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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 mac
romolecules, occurring in all cells and all parts
of cells.
Proteins occur in great variety, ranging in size f
rom relatively small peptides to huge polymers
with molecular weights in the millions.
Proteins are dehydration polymers of amino acids, with
each amino acid residue joined to its neighbor by a s
pecific type of covalent bond (Peptide bond ,肽键 ).
All proteins are constructed from the same ubiquitous s
et of 20 amino acids.
1. Proteins and Amino acids
(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 of proteins
(2) Chemical composition
Simple protein — Contain only amino acid residues.
Conjugated protein – Contain non-amino acid part.
(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. Classification of proteins
(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
Animal
( 1 ) Individual level
Hair and skin (keratins )
Bone and teeth (collagen )
Digestive system Digesting enzymes
Blood
Antibody
( 2 ) Organ level
( 3 ) Cell level
Shape of cell
Supporting body
Structural protein
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 i
mino group in proline) bonded to the same carbon atom, designa
ted 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 bei
ng 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
Nonpolar, aliphatic (hydrophobic) amino acids Aromatic amino acids Polar, uncharged amino acids Negatively and positively charged
according to the properties of their R groups
Gly, G Ala, A Val, V Leu, L Met, M Ile, I
Aliphatic amino acids
Phe, F; Tyr, Y; Trp, W
Aromatic amino acids
They are jointly responsible for the light absorption of
proteins at 280 nm
Ser, S Thr, T Cys, C Pro, P Asn, N Gln, Q
Polar, uncharged amino acids
Asp ,Glu
Negatively and positively charged
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 ami
no 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 StructureFour 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 a
rrangements in space of adjacent amino acid resid
ues 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 ens
ures that critical interactio
ns occur between an amino
acid side chain.
(2) β-pleated sheet
β conformation is the more extended conformation of the polypeptide chains.
Connect the ends of t
wo adjacent segments
of an antiparallel β ple
ated sheet.
(3) β- turn
(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.
αhelix βsheet βturn
Random coil
Protein super-secondary structure
3. Tertiary structure
Tertiary structure refers to the spatial relation
ship among all amino acids in a polypeptide; it is the
complete three-dimensional structure of the polypep
tide.
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. Hydrogen bond ( 氢键 )
2. Electrostatic interaction ( 离子键 )
3. Hydrophobic interaction ( 疏水相互作用 )
4. van der waals force ( 范德华力 )
5. Disulfide bond ( 二硫键 )
A. 三级结构中的作用力
1. Disulfide bond 2. Electrostatic interaction
3. Hydrogen bond 4. Hydrophobic interaction
Primary structure determines secondary, tertiary and quaternary structures
Primary structure
S-S
6. Relationship between all grades structure
Conformational Changes in Hemoglobin Alter Its Oxygen-Binding Capacity
7. Relationship between structure and function of proteins
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
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. 。
Proteins exist as zwitterions
PrNH3
+
COOHPr
NH3+
COO-Pr
NH2
COO-
OH-
H +
OH-
H +
¼æÐÔÀë×ÓpH=pI
ÑôÀë×ÓpH<pI
ÒõÀë×ÓpH>pI
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.
pI and isoionic point ( 等离子点 )
2. Colloidal properties
Solution ( < 1 nm ) Colloid ( 1 – 100 nm ) Suspension (> 100 nm)
Protein 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
+
+
+
+
+
++
带正电荷的蛋白质
- --
--
--
-
带负电荷的蛋白质在等电点的蛋白质
Acid Alkaline
( 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”.
3. Protein 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
Denaturation Urea (尿素)、 β-mercaptoethanol (巯基乙醇)
Renaturation( 复性) Remove Urea 、 β-ME
Unfolded State
( 3 ) Denaturing agents
Physical agents Heat
The temperature at the tran
sition midpoint, where the c
oncentration ratio of native
and denatured states is 1, i
s known either as the meltin
g 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 electrost
atic 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 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.
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)
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。
( NH4 ) 2SO4
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 p
eptides and amino acids. Occurs in the digestion of proteins. Occurs in cells when amino acids are nee
ded 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 wh
en it comes in contact with protein or a substanc
e with peptide bonds.
9. UV absorption of protein
Trp, Tyr and Phe
are responsible fo
r the light absorpt
ion of proteins at
280 nm.