Protein Chemistry

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Protein Chemistry
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• Content
• Introduction of protein
• Amino acids
• Protein Structure
• Protein Properties
• Protein Isolation and Purification

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• I Introduction of Protein
• Proteins are the most abundant biological
  macromolecules, occurring in all cells and all
  parts of cells.
• Proteins occur in great variety, ranging in size
  from relatively small peptides to huge polymers
  with molecular weights in the millions.

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1. Proteins and Amino acids
Proteins are dehydration polymers of amino acids,
with each amino acid residue joined to its
neighbor by a specific type of covalent bond
(Peptide bond,??). All proteins are constructed
from the same ubiquitous set of 20 amino acids.
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2. Chemical composition of proteins

• (1) Elements
• C?H?O?N?P?S
• The nitrogen content of proteins is 15-17,with
  an average of 16,
• ie.1g N 6.25g Pr. Crude Pr. N ? 6.25

(2) Chemical composition Simple protein
Contain only amino acid residues.
Conjugated protein Contain non-amino acid part.
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3. Classification of proteins

• (1) Based on shape
• Globular proteinable to dissolve and
  crystallize
• Fibrous protein--generally water-insoluble
• (2) Based on chemical composition
• Simple protein e.g.lysozyme
• Conjugated protein e.g.hemoglobin
• Glycoproteins, lipoproteins, metalloproteins

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• (3) Based on solubility
• Albumin soluble in water
• Globulin salted out with ammonium sulfate
• Glutelininsoluble in water, dissolve in in
  acidified or alkaline solution
• Gliadin insoluble in water, dissolve in ethanol
  
• Protamineapproximately 80 arginine and
  strongly alkaline
• Histone less alkaline than protamine
• Scleroproteininsoluble proteins of animal organs

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• (4) Based on function
• Active protein (Enzyme and antibody)
• Passive protein (Collagen and keratin)

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4. Biological function of proteins

• Morphological function
• Physiology function
• Nutritional function

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(1)Individual level

• Animal

Hair and skin (keratins)
Bone and teeth (collagen)
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(2)Organ level

• Digestive system
• Digesting enzymes
• Blood
• Antibody

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(3)Cell level

• Shape of cell
• Supporting body
• Structural protein
• Collagen
• Functional protein

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II Amino Acids
1. Hydrolysis of proteins Proteins can be
hydrolyzed by acid, alkali and proteases and
broken down to peptides and mixture of amino
acids. The resulting characteristic
proportion of different amino acids, namely, the
amino acid composition was used to distinguish
different proteins before the days of protein
sequencing.
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• 2. Amino acids structural features
• All natural proteins were found to be built from
  a repertoire of 20 standard ?-amino acids.
• The 20 ?-amino acids share common structural
  features.

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Each has a carboxyl group and an amino group (but
one has an imino group in proline) bonded to the
same carbon atom, designated as the a-carbon.
Each has a different side chain (or R group,
RRemainder of the molecule).
The ?-carbons for 19 of them are asymmetric (or
chiral), thus being able to have two enantiomers.
Glycine has no chirality.
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The two enantiomers of amino acid D-
forms and L- forms
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Align carbon atoms with L-glyceraldehyde, the
amino group is on the left.
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The horizontal bonds project out of the plane of
the paper, the vertical behind.
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3. Classification of amino acids
according to the properties of their R groups

• Nonpolar, aliphatic (hydrophobic) amino acids
• Aromatic amino acids
• Polar, uncharged amino acids
• Negatively and positively charged

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Aliphatic amino acids
Gly, G Ala, A Val, V Leu, L Met, M Ile, I
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Aromatic amino acids
Phe, F Tyr, Y Trp, W
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• They are jointly responsible for the light
  absorption of proteins at 280 nm

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Polar, uncharged amino acids
Ser, S Thr, T Cys, C Pro, P Asn, N Gln, Q
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• Negatively and positively charged

• Asp ,Glu

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Lys, K Arg, R His, H
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4. Acids and Bases properties of Amino Acids
When a crystalline amino acid, such as alanine,
is dissolved in water, it exists in solution as
the dipolar ion, or zwitterion, which can act
either as an acid (proton donor) or as a base
(proton acceptor)
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Isoelectric point of Amino Acids
pI (???) is the pH of an aqueous
solution of an amino acid at which the molecules
on average have no net charge.
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An acidic amino acid pI(pK1pKR)/2
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A basic amino acid pI(pKRpK2)/2
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5. Chemical Reactions of Amino Acids

• Amino groups can be acetylated or formylated
• Carboxyl groups can be esterified

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(1) Peptide formation
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(2) Carboxylic Acid Esterification

• Esterification of the carboxylic acid is usually
  conducted under acidic conditions

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(3) Amine Acylation

• The pH of the solution must be raised to 10 or
  higher so that free amine nucleophiles are
  present in the reaction system.

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(4) Ninhydrin reaction
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III Protein Structure
Four Levels of Architecture in Proteins
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1. Primary structure

• Primary structure is normally defined by the
  sequence of peptide-bonded amino acids and
  locations of disulfide bonds.
• including all the covalent bonds between amino
  acids .
• The relative spatial arrangement of the linked
  amino acids is unspecified.

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2. Secondary structures

• Secondary structure refers to regular, recurring
  arrangements in space of adjacent amino acid
  residues in a polypeptide chain.
• The Peptide Bond Is Rigid and Planar

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(1) ?-Helix
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Four models of ?-helix
(a) right-handed a-helix. (b) The repeat unit is
a single turn of the helix, 3.6 residues. (c)
a-helix as viewed from one end. (d) A
space-filling model of a-helix.
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Factors Affected a- helix stability

• A. steric repulsion is minimized and hydrogen
  bonding is maximized so the helix is stable.

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B. Amino Acid Sequence Affects a Helix Stability

• The twist of an a-helix ensures that critical
  interactions occur between an amino acid side
  chain.

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(2) ß-pleated sheet

• ß conformation is the more extended conformation
  of the polypeptide chains.

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(3) ß- turn

• Connect the ends of two adjacent segments of an
  antiparallel ß pleated sheet.

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(4) Random coil

• A representation of the 3D structure of the
  myoglobin protein. Alpha helices are shown in
  colour, and random coil in white, there are no
  beta sheets shown.

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ßsheet
ßturn
ahelix
Random coil
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Protein super-secondary structure
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3. Tertiary structure

• Tertiary structure refers to the spatial
  relationship among all amino acids in a
  polypeptide it is the complete three-dimensional
  structure of the polypeptide.

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• Globular proteins can incorporate several types
  of secondary structure in the same molecule.
• Enzymes
• Transport proteins
• Peptide hormones
• Immunoglobulins

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4. Quaternary Structure

• The arrangement of proteins and protein subunits
  (???) in three-dimensional complexes constitutes
  quaternary structure.
• The interactions between subunits are stabilized
  and guided by the same forces that stabilize
  tertiary structure multiple noncovalent
  interactions.

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X-Ray Analysis Revealed the Complete Structure of
Hemoglobin (????)
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5. Factors Affecting Protein Structure

1. Hydrogen bond (??)
2. Electrostatic interaction (???)
3. Hydrophobic interaction (??????)
4. van der waals force (????)
5. Disulfide bond (???)

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A.?????????
1. Disulfide bond 2. Electrostatic interaction
3. Hydrogen bond 4. Hydrophobic interaction
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6. Relationship between all grades structure
Primary structure determines secondary, tertiary
and quaternary structures
Primary structure
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7. Relationship between structure and function of
proteins

• Conformational Changes in Hemoglobin Alter Its
  Oxygen-Binding Capacity

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IV Protein Properties

• Isoelectric point of protein
• Colloidal properties
• Protein denaturation
• Protein precipitation
• Protein sedimentation
• Protein hydrolysis
• Color reaction
• UV light absorption

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1. Isoelectric point of protein

• Acidic groups of Amino acids
• ?-COOH group of Glu
• ß-COOH group of Asp
• Phenolic hydroxy group of Tyr
• -SH group of Cys
• Basic groups of Amino acids
• e-NH2 group of Lys
• Imidazolyl group of His
• d-guanidino group of Arg

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Proteins exist as zwitterions
Isoelectric point, pI, is the pH of an aqueous
solution of an amino acid (or protein) at which
the molecules on average have no net charge. ?
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pI and isoionic point (????)

• The Isoionic point is the pH value at which a
  zwitterion molecule has an equal number of
  positive and negative charges.
• pI is the pH value at which the net charge of the
  molecule, including bound ions is zero.
• Whereas the isoionic point is at net charge zero
  in a deionized solution.

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2. Colloidal properties

• Solution (lt 1 nm)
• Colloid (1 100 nm)
• Suspension (gt 100 nm)
• Protein
• Molecular weight of 10,000-1000,000
• Particle size of 220 nm
• Protein solution has colloidal properties.

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Factors affecting the stability of protein
colloidal solution

• Polar surfaces
• pH ?pI
• Same net charges on protein surface
• Repulsion among protein molecules
• Hydration water layer
• Charged amino acid residues
• Water binding capacity of protein

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Polar surfaces and water hydration layer of
proteins
Alkaline
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3. Protein denaturation

• (1)Protein denaturation
• Subtle changes in structure are usually regarded
  as conformational adaptability
• Major changes in the secondary, tertiary, and
  quaternary structures without cleavage of
  backbone peptide bonds are regarded as
  denaturation.

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• (2)Reversibility of protein denaturation
• (???)
• Reversible
• The proteins can regain their native state when
  the denaturing influence is removed.
• Irreversible
• Renaturation

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Native State
Renaturation(??) Remove Urea?ß-ME
Denaturation Urea (??)? ß-mercaptoethanol (????)
Unfolded State
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(3)Denaturing agents

• Physical agents
• Heat
• The temperature at the transition midpoint, where
  the concentration ratio of native and denatured
  states is 1, is known either as the melting
  temperature Tm.
• Hydrostatic pressure
• Shear

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• Chemical agents
• pH and denaturation
• Proteins are more stable against denaturation at
  their isoelectric point than at any other pH.
• At extreme pH values, strong intramolecular
  electrostatic repulsion caused by high net charge
  results in swelling and unfolding of the protein
  molecule.
• Organic solvents and denaturation
• Detergents and denaturation
• Chaotropic Salts and Denaturation

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(4)Changes in physical and chemical properties
during protein denaturation
For most proteins, as denaturant concentration is
increased, the value of y remains unchanged
initially, and above a critical point its value
changes abruptly from yN to yD.
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(5) Application of protein denaturation

• In favor of denaturation
• Sterilization with alcohol
• High pressure pasteurization
• Prevention of denaturation
• Storage at low temperature
• Replacement

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4. Allosteric effect

• Hemoglobulin
• Once the first heme-polypeptide subunit binds an
  O2 molecule, the remaining subunits respond by
  greatly increasing their oxygen affinity. This
  involves a change in the conformation of
  hemoglobin.

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5. Precipitation of proteins
Changes in environmental conditions of protein
colloidal solution might damage the hydration
layer and surface charges and result in
precipitation of proteins.
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Salting-in (??)
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Salting out(??)
(NH4)2SO4
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6.Protein sedimentation Sedimentation is the
tendency for molecules in solution to settle out
of the fluid. This is due to their motion in
response to the forces acting on them gravity,
centrifugal acceleration or electromagnetism.
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• 7.Protein hydrolysis
• Splits the peptide bonds to give smaller peptides
  and amino acids.
• Occurs in the digestion of proteins.
• Occurs in cells when amino acids are needed to
  synthesize new proteins and repair tissues.

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8. Color reaction of protein

• Color reaction of amino acids
• Special color reaction of proteins
• Biuret protein assay
• A chemical test for proteins
• Biuret reagent is usually blue but turns violet
  when it comes in contact with protein or a
  substance with peptide bonds.

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9. UV absorption of protein
Trp, Tyr and Phe are responsible for the light
absorption of proteins at 280 nm.