1、氨基酸、多肽與蛋白質(Amino Acids, Peptides & Proteins)趙 穎2012. 9.18R: W334 Tel: 63165211E-mail: Contents Introduction Composition of ProteinsAmino Acids Classification of Proteins Physio-chemical Properties of Proteins Molecular Structures of Proteins Structure & Function Relationship of Proteins Isol

2、ation, purification and identification of Proteins & Peptides Determination of Primary Structures of Proteins Determination of Stereo Structures of Proteins Chemical synthesis and Biosynthesis of Peptides & ProteinsIntroduction ProteiosProtein 首要，第一 Biomacromolecules 生物大分子 Proteins and pepti

3、des 蛋白質，多肽 Nuleosides & Nucleotides 核酸，核苷酸 Polysaccharides 多糖 Lipids 脂質 Why Protein? Importance of Proteins Proteinoriginated from Greek “Proteios” Proteins are crucial to all aspects of life process, including enzymes which catalyze biochemical reactions for energy transportation and storage, c

4、ell motion and division, etc. Pharmaceutical uses: Insulin, Hirudin, Melittin, Bombesin, Dolastatins, etc. Cyclic peptides and PeptidomimeticsProtein as Medicines Insulin 胰島素 Urokinase 尿激酶 Problem: biodegradable, unstable Cyclamide 環肽, peptidomimetics 擬肽 Proteins as drug target SARS virus Microtubul

5、e 微管 作業：簡述1-2種你所了解的藥物蛋白質靶點及作用機制Peptides, Proteins: Structures & FunctionsStructures Structures1. Primary structures2. Advanced structures Methods1. Extraction & purification2. Structure determinationFunctions FunctionsStructural proteinsEnzymes, etc. MethodsSpectroscopyStructures of Amino Ac

6、ids Amino acids are the building blocks of naturally occurring proteins. It is widely recognized that only 20 L-a-amino acids constitute all proteins. 氨基酸：蛋白質的基本組成單位 20種基本氨基酸：由密碼子編碼CRHH3N+COO-Side ChainAlpha carbonClassification of Amino Acids Based on polarity -Neutral (NonpolarPolar, uncharged)中性-

7、Acidic 酸性-Basic 堿性 Based on chemical structures of side chain-alkyl 脂肪族-Aromatic 芳香族-Heteroaromatic 雜環族Structures of Amino AcidsHCH3SHSOHOHOHNGlyAlaValLeuIlePheCysMetSerThrTyrTrpH2NCO2HR(L-AA)Structures of Amino AcidsNH2NHHNNH2NNHNCOOHHCOOHCOOHCONH2CONH2AspAsnGluGlnLysArgHisProH2NCO2HR(L-AA)New Natu

8、ral L-a-Amino AcidsHOONH2HNOXH2NCO2HSeHN1-pyrrolysine(coded by UAG,found in 2002)selenocystine(coded by UGA,found in 1986)(X = Me or NH2) 硒代半胱氨酸 1-吡咯基賴氨酸Uncommon Amino AcidsModified AA in the Peptide Chains During Post-translationNOCOOHPyroglutamic acid(found in a light-drivenproton-pumping protein)

9、H2NCOOHOIIIOHI(found in thyroglobulin from thryoid)COOHH2NCOOH(found in proteins from corn)NH2OHNOHCOOHH2NCOOH5-hydroxylysine4-hydroxyproline(found in connective-tissue proteins)AA not found in proteinsH2NCOOHNNHNH2NHNH2HOH2NCOOHGABAHistamineSerotonin-alanine-氨基丁酸組織胺5-羥色胺-丙氨酸Physiochemical Propertie

10、s of AAHCCOOHNH3+RHCCOO-NH3+RHCCOO-NH2Rcationic formzwetterion formanionic form(1)Ionization:兼性離子pI 等電點：氨基酸呈電中性的pHPhysiochemical Properties of AA(2) Ionization of side chain:-Acidic: Glu, Asp(COOH), Tyr(phenol), Cys (thiol)for Asp: pKa(-COOH) pKa(-COOH)-Basic: For Lys: pKa(-NH2) pKa(-NH2)H2NHOOCCOOH

11、(a)()H2NHOOC(a)()NH2Chemical reactions of AAMost determination methods are based on amino group reactions(1)Ninhydrin reactionOOOHOHHCCOOHNH2R+H+OOHOHRCHO+CO2NH32ndOOOHOH(hydrindantin)(ninhydrin)OOOONH(Ruhemanns purple)NOOOO茚三酮反應Chemical reactions of AASO2ClNMe2HCCOOHNH2RbaseSO2NHNMe2CCOOHHRNO2FO2NH

12、CCOOHNH2RNO2O2NCCOOHHR(2)Acylation by dansyl group+(3)Alkylation with Sangers reagent (2,4-dinitrofluorobenzene, DNFB)+baseNH丹磺酰氯: DNS-Cl二硝基氟苯二甲氨萘磺酰-aaChemical reactions of AAHCCONHRNH2RNCSPhHCCONHRHNRHNCPhSNNHSPhOR(4)Reaction with phenylisothiocynate (PITC)+(Edmen degradation)苯異硫氰酸酯苯氨基硫甲酰-aa苯乙內酰硫脲C

13、hemical reactions of AAOOH2NCHRCOOHCOOHNOCHRHOOC+(5) Reaction with Fluorescamine熒光胺Separation and Purification Methods Extracted with water or diluted alcohol Ion exchanged chromatography 離子交換層析strongly acidicsulphopropyl (SP) Sepharoseweakly acidiccarboxymethyl (CM) cellulosestrongly basicquaternar

14、y ammonium (Q) Sepharoseweakly basicdimethylaminoethyl (DEAE) cellulose -ninhydrin, OPA (o-phthaldehyde 鄰苯二甲醛), fluorescamine derivatives GC and HPLCGC: PTH-aa TMS 三甲基硅 derivativesHPLC: DNFB, OPA, DNS, PITC, fluorescamine derivativesSeparation and Purification StrategyMixtures of Amino AcidsSolution

15、AdsorbentSi gel column orWeakly Acidic Ion ExchangeAdsorbentbasic AAor mono-saccharideSolutionAdsorbentSolutionAcidic AAor oligo-saccharideAromatic typeAA and peptidecontaining themneutral aliphaticAA and peptidecontaining themWashed with Acids, Al2O3 Columnor Weakly Basic Ion ExchangeActivated Carb

16、onPreparation of AA Extracted from biological materials Fermentation Enzymatic synthesis Chemical synthesisStreckers synthesis, Bucherers synthesisHOOCCOOHHOOCCOOHNH3AspartaseNH2HOOCNH2( )L-alanineAsparaginedecarboxylasePreparation of AA Streckers synthesisBucherers synthesisRCHONH3HCNRCHCNNH2aq. HC

17、l100oCRCHCCO2HNH2RCNH2+HCN-RCHO(NH4)2CO3KCNaq. NaOH200oCRCHCCO2HNH2HNNHROO+NH4Cl200oC合成外消旋產物Preparation of AA Resolution-Formation of salts with chiral acids or bases-Formation of diastereoisomeric amide-Formation of diastereoisomeric shiffs base-Separation on chiral HPLC-Enzymatic resolution Asymme

18、tric synthesis-Coreys approach mimicking VB6 catalysed transamination-Transamination catalysed by aminotransferaseAsymmetric Chemical SynthesisCoreys method基于氨基酸生物合成途徑中-酮酸轉氨基作用，設計了模擬VB6轉氨基作用的手性試劑Enzyme Mediated Asymmetric SynthesisCO2HORCORHNH2HOCORHNH2HOCO2HORAminotransferase Enzymatic synthesis of

19、 unusual amino acids from naturally abundant ones.Biosynthesis of amino acids Essential amino acids 必需氨基酸: -Cannot be self-synthesized -In human: Phe, Lys, Leu, Ile, Met, Thr, Trp, Val, His and Arg Nonessential amino acids 非必需氨基酸:-Can be self-synthesized -Plants can synthesize all kinds of amino aci

20、ds Biosynthesis of amino acids originates from key intermediate products of metabolic pathwaysBiosynthesis of amino acidsDegradation of Amino Acid to a-Keto acidVB6Classification of Proteins Shape of the molecules Composition Solubility Function GlycoproteinGlycans is covalently bound to proteins or

21、 peptides.Glycoproteins Versatile functionsenzymes, hormones, interferons, lectins, memberane blocks, blood type recognition Structure characteristicsopen or branched chains of sugar moieties composed of several type of sugars, including pentose (D-xylose), hexose (D-galactose), hexose derivatives (

22、N-acetyl-D-glucosamine), sialic acid, etc. C-1 of sugar covalently bound to OH in amino acid side chains, or amide of asparagine. Solubilitymost of them are water soluble.Physio-chemical Properties of Peptides and Proteins Zwitterionization and isoelectric pointleast soluble at isoelectric point 兼性離

23、子性質 Macromolecular propertiesdiffusion 擴散(centrifugation 應用：離心分離), viscosity 粘度, impermeability 不透過性 Denaturation 變性destroy of non-covalent bonds in proteins Precipitation 沉淀salts, heavy metals, alkaloids, organic solvents, heat Colorful reactions 呈色反應biuret, ninhydrin, dansyl chloride UV adsorption

24、 紫外吸收280nm characteristic bandsChemical Interactions in PeptidesThe primary structure of proteinsNH3+NHNHNHR1OR2OOCOO-R3 The primary structure includes: number of peptide bonds; The N and C terminal of peptide; number, type and sequence of amino acid in each peptide chain 氨基酸如何連接成肽鏈及在肽鏈中的排列順序The sec

25、ondary structure of proteins: a-helixThe secondary structure of proteins: -pleated sheetThe secondary structure of proteins Others: -turn (rich in prolines) hairpin random coils 借助主鏈的氫鍵形成具有周期性的構象Super-secondary structures, domains 結構域and motifs 模體: the structures between secondary and tertiary structuresThe tertiary structure of proteinsThe tertiary structure of proteins A peptide consists of different portions of a-helix, -sheet, -turn and random coil, which folds to a global conformation Non-polar side chains are buried in the hydrophobic core, while polar sid