Chapter 4. Amino Acids, Peptides, and Proteins

1. Chapter 4. Amino Acids, Peptides, and Proteins • Introduction • Amino acids (氨基酸) • Peptides (肽) • Proteins (蛋白质)

2. I. Introduction • Proteins are the most abundant biological macromolecules in cells. • Great variety in size and structure • Diversity of biological functions • Building blocks for widely diverse protein products • Final products of the information pathways • Amino acids  Peptides  proteins • Built up by the same set of 20 amino acids • 1st to be discovered – Asparagine (天冬酰氨) – 1806 last to be discovered – Threonine (苏氨酸) – 1938 • Common names Asparagine (天冬氨酸) – asparagus Tyrosine (酪氨酸) – cheese (tyros) Glycine (甘氨酸) – sweet (glykos)

3. Some functions of proteins • The light produced by fireflies is the result of a reaction involving the protein luciferin and ATP, catalyzed by the enzyme luciferase. • Erythrocytes contain large amounts of the oxygen-transporting protein hemoglobin. • The protein keratin is the chief structural component of hair, scales, horn, wool, nails, and feathers.

4. II. Amino Acids • Classification (分类) • Standard amino acids in proteins • Optical activity (光学活性) • Spectrophotometric properties (光谱性质) • Acid-base properties (酸碱性质) • Chemical properties ( 化学性质) • Separation and analysis of amino acids (分离分析) -Amino acid

5. P.124-129 1.Classification • Standard amino acids – -L-Amino acids – commonly found in proteins • Nonstandard amino acids – less commonly found in proteins • Nonprotein amino acids – present in living organisms but not in proteins -Amino acid >300 amino acids have been found in cells

6. Some uncommon amino acids found in proteins

7. Amino acids involved in regulation of protein activity Not found in proteins

8. P.143 2. Standard amino acids in proteins(蛋白质里的标准氨基酸) 6 5 4 3 2 1 • Common structural features (结构通性) • -Amino acids • L-configuration • Varying in R • Two ways to identify the carbons within an amino acid • -C is always chiral except in glycine (除Gly外所有-C 均为手性) • Classification by R group      甘油醛 丙氨酸 -C

9. Classification of the standard amino acids by R groups a. Nonpolar, alphatic R groups (非极性、脂肪族基团) Gly, Ala, Val, Leu, Ile, Pro b. Polar, uncharged R groups (极性，不带电荷基团) Ser, Thr, Cys, Asn, Gln, Met c. Aromatic R groups (芳香族基团) Phe, Tyr, Trp d. Positively charged (basic) R groups (带正电/碱性基团) Lys, Arg, His e. Negatively charged (acidic) R groups (带负电/酸性基团) Asp, Glu II. Amino Acids Aliphatic vs. aromatic Polar vs. nonpolar Charged vs. uncharged Acidic vs. basic

10. a c 甘氨酸 丙氨酸 缬氨酸 苯丙氨酸 酪氨酸 色氨酸 d 亮氨酸 异亮氨酸 脯氨酸 b 丝氨酸 赖氨酸 精氨酸 组氨酸 苏氨酸 半胱氨酸 e 谷氨酰胺 天冬氨酸 蛋氨酸 谷氨酸 天冬酰胺

11. 丙氨酸 脯氨酸 缬氨酸 甘氨酸 亮氨酸 异亮氨酸 甲硫氨酸

12. 苏氨酸 丝氨酸 半胱氨酸 谷氨酰胺 天冬酰胺

13. O2 Mercaptoethanol (巯基乙醇) Cysteine (半胱氨酸) Cystine (胱氨酸)

14. 芳香族基团 吲哚 苯丙氨酸 酪氨酸 色氨酸 Strong absorbance at 280 nm

15. 带正电/碱性基团 咪唑 胍基 赖氨酸 组氨酸 精氨酸

16. 带负电/酸性基团 天冬氨酸 谷氨酸

17. P.143 3. Optical activity (光学活性) • All -amino acids (except glycine) have chiral centers, and hence are optically active (除甘氨酸外都有手性和旋光性). • Amino acid residues in proteins are L-isomers (L-构型). • Not all L-amino acids are levorotatory (并非都左旋). • Racemate (外消旋物): A pair of enantiomers (e.g. DL-alanine) • A racemic mixture shows no optical rotation (外消旋物无旋光性). • Meso-cystine (内消旋胱氨酸) 甘油醛 丙氨酸

18. 4. Spectrophotometric properties (光谱性质) Absorbance of ultraviolet light by aromatic amino acids: • A280 – widely used for: • Protein characterization (蛋白质的鉴定) • Rough estimation of protein concentration (蛋白浓度的粗略估计) [protein](mg/ml) = 1.45A280–0.74A260

19. P.130 5. Acid-base properties (酸碱性质) • Amino acids can act as acids and bases –Zwitterions (两性离子) • Amino acids have characteristic titration curves (有特定的滴定曲线) • Effect of chemical environments on pKa (化学环境对pKa的影响) • Amino acids differ in their acid-base properties (有不同的酸碱性质) II. Amino Acids

20. Zwitterions (两性离子) Amino acid crystals have high melting points (>200oC) (氨基酸晶体有高熔点) Amino acid as an acid: (氨基酸作为酸) Amino acid as a base: (氨基酸充当碱)

21. H2A+ HA A- Charge: +1 0 1

22. Titration curves (滴定曲线) • Two distinct stages: • Deprotonation of –COOH • Deprotonation of –NH3+ • Information that can be obtained: • pKa • Buffering range (缓冲范围) • pI (等电点) • Electric charge (电荷) pH < pI Positively charged (带正电) pH > pI Negativelycharged (带负电)

24. Isoelectric point (pI, 等电点) Ka1 Ka2 H2A+ HA A- [HA][H+] Ka1 = [H2A+] [A-][H+]2 Ka1 Ka2 = [H2A+] [A-][H+] II. Amino Acids Ka2 = H2A+] = [A-] [HA] Ka1 Ka2 = [H+]2 pI is the characteristic pH for an amino acid at which the net electric charge of the molecule is zero. When pH = pI, [H2A+] = [A-] pH = pI pI = ½(pKa1 + pKa2)

25. Effect of chemical environments on pKa (化学环境对pKa的影响)

26. Different amino acids with different acid-base properties(不同的氨基酸有不同的酸碱性质) • Amino acids with a nonionizable R group (R基团不电离的氨基) — with similar titration curves as that of Gly (滴定曲线与Gly相似) pKa1 = 1.8-2.4 (-COOH group) pKa2 = 8.8-11.0 (-NH3+ group) • Amino acids with an ionizable R group (R基团电离的氨基酸) — with more complex titration curves • Only His provides buffering power near neutral pH because of the R group (pKa = 6.0) (只有His在中性pH附近有缓冲作用)

27. Titration curves of amino acids with an ionizable R group(带电离基团的氨基酸的滴定曲线) Glutamic acid (谷氨酸) Histidine (组氨酸) pI = ½ (pKa1 + pKR) = 3.22 pI = ½ (pKR + pKa2) = 7.59

28. P.133

29. 6. Chemical properties P.135 • Reactions for the -NH2 group • Reactions for the -COOH group • Reactions for both -NH2 and -COOH groups • Reactions for the R group II. Amino Acids

30. Reactions for the -NH2 group P.135 • Reaction with HNO2 • Acylation (酰化) • Alkylation (烃基化): DNFB, PITC • Formation of Schiff’s base (西佛碱) • Deamination (脱氨基) II. Amino Acids

31. Reaction with HNO2  Used for quantitative determination of amino group (用于氨基的定量测定) Acylation (酰基化) Used for protection of the –NH2 group (用于保护氨基) II. Amino Acids Used for labeling of the N-terminal of peptide chain and quantitative determination of amino acids (用于肽链N-端的标记和氨基酸的定量测定) 丹磺酰氯 (DNS)

32. Alkylation (烃基化) Two examples of characterizing the N-terminal of a polypeptide/protein: Sanger’s reaction: DNFB DNP-amino acid,yellow Edman’s reaction: PITC PTH-amino acid,colorless PTC-amino acid Both reactions are useful in amino acid sequence analysis. (用于氨基酸序列分析的两个反应)

33. Formation of Schiff’s base (西佛碱的形成) II. Amino Acids Deamination (脱氨基反应)

34. Reactions for the -COOH group P.138 • Formation of salts and esters (成盐、成酯) • Formation of acyl chloride (酰氯) • Decarboxylation (脱羧) • Formation of azide (叠氮) II. Amino Acids

35. Formation of salts, esters, and amides (成盐成酯成酰胺) Salt (盐) Ester (酯) II. Amino Acids Amide (酰胺) -COOH group– protected (被保护) -NH2 group– activated (被激活)

36. Formation of acyl chloride (成酰氯反应) 保护基 -COOH is activated (被激活). Useful in peptide synthesis (用于肽的合成) Decarboxylation (脱羧基) (氨基酸脱羧酶) II. Amino Acids Formation of an azide (叠氮的生成) -COOH is activated (被激活) Useful in peptide synthesis

37. Reactions for both -NH2 and -COOH groups P.138 Reaction with ninhydrin (茚三酮) Formation of peptides (肽) 产物在 570 nm 处强吸收 (紫色) Pro例外: 440 nm (亮黄色) Used for peptide synthesis (用于肽链的合成) Used for characterization of amino acids (用于氨基酸的鉴定)

38. P.140 Reactions for the R group

39. Quantitative determination of –SH group by Ellman reaction with DTNB (Ellman反应用于巯基的定量分析) P.142 Cysteine (Cys) DTNB (Ellman试剂) DTNB = dithionitrobenzoic acid II. Amino Acids 硫硝基苯甲酸 Strong absorption at 412 nm (at pH 8.0))

40. Formation and cleavage of a disulfide bond(二硫键的形成和断裂) P.142 O2/metal ions Oxidation (氧化) 半胱氨酸 胱氨酸 Reduction (还原) Mercaptoethanol (巯基乙醇) HCOOOH (过甲酸) 氧化态 还原态 内消旋胱氨酸（Meso-cystine） P.143

41. 7. Separation and analysis of amino acids P.148 Eluent (洗脱剂) • Adsorption chromatography (吸附层析) • Filter-paper chromatography (纸层析) • Thin-layer chromatography (薄层层析) • Ion-exchange chromatography (离子交换层析) • Gas-liquid chromatography (气液层析) • High performance liquid chromatography (高效液相层析) Stationary phase (固定相) II. Amino Acids Mobile phase (流动相) Eluate (洗脱液)

42. P.152 Ion-exchange chromatography(离子交换层析) • Ion exchange resins (离子交换树脂 • Principles of ion-exchange chromatography (原理) • Affinity (亲和性) of amino acids for the ion-exchange resins • Electrostatic effect (静电效应) • pH – pH gradient • Ionic strength (离子强度) – salt gradient • Hydrophobic effect(疏水作用) • Separation of amino acid mixtures • Amino acid analyzer (氨基酸自动分析仪) II. Amino Acids

43. The column matrix for ion exchange chromatography is a synthetic polymer (resin) containing bound charged functional groups Cation exchange resins (阳离子交换树脂)： 强酸型 弱酸型 弱酸型 II. Amino Acids Anion exchange resins (阴离子交换树脂): 强碱型 弱碱型

44. II. Amino Acids

45. Separation of amino acid mixtures (氨基酸的分离) • Cation exchange chromatography is usually used (常用阳离子交换层析) • The column is first treated with NaOH. • The sample is adjusted to pH 2~3 and applied to the column (样品调节至pH 2~3后上柱) • pH/salt gradient is employed (洗脱液的pH/离子强度逐渐) • The elution sequence depends on (洗脱顺序取决于): • Electric charge (电荷) of the amino acid -- sign and magnitude • Hydrophobicity (疏水性) of the amino acid II. Amino Acids

46. Electrostatic effect (静电效应) Asp (acidic) Ala (neutral) Lys (basic) 2.09 2.34 2.18 9.82 9.69 8.95 3.86 II. Amino Acids Electric charge sequence (电荷顺序): Asp < Ala < Lys Elution sequence (洗脱顺序): Asp – Ala – Lys 10.93

47. Hydrophobic effect (疏水效应) II. Amino Acids Gly Ala Val Leu Hydrophobicity (疏水性)： Gly < Ala < Val < Leu Elution sequence (洗脱顺序)： Gly – Ala – Val – Leu

48. P.153 Amino acid analysis 1. Hydrolysis (水解) 2. Derivatization (生成衍生物) 3. HPLC separation (分离) 4. Data treatment (数据处理)

50. III. Peptides (肽) • Formation of a peptide bond (肽键的形成) • Formation of a peptide (肽的形成) • Ionization behavior (电离行为) • Chemical properties (化学性质) • Active peptides (活性肽)