新编-02proteinchemistry-精品课件

1、蛋白质化学 Protein Chemistry Content Introduction of proteinAmino 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 variet

2、y, ranging in size from 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 specific type of covalent bond (Peptide bond，肽键). All proteins are constructed from th

3、e same ubiquitous set 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 re

4、sidues. Conjugated protein Contain non-amino acid part. (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, metallopr

5、oteins 3. Classification of proteins(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 al

6、kaline Histone less alkaline than protamine Scleroproteininsoluble 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）In

7、dividual levelHair and skin (keratins）Bone and teeth (collagen）Digestive system Digesting enzymes Blood Antibody（2）Organ level（3）Cell levelShape of cell Supporting body Structural protein Collagen Functional protein II Amino Acids1. Hydrolysis of proteins Proteins can be hydrolyzed by acid, alkali a

8、nd 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 p

9、roteins 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 si

10、de 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- formsAlign carbon atoms with L-glyceraldehyde, the amino group is on the

11、left. The horizontal bonds project out of the plane of the paper, the vertical behind.3. Classification of amino acidsNonpolar, aliphatic (hydrophobic) amino acids Aromatic amino acidsPolar, uncharged amino acidsNegatively and positively chargedaccording to the properties of their R groupsGly, G Ala

12、, A Val, V Leu, L Met, M Ile, IAliphatic amino acidsPhe, F; Tyr, Y; Trp, WAromatic amino acids They are jointly responsible for the light absorption of proteins at 280 nmSer, S Thr, T Cys, C Pro, P Asn, N Gln, QPolar, uncharged amino acidsAsp ,GluNegatively and positively chargedLys, K; Arg, R; His,

13、 H4. Acids and Bases properties of Amino AcidsWhen 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

14、 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)/25. Chemical Reactions of Amino Acids Amino groups can be acetylated or formylated Carboxyl groups can be esterified (1) Peptide forma

15、tion (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 reactionIII Protein S

16、tructureFour Levels of Architecture in Proteins 1. Primary structurePrimary 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

17、is unspecified. 2. Secondary structuresSecondary 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) -HelixFour models of -helix (a) right-handed -helix. (b) The repeat unit is a single turn of

18、the helix, 3.6 residues. (c) -helix as viewed from one end. (d) A space-filling model of -helix. Factors Affected - helix stabilityA. steric repulsion is minimized and hydrogen bonding is maximized so the helix is stable.B. Amino Acid Sequence Affects Helix StabilityThe twist of an -helix ensures th

19、at critical interactions occur between an amino acid side chain. (2) -pleated sheet conformation is the more extended conformation of the polypeptide chains.Connect the ends of two adjacent segments of an antiparallel pleated sheet. (3) - turn(4) Random coilA representation of the 3D structure of th

20、e myoglobin protein. Alpha helices are shown in colour, and random coil in white, there are no beta sheets shown. helix sheet turnRandom coil Protein super-secondary structure 3. Tertiary structure Tertiary structure refers to the spatial relationship among all amino acids in a polypeptide; it is th

21、e 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 Immunoglobulins4. Quaternary StructureThe arrangement of proteins and protein subunits (亚单位) in three-di

22、mensional 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 S

23、tructure 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 2. Electrostatic interaction 3. Hydrogen bond4. Hydrophobic interaction Primary structure determines secondary, tertiary and quate

24、rnary structures Primary structureS-S6. Relationship between all grades structureConformational Changes in Hemoglobin Alter Its Oxygen-Binding Capacity 7. Relationship between structure and function of proteins IV Protein PropertiesIsoelectric point of protein Colloidal properties Protein denaturati

25、on Protein precipitation Protein sedimentation Protein hydrolysis Color reaction UV light absorption1. Isoelectric point of protein Acidic groups of Amino acids -COOH group of Glu -COOH group of Asp Phenolic hydroxy group of Tyr -SH group of Cys Basic groups of Amino acids -NH2 group of Lys Imidazol

26、yl group of His -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 zwitterionsThe Isoionic point is the pH value at which a zwitterion molecule has an equal number of p

27、ositive 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 propertiesSolution （ 100 nm) Protein Molecular weight of 10,000-1

28、000,000 Particle size of 220 nm Protein solution has colloidal properties.Factors affecting the stability of protein colloidal solutionPolar surfaces pH pI Same net charges on protein surface Repulsion among protein molecules Hydration water layer Charged amino acid residues Water binding capacity o

29、f proteinPolar 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AcidAlkaline（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

30、 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 StateDenaturation Urea （尿素）、 -mercaptoethanol （巯基乙醇）Re

31、naturation(复性） Remove Urea、-ME Unfolded State（3）Denaturing agentsPhysical 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 ShearChemical agents pH and denatur

32、ation 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 a

33、nd denaturation Chaotropic Salts and Denaturation（4）Changes in physical and chemical properties during protein denaturationFor 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）

34、Application of protein denaturationIn favor of denaturation Sterilization with alcohol High pressure pasteurization Prevention of denaturation Storage at low temperature Replacement 4. Allosteric effectHemoglobulin 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