allowing a mixture of low molecular weight
organic acids and bases (ampholytes; p. 77) to distribute
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themselves in an electric field generated across the gel. When a
protein mixture is applied, each protein migrates until it reaches
the pH that matches its pI. Proteins with different isoelectric
points are thus distributed differently throughout the gel.
Combining isoelectric focusing and SDS electrophoresis
sequentially in a process called
two-dimensional electrophoresis
permits the resolution of complex mixtures of proteins (Fig. 3-
21). This is a more sensitive analytical method than either
electrophoretic method alone. Two-dimensional electrophoresis
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separates proteins of identical molecular weight that differ in pI,
or proteins with similar pI values but different molecular weights.
