Tymoczko • Berg • Gatto • Stryer

Biochemistry: A
Short Course
Fourth Edition

CHAPTER 5
Techniques in
Protein
Biochemistry

© 2019 Macmillan Learning

 CHAPTER 5
Techniques in Protein Biochemistry
 Chapter 5: Outline

5.1 The Proteome Is the Functional Representation of the

Genome
5.2 The Purification of Proteins Is the First Step in
Understanding Their Function
5.3 Immunological Techniques Are Used to Purify
and Characterize Proteins
5.4 Determination of Primary Structure Facilitates an
Understanding of Protein Function
 Section 5.1 The Proteome Is the Functional
Representation of the Genome
• The proteome is the entire set of proteins expressed and
modified by a cell under a particular set of biochemical
conditions.

• Unlike the genome, the proteome is not an unvarying

characteristic of the cell.
 Section 5.2 The Purification of Proteins Is
the First Step in Understanding Their
Function
Learning objective 4: Explain how proteins can be
purified.

• Proteins can be purified on the basis of differences in their

chemical properties.

– Protein purification requires a test, or assay, that

determines whether the protein of interest is present.
 Lactate Dehydrogenase

• Proteins can be purified on the basis of differences in their

chemical properties.
– An assay for the enzyme lactate dehydrogenase is
based on the fact that a product of the reaction, NADH,
can be detected spectrophotometrically.
 Specific Activity
• Proteins can be purified on the basis of differences in their
chemical properties.
– Protein purifications are monitored in part by
determining the specific activity of the protein being
purified.
– In the case of an enzyme purification, specific activity is
the ratio of enzyme activity to protein concentration.
Specific activity should increase with each step of the
purification procedure.
 Quick Quiz 1

QUICK QUIZ 1
Why is an assay required for protein
purification?
Proteins Must Be Removed from the Cell to
Be Purified
• Cells are disrupted to form a homogenate, which is a
mixture of all of the components of the cell but no intact
cells.
• The homogenate is then centrifuged at low speed to yield
a pellet consisting of nuclei and a supernatant. This
supernatant is then centrifuged at a higher centrifugal force
to yield another pellet and supernatant. This process,
called differential centrifugation, is repeated several more
times to yield a series of pellets enriched in various cellular
materials and a final supernatant called the cytosol.
Model of Differential Centrifugation
 Proteins Can Be Purified According to
Solubility, Size, Charge, and Binding Affinity
• Salting out takes advantage of the fact that the solubility of
proteins varies with the salt concentration. Most proteins
require some salt to dissolve in water, a process called
salting in. As the salt concentration is increased, different
proteins will precipitate at different salt concentrations, a
process called salting out.
Graph of the Dependency of Protein
Solubility on Salt Concentration
 Dialysis
• Proteins can be purified according to solubility, size,
charge, and binding affinity.

– The salt can be removed from a protein solution by

dialysis. The protein solution is placed in a cellophane
bag with pores too small to allow the protein to diffuse
but big enough to allow the salt to equilibrate with the
solution surrounding the dialysis bag.
Diagram of Dialysis
 Separation by Size
• Proteins can be purified according to solubility, size,
charge, and binding affinity.

– Molecular exclusion chromatography (gel filtration

chromatography) allows the separation of proteins on
the basis of size. A column is filled with porous beads.
When a protein solution is passed over the beads, large
proteins cannot enter the beads and exit the column
first. Small proteins can enter the beads and thus have
a longer path and exit the column last.
Model of Gel-filtration Chromatography
 Ion-exchange Chromatography
• Proteins can be purified according to solubility, size,
charge, and binding affinity.

– Ion exchange chromatography allows separation of

proteins on the basis of charge. The beads in the
column are made so as to have a charge. When a
mixture of proteins is passed through the column,
proteins with the same charge as on the column will exit
the column quickly. Proteins with the opposite charge
will bind to the beads and are subsequently released by
increasing the salt concentration or adjusting the pH of
the buffer that is passed through the column.
Model of Ion-exchange Chromatography
 Affinity Chromatography
• Proteins can be purified according to solubility, size,
charge, and binding affinity.

– Affinity chromatography takes advantage of the fact that

some proteins have a high affinity for specific chemicals
or chemical groups. Beads are made with the specific
chemical attached. A protein mixture is passed through
the column. Only protein with affinity for the attached
group will be retained. The bound protein is then
released by passing a solution enriched in the chemical
to which the protein is bound.
Model of Affinity Chromatography
 High-pressure Liquid Chromatography
• Proteins can be purified according to solubility, size,
charge, and binding affinity.
– The resolving power of any chromatographic technique is
related to the number of potential sites of interaction between
the protein and the column beads. Very fine beads allow
more interactions and thus greater resolving power, but flow
rates through such columns are too slow.
– High-pressure liquid chromatography (HPLC) uses very fine
beads in metal columns and high-pressure pumps to move
the liquid through the column. Because of the increased
number of interaction sites, the resolving power of HPLC is
greater than normal columns.
Graph of Gel Filtration by HPLC
 Proteins Can Be Separated by Gel
Electrophoresis and Displayed
• Proteins will migrate in an electrical
field because they are charged. When
the migration occurs in a gel, the
process is called gel electrophoresis.
• Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) allows
accurate determination of mass. SDS
denatures proteins, and for most
proteins, 1 molecule of SDS binds for
every two amino acids. Thus, proteins
have the same charge-to-mass ratio
and migrate in the gel on the basis of
mass only.
Diagram of Polyacrylamide-gel
Electrophoresis
Model of the Sieving Action of a Porous
Polyacrylamide Gel
 Staining the Gel with Dyes to Visualize
Protein
• Proteins can be separated by gel electrophoresis and
displayed.

– Proteins separated by SDS–PAGE are visualized by

staining the gel with dyes such as Coomassie blue.
Image of Stained Proteins After
Electrophoresis
 Isoelectric Focusing
• Proteins can be separated by gel electrophoresis and
displayed.
– Isoelectric focusing allows separation of proteins in a
gel on the basis of their relative amounts of acidic and
basic amino acids. If a mixture of proteins is placed in a
gel with a pH gradient and an electrical field is applied,
proteins will migrate until they reach their isoelectric
point (pI), the pH at which they have no net charge.
Model of Isoelectric Focusing
 Two-dimensional Gel Electrophoresis
• Proteins can be separated by gel electrophoresis and
displayed.

– In two-dimensional gel electrophoresis, proteins are

separated in one direction by isoelectric focusing. This
gel is then attached to an SDS–PAGE gel, and
electrophoresis is performed at a 90 angle to the
direction of the isoelectric focusing separation.
Diagram of Two-dimensional Gel
Electrophoresis
Image of Two-dimensional Gel
Electrophoresis
Images of Alteration in Proteins Levels
Detected by Two-dimensional Gel
Electrophoresis
 A Purification Scheme Can Be
Quantitatively Evaluated
• The effectiveness of a purification scheme is measured by
calculating the specific activity after each separation
technique.
• SDS–PAGE allows a visual evaluation of the purification
scheme.
 Table 5.1 Quantification of a purification
protocol for a hypothetical protein

Step Total protein Total activity Specific activity Yield Purification

(mg) (units) (units mg−1) (%) level
Homogenization 15,000 150,000 10 100 1
Salt fractionation 4600 138,000 30 92 3

Ion-exchange 1278 115,500 90 77 9

chromatography
Molecular exclusion 68.8 75,000 1100 50 110
chromatography
Affinity 1.75 52,500 30,000 35 3000
chromatography
Diagram of Electrophoretic Analysis of a
Protein Purification
 Quick Quiz 2

QUICK QUIZ 2
What physical differences among proteins
allow for their purification?
 Section 5.3 Immunological Techniques Are
Used to Purify and Characterize Proteins
Learning objective 5: Explain how immunological
techniques can be used to purify and identify proteins.

• The estrogen receptor binds the

steroid hormone estradiol tightly and
with great specificity.
• The estrogen receptor has no
enzymatic activity but can be
purified by immunological
techniques and the use of gradient
centrifugation.
 Centrifugation Is a Means of Separating
Proteins
• Ultracentrifugation can be used to examine proteins. When
subjected to a centrifugal force, the rate of movement of
the particle is defined by the sedimentation coefficient, s.
s = m (1 – v̅ ρ) /ƒ
• Where m = mass, v̅ = the partial specific volume (the
reciprocal of the particle density), ρ = density of the
medium, and ƒ = the frictional coefficient of the particle.
 Sedimentation Coefficients
• Centrifugation Is a Means of Separating Proteins
– Sedimentation coefficients are usually expressed as
Svedberg units (S) equal to 10−13 s.
– The smaller the S value, the slower the protein moves
in a centrifugal field.
Table 5.2 S Values and Molecular Weights
of Sample Proteins

Protein S value (Svedberg units) Molecular weight

Pancreatic trypsin inhibitor 1 6520

Cytochrome c 1.83 12,310

Ribonuclease A 1.78 13,690

Myoglobin 1.97 17,800

Trypsin 2.5 23,200

Carbonic anhydrase 3.23 28,800

Concanavalin A 3.8 51,260

Malate dehydrogenase 5.76 74,900

Lactate dehydrogenase 7.54 146,200

Gradient Centrifugation Provides an Assay for
the Estradiol–Receptor Complex
• A density gradient is formed in a centrifuge tube, and a
mixture of proteins in solution is placed on top of the
gradient.
• To identify the estradiol receptor, the protein mixture is first
incubated with radioactive estradiol, which is readily
detected. Only the estradiol receptor will bind to the
steroid. Moreover, the steroid alone is too small to be
influenced by the centrifugal force.
• After the centrifugation is complete, a small hole is made in
the bottom of the centrifuge tube and portions of the
gradient are collected and tested for radioactivity.
Diagram of Zonal Centrifugation
 Diagram of the Gradient Centrifugation
Analysis of the Estradiol–receptor Complex
 Antibodies to Specific Proteins Can Be
Generated
• An antibody is a protein synthesized in response to the
presence of a foreign substance called an antigen.
• The antibody recognizes a particular structural feature on
the antigen called the antigenic determinant or epitope.
Structure of an Antibody
Model of Antigen–Antibody Interactions
 Monoclonal vs Polyclonal Antibodies
• Antibodies to specific proteins can be generated.
– Any antibody-producing cell synthesizes antibodies that
recognize only one epitope. Each antibody-producing
cell thus synthesizes a monoclonal antibody.
– Any antigen may have multiple epitopes. The antibodies
produced to the antigen by different cells are said to be
polyclonal.
Model of Polyclonal and Monoclonal
Antibodies
Monoclonal Antibodies with Virtually Any
Desired Specificity Can Be Readily Prepared
• Immortal cell lines
producing monoclonal
antibodies can be
generated by fusing DID YOU KNOW?
normal antibody- Clinical laboratories use monoclonal
antibodies in many assays. For example, the
producing cells with detection in the blood of enzymes that are
cells from a type of normally localized in the heart points to a
cancer called multiple myocardial infarction (heart attack). Blood
transfusions have been made safer by
myeloma. antibody screening of donor blood for
viruses that cause AIDS, hepatitis, and other
• A monoclonal cell line infections diseases. Monoclonal antibodies
is isolated by screening also find uses as therapeutic agents.
Trastuzumab (Herceptin), for example, is a
for the antibody of
monoclonal antibody useful for treating
interest. some forms of breast cancer.
Diagram of the Preparation of Monoclonal
Antibodies
 The Estrogen Receptor Can Be Purified by
Immunoprecipitation
• A monoclonal antibody for the estrogen receptor can be
isolated by searching for cell lines that produce an
antibody that binds to the receptor.
• If an antibody for the receptor is present, it will bind to the
receptor and alter the sedimentation constant of the
receptor.
 Graph of the Alteration of the
Sedimentation Profile of an Antibody
 Antibody Can be Used to Purify the
Estrogen Receptor
• The estrogen receptor can be purified by
immunoprecipitation

– Once the monoclonal cell line is isolated, the antibody

can be used to purify the estrogen receptor.
 Diagram of Purification by
Immunoprecipitation (1/3)
 Diagram of Purification by
Immunoprecipitation (2/3)
 Diagram of Purification by
Immunoprecipitation (3/3)
 Proteins Can Be Detected and Quantified
with the Use of an Enzyme-Linked
Immunosorbent Assay
• Antibodies are used as a reagent to determine the amount
of a protein or other antigen present. Enzyme-linked
immunosorbent assay (ELISA) quantifies the amount of
protein present because the antibody is linked to an
enzyme whose reaction yields a readily identified colored
product.
Diagram of Indirect and Sandwich ELISA
Diagram of Indirect ELISA
Diagram of Sandwich ELISA
 Western Blotting Permits the Detection of
Proteins Separated by Gel Electrophoresis

• In western blotting or immunoblotting, proteins are

separated in an SDS–PAGE gel, transferred to a sheet of
polymer, and then stained with a fluorescent antibody.
Diagram of Western Blotting
 Quick Quiz 3

QUICK QUIZ 3
What is the biochemical basis for the
power of immunological techniques?
 Section 5.4 Determination of Primary
Structure Facilitates and Understanding of
Protein Function
• A key step in understanding protein function is to
determine the primary structure, or the amino acid
sequence, of the protein. A preliminary step is to determine
the amino acid composition of the protein.
• The protein is hydrolyzed, and the constituent amino acids
are separated on an ion-exchange column. The amino
acids are visualized by reaction with fluorescamine.
Diagram of Fluorescent Derivatives of
Amino Acids
Elution Profile
 Edman Degradation
• The amino acid sequence can be determined by Edman
degradation. The protein is exposed to phenyl
isothiocyanate (PTH), which reacts with the N-terminal
amino acid to form a PTH-derivative. The PTH-amino acid
can be released without hydrolyzing the remainder of the
protein, and the degradation is subsequently repeated.
Diagram of Edman Degradation (1/3)
Diagram of Edman Degradation (2/3)
Diagram of Edman Degradation (3/3)
 Overlap Peptides
• Because the reactions of the Edman degradation
procedure are not 100% effective, it is not possible to
sequence polypeptides longer than 50 amino acids.
• In order to sequence the entire protein, the protein is
chemically or enzymatically cleaved to yield peptides of
fewer than 50 amino acids.
• The peptides are then ordered by performing a different
cleavage procedure in order to generate overlap peptides.
 Table 5.3 Specific Cleavage of
Polypeptides
Reagent Cleavage site
Chemical cleavage
Cyanogen bromide Carboxyl side of methionine residues
O-Iodosobenzoate Carboxyl side of tryptophan residues
Hydroxylamine Asparagine-glycine bonds
2-Nitro-5-thiocyanobenzoate Amino side of cysteine residues
Enzymatic cleavage
Trypsin Carboxyl side of lysine and arginine residues
Clostripain Carboxyl side of arginine residues
Staphylococcal protease Carboxyl side of aspartate and glutamate residues (glutamate only under certain
conditions)
Thrombin Carboxyl side of arginine
Chymotrypsin Carboxyl side of tyrosine, tryptophan, phenylalanine, leucine, and methionine
Carboxypeptidase A Amino side of carboxyl-terminal amino acid (not arginine, lysine, or proline)
Diagram of Overlap Peptides
 Mass Spectrometry Can Be Used to
Determine a Protein’s Mass, Identity, and
Sequence
• Two mass spectrometry techniques can be used to
determine a protein’s mass: matrix-assisted laser
desorption (MALDI) and electrospray ionization (ESI). In
MALDI, proteins are precipitated onto a matrix and a laser
flash releases negatively charged ions.
• Time of flight (TOF) analysis is used to measure how
rapidly the ions move toward a detector.
• Only picomoles or femtomoles of a protein are required for
MALDI-TOF analysis.
Diagram of MALDI-TOF Mass
Spectrometry
MALDI-TOF Mass Spectrum
 Protein Identity
• Mass spectrometry allows determination of a protein’s
identity. For instance, an unknown protein visible in a two-
dimensional gel can be removed from the gel.
• The protein is then cleaved in some fashion and subjected
MALDI-TOF, revealing a series of peptides with known
masses.
• These peptide masses are then compared to proteins in a
database and are “electronically cleaved” by a computer
using the same cleavage technique used to generate the
protein fragments.
 Protein Sequence
• A protein’s sequence can be determined with the use of
tandem mass spectrometry.
Diagram of Peptide Sequencing by
Tandem Mass Spectrometry
 Quick Quiz 4

QUICK QUIZ 4
Differentiate between amino acid
composition and amino acid sequence.
 Amino Acid Sequences Are Sources of
Many Kinds of Insight (1/2)
1. Primary structures from different proteins can be compared
to infer knowledge about structure and function.
2. Primary structure comparison of similar proteins from
different species provides information about evolution.
3. Primary structure can be searched for internal repeats that
may yield information on the history of the individual
protein.
Model of Repeating Motifs in a Protein
Chain
 Amino Acid Sequences Are Sources of
Many Kinds of Insight (2/2)
4. Primary structure can reveal the presence of amino acid
sequences that regulate protein function and location.
5. Primary structure can provide insight into the molecular
basis of disease.
6. Primary structure can be used as a guide to explore
nucleic acid information.
 APPENDIX: Biochemistry in Focus (1/2)
• The development of affinity chromatography
– Pedro Cuatrecasas, Christian Anfinsen, and colleagues
are generally credited with the development of affinity
chromatography.
– Cuatrecasas purified the enzyme chymotrypsin by
attaching an inhibitor of the enzyme D-tryptophan
methyl ester to the Sepharose matrix. Eventually they
improved their results by adding a six-carbon linker to
the inhibitor and then attaching the linker to the matrix.
 APPENDIX: Biochemistry in Focus (2/2)
Graph of chymotrypsin affinity chromatography
 APPENDIX: Problem-Solving Strategies

PROBLEM:
A peptide antibiotic was isolated from one of the nastiest-looking prokaryotes ever
viewed. The following observations were made about the peptide.
A) Acid hydrolysis yielded equal amounts of the following amino acids: L, M, F, I, and V.
B) For what it is worth, the molecular weight of the peptide is about 1100 daltons.
C) When treated with the enzyme carboxypeptidase—an enzyme that degrades
peptides from the carboxyl terminus—the peptide failed to undergo hydrolysis.
D) Treatment with fluorescamine, which reacts with the α-amino group of amino acids,
followed by acid hydrolysis yielded only unmodified amino acids.
E) Partial acid hydrolysis followed by chromatographic separation yielded the following
dipeptides and tripeptides:
L-F F-I-M-L V-M
V-M-L F-I-V I-V-M
Given the above information, determine the amino acid sequence of the peptide.
 APPENDIX: Problem-Solving Strategies
Solution (1/3)
SOLUTION:

• Because it is from a nasty-looking creature, best to wear rubber

gloves while solving this problem.

– Given the amino acid composition, what is the significance of the

lack of carboxypeptidase?

• Carboxypeptidase doesn’t work. This might lead a person to think

there is no free carboxyl terminus. Except …

– Why is it important to know the amino acid composition before

making any conclusions about the lack of carboxypeptidase
activity?

• Because carboxypeptidase will not degrade peptides with arginine,

lysine, or proline as the terminal amino acid. None of these are
present.
 APPENDIX: Problem-Solving Strategies
Solution (2/3)
SOLUTION:
• What is the significance of the absence of modified N-terminal amino acid?
• The obvious answer is that there is no free N-terminal amino acid. (That is
starting to get creepy.)
• Okay, so no free N-terminal and no free carboxyl terminal. No beginning and
no end.
• What physical structure has no beginning and no end?
• A circle! (Circular prokaryotic peptides are not rare.)
• What is the sequence of the peptide?
• Just look for overlaps and then construct the sequence. The sequencing
information suggests the sequence L-F-I-V-M or M-L-F-I-V or F-I-V-M-L.
• You get the picture.
 APPENDIX: Problem-Solving Strategies
Solution (3/3)
SOLUTION:
• What does the fact that the antibiotic has a molecular weight of 1100
daltons tell us?
– Well it tells us that a pentapeptide is too small. The average
amino acid weighs in at somewhere between 100 and 110, so a
pentapeptide would have a molecular weight around 550
daltons.
• What is the significance of the factual observation that all amino
acids are present in equimolar amounts?
– If a pentapeptide were too small, a decapeptide would be just
right.
L-F-I-V-M-L-F-I-V-M
– or some iteration of the sequence. It is a circle after all.