Biochemistry CH 3

LICEO DE CAGAYAN UNIVERSITY
COLLEGE OF MEDICINE
BIOCHEMISTRY 1ST BIMONTHLY
AUGUST 12, 2020
Fernandez, Ann Ross L. 20200733258
PROTEIN DETERMINATION OF PRIMARY STRUCTURE
LEARNING OBJECTIVES:
1. Describe the structure of proteins.

Proteins are polymers of amino acids linked by covalent peptide bonds. Because it is a large
molecule, different conformations or three dimensional structures are possible. However, of these
many possible structure, one or a few have biological activity which we call native conformations.
Because of their complex structures, proteins are defined in terms of four levels of structure.
2. Describe the structural organization of proteins.

The structure of these proteins is often described according to levels called primary, secondary,
tertiary, and quaternary structure.
Primary structure is the linear sequence of amino acid residues joined through peptide bonds to
form a polypeptide chain.
The secondary structure refers to recurring structures that form in short localized regions of the
polypeptide chain.
The tertiary structure is the overall three-dimensional conformation of a protein which is a

summation of its secondary structure.
The quaternary structure is the association of polypeptide subunits in a geometrically specific

manner.The forces involved in a protein folding into its final conformation are primarily
noncovalent interactions. These interactions include the attraction between positively and
negatively charged molecules (ionic interactions), the hydrophobic effect, hydrogen bonding, and
van der Waals interactions (the nonspecific attraction between closely packed atoms).
3. Describe multiple chromatographic methods commonly employed for the isolation of proteins from
biologic materials.
Generally, in column chromatography, the stationary phase matrix consists of small beads loaded
into a cylindrical container of glass, plastic, or steel called a column. Liquid-permeable frits confine
the beads within this space while allowing the mobile-phase liquid to flow or percolate through the
column. As the mobile-phase liquid emerges from the column, it is automatically collected as a
series of small portions called fractions.
Affinity chromatography uses the specific binding properties of proteins for their ligands. It
separates proteins based on their affinity for specific ligands. In theory, only proteins that interact
with the ligand adhere.
Similar to affinity chromatography, ion-exchange chromatography uses a ligand. However, the

interaction between the ligand and the protein is based on net charge (charge-charge interactions.
For example, proteins with a net postive charge at a given pH will tightly adhere to beads with
negatively charged functional groups such as carboxylates or sulfates. The nonadherent proteins
flow throught the matrix and are washed away. After all the nonadherent proteins are eluted, the
eluent (the mobile phase) is changed by raising its ionic strength which weakens the charge-
charge interactions.
High-pressure liquid chromatography (HPLC) enables protein separation faster than a standard
column chromatography. Older column chromatography matrices consisted of long, intertwined
oligosaccharide polymers shaped into spherical beads. With their relatively large size, the
matrices perturbed mobile-phase flow and limited the available surface area for accommodating
added charged or ligand-like groups. Resolution could be increased by reducing particle size.
However, the resistance created by a more tightly packed matrix required the use of very high
pressures that would crush beads made from soft and spongy materials such as polysaccharide
or acrylamide. Eventually, methods were developed to manufacture silicon particles of the
necessary size and shape, to derivatize their surface with various functional groups, and to pack
them into stainless steel columns capable of withstanding pressures of several thousand psi.
Size-exclusion chromatography (also called gel-filtration chromatography) separates proteins on

the basis of their Stokes radii (effective volume occcupied by protein in free solution). This
technique employs porous beads such that when proteins with Stoke radii too large to enter the
pores, they remain in the flowing mobile phase. The proteins emerge in descending order of their
Stokes radii.
Hydrophobic interaction chromatography separates proteins based on their tendency to associate

with a stationary phase matrix coated with hydrophobic groups. The proteins with exposed
hydrophobic surfaces surfaces adhere to the matrix via hydrophobic interactions that are
enhanced by employing a mobile phase of high ionic strength. After nonadherent proteins are
washed away, the polarity of the mobile phase is decreased by gradually lowering its salt
concentration. If the interaction between protein and stationary phase is particularly strong,
ethanol or glycerol may be added to the mobile phase to decrease its polarity and further weaken
hydrophobic interactions.
4. Describe how electrophoresis in polyacrylamide gels can be used to determine a protein’s purity,
relative mass, and isoelectric point.
Electrophoresis separates charged biomolecules based on the rates at which they migrate in an
applied electrical field. The acrylamide (porous matrix) offers more resistance to large molecules.
Because the shape and charge are approximately the same for all proteins in the sample, the size
of the protein becomes the determining factors in the separation: smaller proteins move faster
than large ones. This technique, such as in SDS-PAGE (using polyacrylamide gel), can then be
used to estimate the molecular weights of proteins by comparing the sample with the standard
samples.
In electrophoresis without the use of SDS (an anionic detergent), it would be possible to determine
the isolectric point of a protein. For proteins with similar molecular weights, these will still
travel/migrate differently because of the difference in mass-charge ratio (which is influenced by its
size and isoelectric point). In electrophoresis with SDS, however, the SDS molecules overwhelms
the charge contributions of the amino acid functional groups and renders the charge-to-mass ratio
of the SDS-polypeptide complex equal.
5. Describe the basis on which quadrupole and time-of-flight spectrophotometers determine molecular
mass.
In a simple, single quadrupole mass spectrometer, the sample is imparted a positive charge
through placing it in a vacuum to be vaporized in presence of a proton donor. The ion is then
propelled toward a curved flight tube through using an electrical field. The flight tube has a
magnetic field which deflects the ion at right angles to their original direction of flight. The current
which powers the electromagnetic field is gradually increased until the path of each ion is bent
sufficiently to strike the detector at the end of the flight tube. Molecular mass is proportional to the
force required to bend the path of ions to the same extent (note that this is for ions with identical
net charge). It follows that the greater the mass of the ion, the higher the magnetic field required to
focus it onto the detector.
For time-of-flight (TOF) spectrometers, the flight tube is linear. After vaporization, the ions are
made to accelerate toward the detector by applying the electric field. For molecules of identical
charge, the velocity to which they are accelerated, and the time required to reach the detector, is
inversely proportional to their mass.
6. Give three reasons why mass spectrometry (MS) has largely supplanted chemical methods for the
determination of the primary structure of proteins and the detection of posttranslational
modifications.
Chemical methods present drawbacks in determing primary structure of proteins and one is
that it is a time- and labor-intensive process. Another reason is that every step in the
procedure represented a compromise between efficiency (for each amino acid) and flexibility
(to accommodate all 20 amino acids). In the case of Edman sequencing, its read length varies
from 5 to 30 amino acid residues depending on the quantity and purity of the peptide. Finally,
chemical methods requires large quantities of purified protein (and its purification conditions)
also present a drawback.
With an access to a database of genomes of thousands of organisms, to identify a protein, all

scientists need is the amino acid sequence for some segment (as little as five or six residues)
of the protein. This is also the reason why mass spectrometry (MS) has emerged as the
method of choice for protein identification. MS is superior in sensitivity, speed and versatility
compared to chemical methods for determining the sequences of peptides and proteins.
7. Explain why MS can identify posttranslational modifications that are undetectable by Edman
sequencing or DNA sequencing.
MS exploits the difference in the mass-to-charge ratio (m/z) of ionized atoms or molecules in order
to separate them from each other. This ratio is a characteristic property which can be used to get
structural and chemical information about the molecule and identify them.
MS is significantly more sensitive and tolerant of variations in sample quality compared to the
Edman technique and other chemical methods of sequencing. Because of MS’ sensitivity and the
use of mass-to-charge ratio, MS can be used to analyze metabolites, carbohydrates, and lipids,
and to detect posttranslational modifications such as phosphorylation or hydroxylation that
changes (in increments) the mass of a protein.
REFERENCE:
HARPER’S ILLUSTRATED BIOCHEMISTRY 31ST EDITION
Campbell, M.K., & Farrell, S.O. (2012). Biochemistry (7th edition). Boston, MA: Cengage Learning.
Lieberman, M., & Peet, A. (2015). Marks’ Essentials of Medical Biochemistry (2nd edition).
Philadelphia, PA: Lippincott Williams & Wilkins.

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LICEO DE CAGAYAN UNIVERSITY

Fernandez, Ann Ross L. 20200733258

PROTEIN DETERMINATION OF PRIMARY STRUCTURE

1. Describe the structure of proteins.

2. Describe the structural organization of proteins.

The tertiary structure is the overall three-dimensional conformation of a protein which is a

The quaternary structure is the association of polypeptide subunits in a geometrically specific

Similar to affinity chromatography, ion-exchange chromatography uses a ligand. However, the

Size-exclusion chromatography (also called gel-filtration chromatography) separates proteins on

Hydrophobic interaction chromatography separates proteins based on their tendency to associate

With an access to a database of genomes of thousands of organisms, to identify a protein, all

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