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1、蛋白质化学 Protein Chemistry Content qIntroduction of proteinqAmino acids qProtein Structure qProtein Properties qProtein 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 v
2、ariety, 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，肽键). All proteins are constructed
3、 from the 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
4、 acid residues. 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, m
5、etalloproteins 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 s
6、trongly 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 A
7、nimal （1）Individual levelHair and skin (keratins）Bone and teeth (collagen）nDigestive system qDigesting enzymes n nBlood qAntibody（2）Organ level（3）Cell levelnShape of cell q Supporting body n Structural protein qCollagenn Functional protein II Amino Acids1. Hydrolysis of proteins Proteins can be hydr
8、olyzed 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 structura
9、l 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.
10、 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- formsAlign carbon atoms with L-glyceraldehyde, th
11、e amino group is on the left. The horizontal bonds project out of the plane of the paper, the vertical behind.3. Classification of amino acidsnNonpolar, aliphatic (hydrophobic) amino acids nAromatic amino acidsnPolar, uncharged amino acidsnNegatively and positively chargedaccording to the properties
12、 of their R groupsGly, G Ala, A Val, V Leu, L Met, M Ile, IAliphatic amino acidsPhe, F; Tyr, Y; Trp, WAromatic amino acidsn nThey 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 acidsqAsp ,GlunNegatively and positi
13、vely chargedLys, K; Arg, R; His, 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 poin
14、t of Amino Acids pI (等电点）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)/25. Chemical Reactions of Amino Acids n Amino groups can be acetylated or formylated n Carboxyl gr
15、oups can be esterified (1) Peptide formation (2) Carboxylic Acid Esterification nEsterification of the carboxylic acid is usually conducted under acidic conditions (3) Amine Acylation nThe pH of the solution must be raised to 10 or higher so that free amine nucleophiles are present in the reaction s
16、ystem. (4) Ninhydrin reactionIII Protein StructureFour Levels of Architecture in Proteins 1. Primary structurenPrimary structure is normally defined by the sequence of peptide-bonded amino acids and locations of disulfide bonds. nincluding all the covalent bonds between amino acids .n The relative s
17、patial arrangement of the linked amino acids is unspecified. 2. Secondary structuresnSecondary structure refers to regular, recurring arrangements in space of adjacent amino acid residues in a polypeptide chain. nThe Peptide Bond Is Rigid and Planar (1) -HelixFour models of -helix (a) right-handed -
18、helix. (b) The repeat unit is a single turn of the helix, 3.6 residues. (c) -helix as viewed from one end. (d) A space-filling model of -helix. Factors Affected - helix stabilitynA. steric repulsion is minimized and hydrogen bonding is maximized so the helix is stable.B. Amino Acid Sequence Affects
19、Helix StabilitynThe twist of an -helix ensures that critical interactions occur between an amino acid side chain. (2) -pleated sheetn conformation is the more extended conformation of the polypeptide chains.qConnect the ends of two adjacent segments of an antiparallel pleated sheet. (3) - turn(4) Ra
20、ndom coilnA 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. helix sheet turnRandom coil Protein super-secondary structure 3. Tertiary structuren Tertiary structure refers to the spatial relation
21、ship among all amino acids in a polypeptide; it is the complete three-dimensional structure of the polypeptide. nGlobular proteins can incorporate several types of secondary structure in the same molecule. qEnzymes qTransport proteins qPeptide hormones qImmunoglobulins4. Quaternary StructurenThe arr
22、angement of proteins and protein subunits (亚单位亚单位) in three-dimensional complexes constitutes quaternary structure. nThe interactions between subunits are stabilized and guided by the same forces that stabilize tertiary structure: multiple noncovalent interactions. X-Ray Analysis Revealed the Comple
23、te Structure of Hemoglobin （血红蛋白）（血红蛋白）5. Factors Affecting Protein Structure 1.Hydrogen bond (氢键氢键) 2.Electrostatic interaction (离子键离子键) 3.Hydrophobic interaction (疏水相互作用疏水相互作用) 4.van der waals force (范德华力范德华力) 5.Disulfide bond (二硫键二硫键)A.三级结构中的作用力三级结构中的作用力 1. Disulfide bond 2. Electrostatic interac
24、tion 3. Hydrogen bond4. Hydrophobic interaction Primary structure determines secondary, tertiary and quaternary structures Primary structureS-S6. Relationship between all grades structureConformational Changes in Hemoglobin Alter Its Oxygen-Binding Capacity 7. Relationship between structure and func
25、tion of proteins IV Protein PropertiesnIsoelectric point of protein nColloidal properties nProtein denaturation nProtein precipitation nProtein sedimentation nProtein hydrolysis nColor reaction nUV light absorption1. Isoelectric point of protein Acidic groups of Amino acids u-COOH group of Glu u-COO
26、H group of Asp u Phenolic hydroxy group of Tyr u -SH group of Cys u Basic groups of Amino acids u-NH2 group of Lys u Imidazolyl group of His u-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 ch
27、arge. 。Proteins exist as zwitterionsThe 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
28、deionized solution. pI and isoionic point (等离子点等离子点)2. Colloidal propertiesnSolution （ 100 nm) nProtein qMolecular weight of 10,000-1000,000 qParticle size of 220 nm qProtein solution has colloidal properties.Factors affecting the stability of protein colloidal solutionnPolar surfaces qpH pI qSame n
29、et charges on protein surface qRepulsion among protein molecules nHydration water layer qCharged amino acid residues qWater binding capacity of proteinPolar surfaces and water hydration layer of proteins+带正电荷的蛋白质带正电荷的蛋白质带负电荷的蛋白质带负电荷的蛋白质在等电点的蛋白质在等电点的蛋白质AcidAlkaline（1）Protein denaturation nSubtle chan
30、ges in structure are usually regarded as “conformational adaptability” nMajor 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）（可逆性） Reversible
31、 The proteins can regain their native state when the denaturing influence is removed. Irreversible Renaturation Native State Native StateDenaturationDenaturationUreaUrea（尿素）、（尿素）、 -mercaptoethanolmercaptoethanol（巯基乙基乙醇）醇）Renaturation(Renaturation(复性）复性） Remove UreaRemove Urea、-ME -ME Unfolded StateU
32、nfolded State（3）Denaturing agentsnPhysical agents qHeat nThe temperature at the transition midpoint, where the concentration ratio of native and denatured states is 1, is known either as the melting temperature Tm. n qHydrostatic pressure qShearnChemical agents qpH and denaturation nProteins are mor
33、e stable against denaturation at their isoelectric point than at any other pH. nAt extreme pH values, strong intramolecular electrostatic repulsion caused by high net charge results in swelling and unfolding of the protein molecule. qOrganic solvents and denaturation qDetergents and denaturation qCh
34、aotropic 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） Application of pro
35、tein denaturationnIn favor of denaturation qSterilization with alcohol qHigh pressure pasteurization nPrevention of denaturation qStorage at low temperature nReplacement 4. Allosteric effectnHemoglobulin qOnce the first heme-polypeptide subunit binds an O2 molecule, the remaining subunits respond by
36、 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
37、(Salting-in (盐溶）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 outSalting out（盐析）（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。疏水区域，它们发生相互作用而沉淀。（NHNH4 4）2 2SOS
38、O4 4 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.60000800006000080000转转/ /分分 重力重力6060万万8080万倍万倍 7.Protein hydrol
39、ysis nSplits the peptide bonds to give smaller peptides and amino acids. nOccurs in the digestion of proteins. nOccurs in cells when amino acids are needed to synthesize new proteins and repair tissues.8. Color reaction of proteinnColor reaction of amino acids nSpecial color reaction of proteins qBiuret protein assay qA chemical test for proteins qBiuret reagent is usually blue but turns violet when it comes in contact with protein or a substance with peptide bonds.9. UV absorption of proteinTrp, Tyr and Phe are responsible for the light absorption of proteins at 280 nm.
