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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.