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BIO 203 Biochemistry Iby
Seyhun YURDUGL,Ph.D.
Lecture 5Proteins
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Content Outline
General properties of the proteins
Protein primary structure Protein secondary structure
The Super-secondary structure
Tertiary structure Quaternary structure
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General properties of proteins The classification of proteins:
carried out according to their biologicalroles.
Eight(8) types: present.
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1. Enzymes The most varied and most highly
specialized proteins:
with catalytic activity. Many thousands of different enzymes
capable of catalyzing different reactions:
present in different organisms. e.g. hydrogen peroxidase,
F-galactosidase(lactase) etc
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2. Transport proteins: In blood plasma
bind and carry specific molecules or ions from one
organ into another: E.g. hemoglobin binds oxygen as the blood passes
through the lungs,
Carries it to peripheral tissues and there releases it, to participate in the energy yielding oxidation of
nutrients.
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3. Nutrient and storage proteins: The seeds of many plants:
store these proteins required for the growthof the germinating seedling.
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3. Nutrient and storage proteins: e.g. seed proteins of wheat, corn and rice.
e.g. ovalbumin, the major protein of eggwhite
e.g. casein, the major protein of milk.
e.g. ferritin in some bacteria and in plantand animal tissues stores iron.
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4. Contractile or motile proteins Some proteins provide cells and organisms
with the ability to contract,
to change shape;
or to move about.
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4. Contractile or motile proteins e.g. actin and myosin: in the contractile
system of skeletal muscle.
Microtubules in cilia and flagella; built bytubulin:
a contractile protein.
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5.Structural proteins: Many proteins serve as supporting
filaments,
to give biological structures strength orprotection.
The major component of tendons and
cartilage: collagen; a typical example. Keratin(hair, fingernails); and fibroin of thespiders web(other examples)
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6.Defense proteins: Many proteins defend organisms against
invasion by other species;
or protect them from injury.
e.g. immunoglobulins, fibrinogen and fibrinin blood clotting.
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7.Regulatory proteins: Some proteins help regulate cellular or
physiological activity.
E.g. many hormones (insulin etc.).
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8.Other proteins The proteins not easily classified:
included in this group.
Examples:
Monellin(isolated from an Africanplant),has an intensive sweet taste,
used as a food sweetener.
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8.Other proteins The blood plasma of some Antarctic fish:
contains anti-freeze proteins.
Protect the blood of the fish from freezing.
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Protein primary structure:
refers to the linear number and order of the amino acids
present. The convention for the designation of the order ofamino acids:
the N-terminal end (i.e. the end bearing the residue withthe free -amino group) is to the left (and the number1
amino acid) and the C-terminal end (i.e. the end with the residue
containing a free alpha-carboxyl group) is to the right.
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How can be the further conformation
of proteins defined? the partial double-bond character of the peptide
bond:
that defines the conformations a polypeptide chainmay assume.
Within a single protein:
different regions of the polypeptide chain mayassume different conformations;
determined by the primary sequence of the aminoacids.
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Protein secondary structure
The ordered array of amino acids in a protein conferregular conformational forms upon that protein.
These conformations constitute the secondarystructures of a protein.
In general proteins fold into two broad classes ofstructure termed:
globular proteins
fibrous proteins.
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Protein secondary structure: Globular proteins:
compactly folded and coiled,
whereas, fibrous proteins are more filamentousor elongated.
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The alpha-helix
a common secondary structure encountered in
proteins of the globular class. The formation of the -helix is spontaneous. stabilized by H-bonding between amide
nitrogens and carbonyl carbons of peptide
bonds spaced four residues apart.
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The alpha-helix
This orientation ofH-bondingproduces a helical coiling of thepeptide backbone:
such that the R-groups lie on the
exterior of the helix andperpendicular to its axis.
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The alpha-helix Not all amino acids favor the formation of
the -helix:
due to steric constraints of the R-groups. imino acid (HN=) whose structure
significantly restricts movement of peptide
bond: thereby, interfering with extension of thehelix.
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The alpha-helix The disruption of the helix is important as:
introduces additional folding of thepolypeptide backbone:
to allow the formation of globular proteins.
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-sheets
Whereas an -helix is composed of a single
linear array of helically disposed amino acids, -sheets are composed of 2 or more different
regions of stretches of at least 5-10 aminoacids.
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-sheets The folding and alignment of stretches of the
polypeptide backbone aside one another to form -sheets:
stabilized by H-bonding between amide nitrogens andcarbonyl carbons.
However, the H-bonding residues are present inadjacently opposed stretches of the polypeptide
backbone:
as opposed to a linearly contiguous region of thebackbone in the -helix.
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-sheets -sheets are said to be pleated:
due to positioning of the -carbons of the
peptide bond which alternates above and below the plane of
the sheet.
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-sheets -sheets are either:
parallel
or antiparallel. In parallel sheets: adjacent peptide chains proceed
in the same direction (i.e. the direction of N-
terminal to C-terminal ends is the same), whereas, in antiparallel sheets,
adjacent chains are aligned in opposite directions.
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Super-secondary structure
Some proteins contain an ordered organization
of secondary structures that form: distinct functional domains;
or structural motifs.
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Super-secondary structure: e.g. the helix-turn-helix domain of bacterial
proteins that regulate transcription
and the leucine zipper, helix-loop-helix and zinc finger domains of eukaryotic
transcriptional regulators.
These domains are termed super-secondarystructures.
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Helix turn helix Helix loop helix
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Zinc Finger
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Tertiary structure:
refers to the complete three-dimensional structure ofthe polypeptide units of a given protein.
Included in this description: the spatial relationship of different secondarystructures to one another within a polypeptide chain;
and how these secondary structures themselves foldinto the three-dimensional form of the protein.
Secondary structures of proteins often constitutedistinct domains.
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A hemolysin from the bacterium Staphylococcus aureus
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Tertiary Structure also describes the relationship of different
domains to one another within a protein.
The interactions of different domains : governed by several forces: include hydrogen bonding, hydrophobic interactions,
electrostatic interactions and van der Waals forces.
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Forces controlling protein
tertiary structure:
Hydrogen bonding:
Polypeptides contain numerous proton donors and acceptorsboth in their backbone
and in the R-groups of the amino acids.
The environment in which proteins are found
contains the H-bond donors and acceptors of the water
molecule. H-bonding, occurs not only within and between polypeptide
chains
but with the surrounding aqueous medium.
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Hydrophobic forces:
Proteins are composed of amino acids:
that contain either hydrophilic
or hydrophobic R-groups. It is the nature of the interaction of the different
R-groups with the aqueous environment:
that plays the major role in shaping proteinstructure.
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Hydrophobic Forces: The spontaneous folded state of globular
proteins:
is a reflection of a balance between theopposing energetics ofH-bonding betweenhydrophilic R-groups
and the aqueous environment and the repulsion
from the aqueous environment by thehydrophobic R-groups.
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Hydrophobic Forces: The hydrophobicity of certain amino acid R-
groups :
tends to drive them away from the exterior of proteins
and into the interior.
This driving force restricts the availableconformations into which a protein may fold.
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Electrostatic forces:
Electrostatic forces are mainly of three types;
charge-charge, charge-dipole
and dipole-dipole.
Typical charge-charge interactions that favorprotein folding are those between oppositelycharged R-groups.
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Electrostatic forces: A substantial component of the energy involved
in protein folding is charge-dipole interactions:
refers to the interaction of ionized R-groups ofamino acids;
with the dipole of the water molecule.
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Electrostatic Forces: The slight dipole moment;
exist in the polar R-groups of amino acid also
influences their interaction with water. So the majority of the amino acids;
found on the exterior surfaces of globularproteins:
contain charged or polar R-groups.
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van der Waals Forces:
There are both
attractive
and repulsive van der Waals forces that control proteinfolding.
extremely weak forces
relative to other forces governing conformation,
it is the huge number of such interactions that occur inlarge protein molecules
that make them significant to the folding of proteins.
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Attractive van der Waals Forces:
involve the interactions;
among induced dipoles that arise from
fluctuations in the charge densities: that occur between adjacent uncharged non-
bonded atoms.
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Repulsive van der Waals forces involve the interactions;
that occur when uncharged non-bonded atoms
come very close together,but do not induce dipoles.
The repulsion is:
the result of the electron-electron repulsion, that occurs as two clouds of electrons begin to
overlap.
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Quaternary structure:
Many proteins contain 2 or more differentpolypeptide chains:
that are held in association by the same non-covalentforces;
that stabilize the tertiary structures of proteins.
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Quaternary structure:
Proteins with multiple polypeptide chains are termedoligomeric proteins.
The structure formed by monomer-monomerinteraction in an oligomeric protein:
is known as quaternary structure.
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Hemoglobin structure
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Quaternary structure:
Oligomeric proteins can be composed of multipleidentical polypeptide chains
or multiple distinct polypeptide chains.
Proteins with identical subunits are termedhomooligomers.
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Quaternary structure:
Proteins containing several distinct polypeptidechains are termed heterooligomers.
e.g. Hemoglobin, the oxygen carrying protein of theblood,
contains two alpha and two beta subunits arrangedwith a quaternary structure in the form,
alpha-2 beta-2.
Hemoglobin is, therefore, a hetero-oligomericprotein.
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Complex protein structures:
Proteins also are found to be covalently conjugatedwith carbohydrates.
These modifications occur following the synthesis(translation) of proteins and are,
therefore, termed post-translational modifications.
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Complex protein structures
These forms of modification: impart specialized functions upon the resultant
proteins.
Proteins covalently associated with carbohydratesare termed glycoproteins.
Glycoproteins are of two classes,
N-linked
and O-linked, referring to the site of covalent attachment of the
sugar moieties.
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Complex protein structures:
N-linked sugars are attached to the amide nitrogenof the R-group of asparagine;
O-linked sugars are attached to the hydroxyl groupsof either serine:
or threonine;
and occasionally to the hydroxyl group of themodified amino acid, hydroxylysine.
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Complex protein structures:
There are extremely important glycoproteins:
found on the surface of erythrocytes.
It is the variability: in the composition of the carbohydrate portions of
many glycoproteins and glycolipids of erythrocytes
that determines blood group specificities.
There are at least 100 blood group determinants, most of which:
due to carbohydrate differences.
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The techniques to characterize
the protein structure in detail
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Amino-Terminal Sequence
Determination
Prior to sequencing peptides:
it is necessary to eliminate disulfide bonds within peptidesand between peptides.
Several different chemical reactions can be used: in order to permit separation of peptide strands,
and prevent protein conformations that are dependentupon disulfide bonds.
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Amino-terminal sequence
determination The most common treatments are to use either:
2-mercaptoethanol
or dithiothreitol.
Both of these chemicals reduce disulfide bonds. To prevent reformation of the disulfide bonds,
the peptides are treated with iodoacetic acid:
in order to alkylate the free sulfhydryls.
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Amino-terminal sequence
determination
There are three major chemical techniques:
for sequencing peptides and proteins from the N-terminus. These are:
the Sanger,
Dansyl chloride
and Edman techniques.
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Sanger's reagent:
sequencing technique:
utilizes the compound,
2,4-dinitrofluorobenzene (DNF):
which reacts with the N-terminal residueunder alkaline conditions(derivatization).
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Sanger's reagent:
The derivatized amino acid can be hydrolyzed:
and will be labeled with a dinitrobenzene group
that imparts a yellow color to the amino acid. Separation of the modified amino acids (DNP-derivative) by electrophoresis;
and comparison with the migration of DNP-
derivative standards: allows for the identification of the N-terminal
amino acid.
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Dansyl chloride:
Like DNF, dansyl chloride reacts with the N-terminal residue
under alkaline conditions. Analysis of the modified amino acids is similar tothe Sanger method;
except that the dansylated amino acids are
detected by fluorescence. This imparts a higher sensitivity into thistechnique over that of the Sanger method.
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Edman degradation:
it allows for additional amino acid sequenceto be obtained from the N-terminus inward.
Using this method: to obtain the entire sequence of peptides.
This method utilizes phenylisothiocyanate
(PITC) to react with the N-terminal residueunder alkaline conditions.
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Edman degradation:
The resultant phenylthiocarbamyl derivatized amino acid:is hydrolyzed in anhydrous acid.
The hydrolysis reaction:
results in a rearrangement of the released N-terminalresidue to a phenylthiohydantoin derivative.
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Edman degradation:
As in the Sanger and Dansyl chloridemethods,
the N-terminal residue is tagged with anidentifiable marker,
however, the added advantage of the Edmanprocess is that:
the remainder of the peptide is intact.
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Edman degradation:
The entire sequence of reactions:
can be repeated over and over:
to obtain the sequences of the peptide.
This process has subsequently beenautomated:
to allow rapid and efficient sequencing ofeven extremely small quantities of peptide.
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Protease digestion
Due to the limitations of the Edman degradationtechnique,
peptides longer than around 50 residues can not besequenced completely.
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Protease digestion
The ability to obtain peptides of this length, fromproteins of greater length,
is facilitated by the use of enzymes,
endopeptidases,
that cleave at specific sites within the primarysequence of proteins.
The resultant smaller peptides can bechromatographically separated and subjected toEdman degradation sequencing reactions.
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Specificities of Several Endoproteases
Enzyme Source Specificity Additional Points
Trypsin Bovine pancreas
peptide bond C-terminal to R, K,
but not if next toP
highly specificfor positivelycharged residues
Chymotrypsin Bovine pancreas
peptide bond C-terminal to F, Y,
W but not if nextto P
prefers bulkyhydrophobic
residues, cleavesslowly at N, H, M,
L
Elastase Bovine pancreas
peptide bond C-terminal to A, G,
S, V, but not ifnext to P
ThermolysinBacillus
thermoproteolyticus
peptide bond N-terminal to I, M, F,
W, Y, V, but not ifnext to P
prefers smallneutral residues,
can cleave at A,D, H, T
PepsinBovine gastric
mucosa
peptide bond N-terminal to L, F,
W, Y, but whennext to P
exhibits littlespecificity,requires low pH
EndopeptidaseV8
Staphylococcusaureus
peptide bond C-terminal to E
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Carboxy-terminal sequence
determination
No reliable chemical techniques exist for sequencingthe C-terminal amino acid of peptides.
However, there are enzymes, exopeptidases, thathave been identified that cleave peptides at the C-terminal residue
which can then be analyzed chromatographically
and compared to standard amino acids. This class of exopeptidases are called,
carboxypeptidases.
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Sources of several exopeptidases
Enzyme Source
Carboxypeptidase A Bovine pancreasCarboxypeptidase B Bovine pancreas
Carboxypeptidase C Citrus leaves
Carboxypeptidase Y Yeast
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Chemical digestion of proteins
The most commonly utilized chemical reagent that cleaves peptide bonds by recognition of specific amino acid
residues: is cyanogen bromide (CNBr).
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Chemical digestion of proteins-
CNBr This reagent causes specific cleavage at the C-terminal side of M
residues. The number of peptide fragments that result from CNBr cleavage is equivalent to one more than the number of M residues in a
protein. The most reliable chemical technique forC-terminal residue
identification is hydrazinolysis.
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Chemical Digestion of Proteins
A peptide is treated with hydrazine, NH2-NH2, at hightemperature (90oC) for an extended length of time (20-100hr).
This treatment cleaves all of the peptide bonds yieldingamino-acyl hydrazides of all the amino acids excluding theC-terminal residue
which can be identified chromatographically compared toamino acid standards.
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Chemical digestion of proteins-
Hydrazine Due to the high percentage of hydrazine induced side
reactions:
only used on carboxypeptidase resistant peptides
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Centrifugation of proteins
Proteins will sediment through a solution in a centrifugal fielddependent upon their mass.
Analytical centrifugation measure the rate that proteinssediment. The most common solution utilized is a linear gradient of
sucrose (generally from 5-20%).
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Centrifugation of proteins:
Proteins are layered atop the gradient in an ultracentrifuge tubethen subjected to centrifugal fields in excess of100,000 x g.
The sizes of unknown proteins can then be determined bycomparing their migration distance:
in the gradient with those of known standard proteins.
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Electrophoresis of proteins
Proteins also can be characterized
according to
size and charge by separation in an electric current(electrophoresis) within solid sieving gels madefrom polymerized and cross-linked acrylamide.
The most commonly used technique is termed SDSpolyacrylamide gel electrophoresis (SDS-PAGE).
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Use for separation and molecular weight determinationof all types of proteins
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Electrophoresis of Proteins
The gel is a thin slab of acrylamide polymerizedbetween two glass plates.
This technique utilizes a negatively chargeddetergent (sodium dodecyl sulfate) to denature andsolubilize proteins.
SDS denatured proteins have a uniform negativecharge such that all proteins will migrate through the
gel in the electric field based solely upon size.
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Electrophoresis of proteins:
The larger the protein the more slowly it will movethrough the matrix of the polyacrylamide.
Following electrophoresis the migration distance ofunknown proteins relative to known standard
proteins
is assessed by various staining or radiographicdetection techniques.
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Electrophoresis of proteins
The use of polyacrylamide gel electrophoresis also can be used todetermine the isoelectric charge of proteins (pI).
This technique is termed isoelectric focusing. Isoelectric focusing utilizes a thin tube of polyacrylamide made in the
presence of a mixture of small positively and negatively chargedmolecules termed ampholytes.
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Electrophoresis of proteins:
The ampholytes have a range of pIs thatestablish a pH gradient along the gel whencurrent is applied.
Proteins will, therefore, cease migration inthe gel when they reach the point
where the ampholytes have established a pHequal to the proteins pI.
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Size exclusion chromatography
This chromatographic technique is based upon the use of aporous gel in the form of insoluble beads placed into a column.
As a solution of proteins is passed through the column, smallproteins can penetrate into the pores of the beads and,
therefore, are retarded in their rate of travel through thecolumn.
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Size exclusion chromatography
The larger proteins a protein is the less likely it will enter thepores.
Different beads with different pore sizes can be used
depending upon the desired protein size separation profile.
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Affinity chromatography
Proteins have high affinities for their substrates or co-factors orprosthetic groups or receptors or antibodies raised againstthem.
This affinity can be exploited in the purification of proteins.
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Affinity chromatography
A column of beads bearing the high affinity compound can beprepared and a solution of protein passed through the column.
The bound proteins are then eluted by passing a solution of
unbound soluble high affinity compound through the column.
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LITERATURE CITED
Devlin,T.M. Textbook of Biochemistry with ClinicalCorrelations,Fifth Edition,Wiley-Liss Publications,NewYork, USA, 2002.
Lehninger, A. Principles of Biochemistry, Second edition,Worth Publishers Co., New York, USA, 1993.
Matthews, C.K. and van Holde, K.E., Biochemistry,Second edition, Benjamin / Cummings Publishing
Company Inc., San Francisco, 1996. Segel, I.H., Biochemical Calculations,Wiley
Publications, New York, USA, 1976.