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蛋白质化学
Protein Chemistry
Content

Introduction of protein

Amino acids

Protein Structure

Protein Properties

Protein Isolation and Purification
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.
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，肽键).
All proteins are constructed from the same ubiquitous
set of 20 amino acids.
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.
3. Classification of proteins
(1) Based on shape

Globular protein—able 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
(3) Based on solubility

Albumin∶ soluble in water

Globulin∶ salted out with ammonium sulfate

Glutelin∶insoluble in water, dissolve in in
acidified or alkaline solution

Gliadin ∶insoluble in water, dissolve in ethanol

Protamine∶approximately 80% arginine and
strongly alkaline

Histone ∶less alkaline than protamine

Scleroprotein∶insoluble proteins of animal organs
(4) Based on function

Active protein (Enzyme and antibody)

Passive protein (Collagen and keratin)
4. Biological function of proteins
 Morphological function
 Physiology function
 Nutritional function
（1）Individual level
 Animal
Hair and skin (keratins）
Bone and teeth (collagen）
（2）Organ level

Digestive system
 Digesting enzymes


Blood
 Antibody
（3）Cell level

Shape of cell


Structural protein


Supporting body
Collagen
Functional protein
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.
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.
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, R=“Remainder of the
molecule”).
The -carbons for 19 of them are asymmetric (or chiral), thus
being able to have two enantiomers. Glycine has no chirality.
The two enantiomers of amino acid :
D- forms and L- forms
Align carbon atoms with L-glyceraldehyde, the amino
group is on the left.
The horizontal bonds project out of the plane of
the paper, the vertical behind.
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
Aliphatic amino acids
Gly, G
Ala, A
Val, V
Leu, L
Met, M
Ile, I
Aromatic amino acids
Phe, F; Tyr, Y; Trp, W


They are jointly responsible for the light absorption of
proteins at 280 nm
Polar, uncharged amino acids
Ser, S
Thr, T
Cys, C
Pro, P
Asn, N
Gln, Q

Negatively and positively charged

Asp ,Glu
Lys, K; Arg, R; His, H
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):
Isoelectric point of Amino Acids
pI (等电点） is the pH of an aqueous
solution of an amino acid at which the molecules
on average have no net charge.
An acidic amino acid pI=(pK1+pKR)/2
A basic amino acid pI=(pKR+pK2)/2
5. Chemical Reactions of Amino Acids
 Amino

groups can be acetylated or formylated
Carboxyl groups can be esterified
(1) Peptide formation
(2) Carboxylic Acid Esterification

Esterification of the carboxylic acid is usually
conducted under acidic conditions
(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.
(4) Ninhydrin reaction
III Protein Structure
Four Levels of Architecture in Proteins
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.
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
(1) -Helix
Four models of -helix
(a) right-handed α-helix.
(b) The repeat unit is a single turn of the helix, 3.6 residues.
(c) α-helix as viewed from one end.
(d) A space-filling model of α-helix.
Factors Affected α- helix stability

A. steric repulsion is minimized and hydrogen
bonding is maximized so the helix is stable.
B. Amino Acid Sequence Affects α Helix Stability

The twist of an α-helix
ensures that critical
interactions occur between
an amino acid side chain.
(2) β-pleated sheet

β conformation is the more extended conformation of
the polypeptide chains.
(3) β- turn

Connect the ends of
two adjacent segments
of an antiparallel β
pleated sheet.
(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.
βturn
βsheet
αhelix
Random coil
Protein super-secondary structure
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.


Globular proteins can
incorporate several
types of secondary
structure in the same
molecule.




Enzymes
Transport proteins
Peptide hormones
Immunoglobulins
4. Quaternary Structure


The arrangement of proteins and protein subunits (亚单位)
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.
X-Ray Analysis Revealed the Complete Structure of
Hemoglobin （血红蛋白）
5. Factors Affecting Protein Structure
1.
2.
3.
4.
5.
Hydrogen bond (氢键)
Electrostatic interaction (离子键)
Hydrophobic interaction (疏水相互作用)
van der waals force (范德华力)
Disulfide bond (二硫键)
A.三级结构中的作用力
1. Disulfide bond
3. Hydrogen bond
2. Electrostatic interaction
4. Hydrophobic interaction
6. Relationship between all grades structure
Primary structure determines secondary, tertiary and
quaternary structures
S-S
Primary
structure
7. Relationship between structure and function
of proteins

Conformational Changes in Hemoglobin Alter Its
Oxygen-Binding Capacity
IV Protein Properties

Isoelectric point of protein

Colloidal properties

Protein denaturation

Protein precipitation

Protein sedimentation

Protein hydrolysis

Color reaction

UV light absorption
1. Isoelectric point of protein
 Acidic groups of Amino acids∶
γ-COOH
group of Glu
β-COOH group of Asp
 Phenolic hydroxy group of Tyr
 -SH group of Cys

 Basic groups of Amino acids ∶
ε-NH2
group of Lys
 Imidazolyl group of His
δ-guanidino group of Arg
Proteins exist as zwitterions
NH3+
Pr
COOH
ÑôÀë×Ó
pH<pI
OH
H
NH 3+
-
+
Pr
OH
-
COO
¼æÐÔÀë×Ó
pH=pI
H
NH2
-
+
Pr
COO
ÒõÀë×Ó
pH>pI
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. 。
-
pI and isoionic point (等离子点)

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.
2. Colloidal properties




Solution
Colloid
Suspension
Protein



（< 1 nm）
（1 – 100 nm）
(> 100 nm)
Molecular weight of 10,000-1000,000
Particle size of 2~20 nm
Protein solution has colloidal properties.
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
Polar surfaces and water hydration layer of
proteins
+
+
－
+
Acid
+
Alkaline
+
+
－
+
带正电荷的蛋白质
－
在等电点的蛋白质
－
－
－－
－
带负电荷的蛋白质
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”.
（2）Reversibility of protein denaturation
（可逆性）
Reversible
The proteins can regain their native state when
the denaturing influence is removed.
Irreversible
Renaturation
Native State
Renaturation(复性）
Remove Urea、β-ME
Denaturation
Urea （尿素）、
β-mercaptoethanol （巯基乙醇）
Unfolded State
（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

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
（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.
（5） Application of protein denaturation

In favor of denaturation
Sterilization with alcohol
 High pressure pasteurization


Prevention of denaturation


Storage at low temperature
Replacement
4. Allosteric effect

Hemoglobulin

Once the first hemepolypeptide subunit binds
an O2 molecule, the
remaining subunits
respond by greatly
increasing their oxygen
affinity. This involves a
change in the
conformation of
hemoglobin.
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.
Salting-in (盐溶）
盐溶
蛋白质分子在等电点时，容易互相吸引，聚合沉淀；加入
盐离子会破坏这些静电相互作用，使分子散开而溶于水
盐析
Salting out（盐析）
（NH4）2SO4
蛋白质分子表面的疏水区域，聚集了许多水分子，盐浓度
高时，这些水分子被盐抽出（水化层被破坏），暴露出的
疏水区域，它们发生相互作用而沉淀。
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.
60000~80000转/分
重力60万～80万倍
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.
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.
9. UV absorption of protein
Trp, Tyr and Phe
are responsible
for the light
absorption of
proteins at 280
nm.