生物化学精品课程 Amino acids Chapter 2 The Same 20 Amino Acids Serve as Building Blocks for All Proteins in Nature.

生物化学精品课程

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Chapter 2

The Same 20 Amino Acids Serve as

Building Blocks for All Proteins in Nature


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1. Proteins are extremely versatile (通用的 ) in function and crucial in virtually all biological processes.

1.1 Almost all chemical reactions occurring in living organisms are catalyzed by enzymes.

1.1.1 Many thousands of enzymes have been discovered, each catalyzing a different kind of chemical reaction.

1.1.2 Life would not occur without enzyme catalysis.

1.1.3 Enzymes are the most varied and most highly specialized proteins.


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1.2 Many small molecules and ions are transported by specific proteins.

1.2.1 Some proteins transport molecules from one organ to another, many exist in the blood plasma, e.g., hemoglobin (oxygen), serum albumin (fatty acids), lipoproteins (lipids), and transferrin (iron).

1.2.2 Some proteins transport molecules across plasma membrane or organelle membranes (glucose, amino acids, nucleotides, chloride, potassium, sodium ions but not water).


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1.3 Some proteins function as nutrient or storage proteins. For

example, many of such kind exist in plant seeds, animal eggs

(Ovalbumin of egg white, casein of milk). Ferritin （铁蛋白） in animal tissue function to store ion (about 4500 ions

are stored in the cavity of each ferritin complex!).

1.4 Some proteins are responsible for the coordinated协调运动 motions (contraction, changing shape, moving about). For

example, actin and myosin in muscle and nonmuscle cells.

Tubulin and dynein in eukaryotic flagella and cilia.


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1.5 Some proteins are responsible for mechanical support (strength and protection)

1.5.1 Collagen in tendons ( 筋腱 ), cartilage （软骨） , and leather.

1.5.2 Elastin in ligaments ( 韧带 ).

1.5.3 Keratin 角蛋白 in hair, fingernails, feathers.

1.5.4 Fibroin in silk fibers and spider webs.

1.5.5 Resilin （节肢弹性蛋白） in wing hinges of some insects.


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1.6 Some proteins function to defend the organisms

1.6.1 Immunoglobins in vertebrates

1.6.2 Fibrinogen 纤维蛋白原 and thrombin ( 凝血酶 ) in blood-clotting

1.6.3 Snake venom, bacterial toxins, and toxic plant proteins (ricin, 篦麻毒素 , 篦麻蛋白 ).

1.7 Some proteins help regulate cellular or physiological activity

1.7.1 Growth factors and transcription factors regulate cell growth and differentiation.

1.7.2 Hormones (e.g., insulin, growth hormon) coordinate activities of different cells in multi-cellular organisms.


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1.8 Some proteins are responsible for the generation and

transmission of nerve impulses.

1.8.1 Rhodopsin ( 视紫红质 , 视网膜色素 ) responses to

light to generate vision.

1.8.2 Acetylcholine ( 乙酰胆碱 ) receptor are responsible

for transmitting nerve impulses at synapses ( 神经突触 ).

1.9 Many proteins have other functions. Antifreeze proteins protect

the blood of Antarctic fish from freezing. Special heat stable

proteins in thermophile （嗜热生物） bacteria.


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Chymotrypsin with its active site


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Fireflies emit light catalyzed by luciferase with ATP


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Erythrocytes contain a large amount of hemoglobins, the oxygen-transporting protein.


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The protein keratin （角蛋白） is the chief structural components of hair, scales, horn, wool, nails and feathers.


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2. All natural proteins were found to be built from a repertoire （保留剧目） of 20 standard -amino acids

2.1 The earliest studies of proteins focused on the free amino acids derived from these proteins.

2.1.1 The 1st amino acid (asparagine) was discovered in 1806 from asparagus （芦笋， a green vegetable).

2.1.2 The last (threonine) was not identified until 1938!

2.1.3 All the amino acids were given a trivial (common) name. Glutamate from wheat gluten (sticky). Tyrosine from cheese (“tyros” in Greek).


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2.2 The 20 -amino acids share common structural features.

2.2.1 Each has a carboxyl group and an amino group (but one has an imino 亚氨基 group in proline) bonded to the same carbon atom, designated as the -carbon.

2.2.2 Each has a different side chain (or R group, R=“Remainder of the molecule”).

2.2.3 The -carbons for 19 of them are asymmetric (or chiral), thus being able to have two enantiomers （对映体） . Glycine has no chirality.


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In protein chemistry, we use Greek letter nomenclature.


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2.3 The two enantiomers of each amino acid defined by the -carbon are designated D- and L- forms (D for Dextrorotary, L for Levorotary)

2.3.1 The D- and L-forms of amino acids are named in reference to the absolute configuration of D- and L- glyceraldehydes (whose structure was orignally assumed and confirmed by X-ray crystallography later).

2.3.2 Only the L-amino acids have been found in proteins (D-isomers have been found only in small peptides of bacteria cell walls and in some peptide antibiotics).

2.3.3 The correlation of structure (or configuration) with optical rotation is very complex and has not been successful to date! (i.e., the D- and L-signs do not tell anything about their optical rotation!)


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2.4 The amino acids ionize in aqueous solutions.

2.4.1 Crystalline amino acids (in neutral aqueous solutions) have melting points much higher than those of other organic molecules of similar size.

2.4.2 The amino acids ionize to various states depending on pH values.

2.4.3 The amino acids (of neutral side chains) exist predominantly as dipolar ions, known as zwitterions (German for “hybrid ions”).


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2.5 Each amino acid is given a three-letter abbreviation and a one-

letter symbol. They often the first three letter and the first letter.

When there is confusion, an alternative is used. They must be

remembered. (fig.)

2.6 All proteins in all species (from bacteria to human) are

constructed from the same set of 20 amino acids.

2.6.1 All proteins, no matter how different they are in

structure and function, are made from the 20 standard amino acids.

2.6.2 This fundamental alphabet of the protein language is at

least two billion years old.


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Stryer’s method: walk from the amino group to the carboxyl

group, the hydrogen atom is on your left. L-Alanine


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Align carbon atoms with L-glyceraldehyde, the amino group

is on the left.


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The horizontal bonds project out of the plane of the paper,

the vertical behind.


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Lined up by similarity: chiral to chiral, COO to CHO


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Gly, GAla, AVal, VLeu, LMet, MIle, I


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Phe, F; Tyr, Y; Trp, W


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Ser, SThr, TCys, CPro, PAsn, NGln, Q


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Lys, K; Arg, R; His, H


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Asp, D; Glu, E


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3. The 20 amino acids are usually grouped according to the properties (mainly polarity) of their R groups

3.1 Six amino acids have nonpolar, aliphatic （脂肪族的） (hydrophobic) R groups.

3.1.1 They are Gly, Ala, Val, Leu, Ile, and Pro.

3.1.2 Gly has a hydrogen as its R group, having minimal steric hindrance.

3.1.3 Pro has an imino group, instead of an amino group, forming a five-membered ring structure, being rigid in conformation.


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3.1.4 Pro is often found in the bends of folded protein

chains and often present on the surface of proteins.

3.1.5 In protein structure Gly offers the most flexibility,

while Pro the least!

3.1.6 Ala, Val, Leu, and Ile, have hydrocarbon R groups,

often involved in hydrophobic interactions.


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3.2 Phe, Tyr, and Trp have aromatic （芳香族的） R groups

3.2.1 Phe and Tyr both have benzene rings.

3.2.2 Tryptophan has an indole ring.

3.2.3 All three participate in hydrophobic interactions.

3.2.4 The -OH group in Tyr is an important functional group

in proteins. (phosphorylation, hydrogen bond, etc)

3.2.5 They are jointly responsible for the light absorption of

proteins at 280 nm

A=Log Io/I = cl Lambert-Beer’s law

extinction coefficient; c, concentration; l, optical length


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3.3 Ser, Thr, Asn, Gln, Cys, and Met have polar, uncharged R groups.

3.3.1 The R groups are more hydrophilic, due to the presence of hydroxyl groups, sulfur atoms, or amide groups.

3.3.2 -SH group of two Cys in proteins can be oxidized to form a covalent disulfide bond.

3.3.3 Cys and Met often participate in hydrophobic interactions.


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3.4 Asp and Glu have carboxyl in their R groups. They have net

negative charge at pH 7.0, thus usually named as aspartate and

glutamate (conjugate base names, instead of aspartic acid and

glutamic acid, un-ionized form).

3.5 Arg, Lys, and His have positively charged R groups at pH 7.0.

3.5.1 Their R groups contain guanidino, amino, imidazole

groups respectively.

3.5.2 The side chain of His can be positively or uncharged

depending on the local environment near pH 7.0.


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3.6 The extent of hydrophobicity and hydrophilicity of the side

chains is reflected by their hydropathy index values. “-” values

usually mean hydrophilic, “+” values hydrophobic.

3.7 Nonstandard amino acids are found in certain proteins,

generally as a result of post-translational modifications.

3.7.1 These modifications are made after the standard

amino acids have been incorporated into proteins.

3.7.2 4-Hydroxyglutamate and 5-hydroxylysine in collagen.


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3.7.3 -carboxyglutamate is found in the blood-clotting prothrombin ( 凝血素， an enzyme).

3.7.4 Desmosine is a covalent linkage made from four Lys side chains in elastin （弹性蛋白） .

3.7.5 Selenocysteine is found in many enzymes (having been recognized as the 21st amino acid in ribosome-mediated protein synthesis!).

3.7.6 Many additional nonstandard amino acids are found in cells, but not in proteins (e.g., ornithine 鸟氨酸 and citrulline 瓜氨酸 , intermediates in amino acid metabolism).


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Nonstandard amino acids not found in protein


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Nonstandard amino acids not found in protein


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4. Amino acids, being both weak acids and bases, have

characteristic titration curves and pKa values

4.1 Amino acids can act both as acids and bases. The zwitterion form of amino acids are ampholytes 两性电解质 . Amino acids can be diprotic （双质子） and triprotic （三质子） acids.4.2 Monoamino monocarboxylic α-amino acids (e.g., Gly, Ser, Phe with no ionizable groups) all have similar two-stage titration curves.

4.2.1 The first stage reflects the deprotonation （去质子化） of the α-COOH group (pK1).

4.2.2 The second stage reflects the deprotonation of the α -NH 3 + group (pK2).

4.2.3 The pKα value of the a-COOH is more than 2.0 units smaller than that of acetic acid (pK α of 4.76), that is, a stronger (weak) acid.

4.2.4 These amino acids have two buffering power regions.


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4.3 Acidic and basic amino acids have three-stage titration curves. The additional stage is for the ionizable group on the side chains (pKR).

4.4 There is a specific pH (designated pI) at which an amino acid has equal positive and negative charge.

4.4.1 An amino acid does not move in an electric field at its pI, called isoelectric point.

4.4.2 The pI of monoamino monocarboxylic amino acids reflects a status at which the -COOH group is fully deprotonated, but the -NH3

+ group has not yet started deprotonating

pI = (pK1+pK2)/2


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4.4.3 The pI point of an acidic amino acid reflects a status at which the α-COOH is fully deprotonated, but the side chain -COOH and the a-NH3

+ group have not yet started deprotonating

pI = (pK1+pKR)/2

4.4.4 The pI point of a basic amino acid reflects a status at which the α -COOH and the side chain -NH3

+ or -NH+= group have fully

deprotonated, but the NH3+ group not yet deprotonated

pI = (pKR+pK2)/2

4.4.5 The amino acids are positively charged at pH smaller than their pI values, negatively charged at pH larger than their pI values.


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An acidic amino acid pI=(pK1+pKR)/2


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A basic amino acid pI=(pKR+pK2)/2


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5. The 20 amino acids can be separated from each other by ion-exchange chromatography

5.1 Each of the amino acids has a different pI value. Therefore, each amino acid has a different net charge at a given pH.

5.2 The variously charged amino acids bind to charged synthetic resins with various affinities.

5.2.1 When the resin is positively charged, negatively charged amino acids (or other anions) will bind, and vice versa. 5.2.2 Amino acids having the same charge as the resin will not bind. 5.2.3 The positively charged resin is called anion-exchange resin. The negatively charged called cation-exchange resin (e.g., the sulfonated polystyrene 聚苯乙烯 ). 5.2.4 The resin (serving as the stationary phase) is usually packed in a column (providing the mechanical support and stable fluid flow).


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5.3 The bound amino acids can be eluted by running a pH or salt gradient (serving as the mobile phase).

5.3.1 Amino acids will be eluted out in the order of their binding affinity (strongly bound ones being eluted out later).

5.3.2 This way of separating amino acids (or other charged biomolecules) is called ion-exchange chromatography.

5.3.3 Chromatography is a method of separating substances by allowing them to partition between two phases, one mobile, one stationary (differences in charge, size, hydrophobic interactions, specific interactions can be exploited for substance separation with chromatography).

A.J. Martin and R.L. Synge won the Nobel Prize in 1952 for inventing chromatography.


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5.4 The mobile phase can percolate （过滤） through the column at low pressure or high pressure.

5.4.1 To operate under high pressure, specially designed resins and apparatus (the pumps and the plumbing system) are needed.

5.4.2 Using high pressure allows better separation in a much shorter period of time, thus named High Performance Liquid Chromatography (HPLC).5.5 Quantitative estimates of each amino acid in a mixture can be efficiently carried out using a fully automated Amino Acid Analyzer.

5.5.1 Addition of sample mixture, elution of each amino acid, collection and analysis of fractions, and data recording are all fully automated!

5.5.2 HPLC (ion exchange) column is used in the machine.


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Cation-exchangecolumn


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6. The amino acids can be detected using various chemical reagents

6.1 The free α-amino group of any amino acids will react with

ninhydrin[ 化 ]( 水合 ) 茚三酮 to form a purple product.

6.1.1 Detects amino acids nonspecifically.

6.1.2 The imino group of Pro gives a yellow color.

6.1.3 The concentration/amount (micro gram) of amino

acid can be determined by measuring optical absorbance (at 440

nm for purple or 550 nm for yellow).


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6. The amino acids can be detected using various chemical reagents

6.2 Other reagents also react with the α-amino group, keeping the

R group part of the products

6.2.1 1-Fluoro-2,4-dinitrobenzene and dabsyl chloride react

to form colored derivatives that are stable under harsh conditions

(heating in 6N HCl at 110C for 24 hours!)

6.2.2 Fluorescent derivatives, permitting the detection of

nanogram amount of amino acids.

6.2.3 Identity of amino acids can be revealed by comparing

with a standard.


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7. Amino acids covalently join one another to form peptides

7.1 The a-carboxyl group of one amino acid joins with the α-amino

group of another amino acid by a peptide bond (actually an amide

bond)

7.1.1 This is a condensation reaction where a water

molecule is liberated or eliminated.

7.1.2 G of the condensation reaction is about 5 kcal/mol,

not being able to occur spontaneously (an endergonic 吸能 reaction).

7.1.3 The condensation reaction can occur repeatedly to

form oligopeptides (with less than 50 aa), polypeptides (bwt 50-100

aa), and proteins (longer).


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7.2 The peptide chain is directional.

7.2.1 An amino acid unit in a peptide chain is called a

residue.

7.2.2 The end having a free α-amino group is called amino-

terminal or N-terminal.

7.2.3 The end having a free a-carboxyl group is called

carboxyl-terminal or C-terminal.

7.2.4 By convention, the N-terminal is taken as the

beginning of the peptide chain, and put at the left (C-terminal at the

right). Biosynthesis starts from the N-terminal.


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7.2.5 The peptide chain consist of a regularly

repeating main chain (or backbone) and the variable side

chains of the residues.

7.2.6 Amino acid residues have an order or

sequence on a peptide.

7.2.7 The 20 amino acids are analogous to the 26

letters in English ； the number of different peptides

made of them is unlimited.


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7.3 The size of a peptide can be described by its total number of

residues (e.g., a pentapeptide, a octapeptide 八肽 ) or relative

molecular mass (molecular weight).

7.3.1 The mean molecular weight of an amino acid

residue in a peptide is ~110 dalton.

7.3.2 Most natural polypeptide chains contain between

50 and 2000 amino acid residues, thus having relative molecular

mass between 5500 daltons and 220,000 daltons, or 5.5 kDa and

220 kDa, respectively.


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7.4 Each peptide has a characteristic titration curve and a

isoelectric point (pI).

7.4.1 The titration curve of a peptide reflect the collective

behavior of all the acid-base groups.

7.4.2 The peptide would not move in an electric field at its

pI. (determined by IEF, isoelectric focusing gel).

7.4.3 2D gel in proteomics: one dimension is IEF

separation by pI; the other SDS-PAGE separation by molecular

weight.


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8. Many short peptides have important biological activities

8.1 Some short peptides (neuropeptides 神经肽 ) act as

neurotransmitters, neurohormones, and neuromodulators.

8.1.1 These peptides are secreted by the neurons.

8.1.2 The LHRH-like decapeptide (10-residue) act as a

neurotransmitter for the frog sympathetic ganglia 神经中枢 .

8.1.3 Thyrotropin-releasing factor (3-residue) is formed

in the hypothalamus 视丘下部 and stimulates the release of

thyrotropin 促甲状腺素 from the anterior 前面的 pituitary 垂体 gland.


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8.1.4 Oxytocin ([ 化 ] 脑下垂体后叶荷尔蒙之一种 9-

residue) is secreted by the posterior pituitary and stimulates

uterine contraction.

8.1.5 The opioid peptides (including mainly enkaphalins,

endorphins, and dynorphins) have been implicated in the control

of pain, responses to stress, and other functions.

8.1.6 Some drugs, like morphine and heroin, generate

their addictive effect by binding to opioid peptide receptors!


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8.2 Some short peptides act as antibiotics

8.2.1 Gramicidin A (15-residue) is a well studied peptide antibiotic (from Bacillus brevis). Its structure has been determined.

8.2.2 It contains alternating L- and D-amino acid residues.

8.2.3 It is not synthesized on ribosomes!

8.2.4 Gramicidin S ([ 生化 ] 短杆菌肽 10-residue, circular) is another antibiotic also from Bacillus 杆状菌 brevis.

8.2.5 Peptide antibiotics have also been found in frog skins, neutrophile 嗜中性 cells, and insects.


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8.3 Many short peptides are used as defensive poisons.

8.3.1 α-Amantin (8-residue, circular) in mushroom

is an extremely toxic peptide (inhibiting RNA

polymerases II and III at picomolar levels!)

8.3.2 Very toxic short peptides are also found in

snake venom, spider.


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8.4 Many vertebrate hormones are small polypeptides.

8.4.1 Insulin (51-residue) is produced by the pancreas and acts to lower blood glucose level, after food intake.

8.4.2 Glucagon ( 胰高血糖素，胰增血糖素， 29-residue) is also produced by the pancreas and acts to increase the blood glucose level.

8.4.3 Corticotropin ( 促肾上腺皮质激素， or adrenocorticotropin, 39-residue) is produced by the anterior pituitary gland and stimulates the growth of adrenal cortex and secretion of corticosteroid.

8.4.4 Vasopressin ( 后叶加压素，抗利尿激素， 9-residue) stimulates the reabsorption of water in the distal tubules of the kidney (diabetes insipidus patients have deficient vasopression)


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8.5 Many such bioactive peptides are present in exceedingly small

amounts (thus difficult to discover!) and acts at very low

concentrations.

Bioactive short peptides can be selected by making random

peptide libraries (through chemical synthesis, combinatory 可组合的， chemistry, or phage display). Very little is known about the

receptors of these bioactive peptides. They should be good

potential drug targets.


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9. Peptides can be synthesized chemically

9.1 Peptides of up to 150 residues can be synthesized by automated

solid-phase methods mainly invented by R. Bruce Merrifield (who

won the 1984 Nobel Prize in Chemistry for this).

9.1.1 Amino acids are added stepwise to a growing peptide

chain that is linked to an insoluble matrix, such as polystyrene 聚苯乙烯 beads.

9.1.2 A major advantage is that the desired product at each

stage is bound the insoluble beads with other chemicals easily

filtered and washed away.


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9.1 Peptides of up to 150 residues can be synthesized by automated

solid-phase methods mainly invented by R. Bruce Merrifield (who

won the 1984 Nobel Prize in Chemistry for this).

9.1.1 Amino acids are added stepwise to a growing peptide

chain that is linked to an insoluble matrix, such as polystyrene 聚苯乙烯 beads.

9.1.2 A major advantage is that the desired product at each

stage is bound the insoluble beads with other chemicals easily

filtered and washed away.


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9.1.3 The synthesis starts with fixing the C-terminal amino acid on the insoluble beads through its a-carboxyl group. This is in the reverse direction of biosynthesis.

9.1.4 The a-amino group of the next amino acid to be added is protected and its carboxyl group activated. The amino group is protected by the t-butyloxycarbonyl group (t-boc) and deprotected by CF3COOH. The carboxyl group is activated by dicyclohexylcarbodiimide (DCC).

9.1.5 The peptide bond is formed by the free a-amino group (deprotected) of the fixed C-terminal residue attacking the DCC activated α-carboxyl group of the free amino acid in solution.

9.1.6 After washing away unreacted free amino acids and other reagents, step 9.1.4 and 9.1.5 are repeated. The synthesized peptide is eventually cleaved off from the resin by adding HF.


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9.2 The efficiency of this solid phase synthesis is much lower than biosynthesis in living organisms.

9.2.1 Synthesizing a 100 amino acid peptide will take about 4 days to finish with a fully automated machine with reasonable 合理的 yield.

9.2.2 The same peptide would be synthesized with exquisite fidelity in about 5 seconds in a bacterial cell!

9.3 Peptides having natural activity have been synthesized chemically.

9.3.1 The complete bovine insulin was first synthesized and show to be the same as the natural insulin in China in 1965!

9.3.2 Merrifield also synthesized the interferon 干扰素 (155 aa) and ribonuclease (124 aa).


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Homework

1. The four families of amino acid R groups can be classified as being either

hydrophobic or hydrophilic. What properties of the R group determine this?

Classify each family as hydrophobic or hydrophilic.

2. Compare the structures at physiological pH of the R groups of the following

pairs of amino acids. Determine which member of the pair is hydrophobic and

which is hydrophilic. Justify your choice.

(a) alanine/serine ；(b) phenyalanine/tyrosine ；(c) isolecuine/glutamate ；(d) praline/histidine.

3. Consider neutral (pH 7) solutions of the amino acids alanine., arginine,

asparagine, aspartate, glutamate, lysine, phenylalanine, and serine. Classify each

amino acids as one of the following: nonpolar, polar uncharged, negatively

charged, or positively charged.


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Homework

4. Why does the neutral amino acid phenyalanine exist as a dipolar molecule?

Determine whether the following statements are true or false. If false, explain

why.

(a) All 20 standard amino acids found in proteins have at least one asymmetric

carbon atom.

(b) An epuimolar mixture of D- and L-alanine does not rotate the plane of

polarized light.

(c) The observed optical rotation of an amino solution is directly proportional to

the length of the light path through the polarimeter tube and the concentration of

the amino acid solution.

(d) Alanine obtained from a protein hydrolysate has the same absolute

configuration as D-glyceraldehyde.

5. (a) Which amino acid has good buffering capacity against added acid in the pH

region of intracellular fluids and bloods?

(b) Which amino acid is always positively charged at all pH values below 12?

(c) Which amino acid are negatively charged at all pH values above 4?


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Homework

6. Amino acids are frequently separated by either paper electrophoresis or ion

exchange chromatography.

(a) What is the basis for these separation techniques?

(b) Do both techniques use the acid-base properties of the amino acids to effect a

separation? How?

(c) How are the locations of the amino acids on the chromatogram detected?

(d) Can these techniques be used to separate peptides? Explain.

7. Glycine at Physiological pH: Distribution Between Zwitterion, Anion, and

Cation. At physiological pH, glycine exists predominantly as the zwitterion.

Calculate the percentage distribution of zwitterion, anion, and cation of a solution

of glycine at pH 7.4:


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Homework

8. A mixture of serine, glutamic acid, and lysine is separated by paper

electrophoresis in two experiments, one at pH 1 and one at pH 12. After the

separation, the papers are treated with ninhydrin to reveal the location of the

amino acids (purple spots). The results are shown in Figure. Match the

electrophoresis strips with the appropriate pH and identify all the spots. Draw the

strip corresponding to a separation at pH 7.

Cathode

Cathode

Anode

Anode

Origin

生物化学精品课程 Amino acids Chapter 2 The Same 20 Amino Acids Serve as Building Blocks for All Proteins in Nature.

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