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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.