Amino Acids & Peptides

Theory — Amino Acids & Peptides

1. Amino acid structure

Every standard amino acid has a central α-carbon (Cα) bonded to four groups: an α-amino group (-NH₂), an α-carboxyl group (-COOH), a hydrogen, and a side chain (R) that varies among the 20 standard amino acids. With the exception of glycine (R = H, achiral), all standard amino acids are chiral; biological proteins use exclusively the L-enantiomer.

2. Zwitterions and isoelectric point (pI)

At physiological pH (~7.4), the α-amino group is PROTONATED (-NH₃⁺) and the α-carboxyl group is DEPROTONATED (-COO^-). The molecule has BOTH a positive AND a negative charge \u2014 a zwitterion. Net charge = 0. Despite both ends being charged, the overall molecule is neutral.

The isoelectric point (pI) is the pH at which the amino acid carries no NET charge (neutral overall, even if charged groups are present). For a simple amino acid (no charged side chain): pI = (pKa₁ + pKa₂)/2. For amino acids with charged side chains: pI = average of the two pKa values that flank the neutral form.

3. The 20 standard amino acids by classification

Class	Amino acids (3-letter / 1-letter)	Side-chain feature
Nonpolar / hydrophobic	Gly (G), Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Met (M)	Aliphatic, no charges; bury in protein interior
Aromatic	Phe (F), Tyr (Y), Trp (W)	Aromatic ring; UV absorption at 280 nm; Tyr has -OH (mildly polar)
Polar uncharged	Ser (S), Thr (T), Cys (C), Asn (N), Gln (Q)	H-bond donors/acceptors; Cys has -SH (thiol)
Acidic / negatively charged	Asp (D), Glu (E)	Side-chain -COOH; deprotonated at neutral pH
Basic / positively charged	Lys (K), Arg (R), His (H)	Side-chain -NH₂ or guanidine or imidazole; protonated at neutral pH

4. pKa values and titration curves

Amino acid	pKa₁ (\u03b1-COOH)	pKa₂ (\u03b1-NH₃⁺)	pKa_R (side chain)	pI
Glycine	2.34	9.60	\u2014	5.97
Alanine	2.34	9.69	\u2014	6.01
Aspartate	1.88	9.60	3.65	2.77
Glutamate	2.19	9.67	4.25	3.22
Histidine	1.82	9.17	6.00	7.59
Lysine	2.18	8.95	10.53	9.74
Arginine	2.17	9.04	12.48	10.76
Cysteine	1.96	10.28	8.18	5.07
Tyrosine	2.20	9.11	10.07	5.66

A titration curve plots pH vs. equivalents of base added. For a simple amino acid (e.g., glycine), the curve is sigmoidal with TWO inflection points (pKa₁ and pKa₂) and TWO buffering regions (flat plateaus around each pKa). For amino acids with ionisable side chains, there are THREE pKa values and THREE buffering regions.

Henderson-Hasselbalch & pI calculation pH = pKa + log([A⁻]/[HA]) (Henderson-Hasselbalch)
For neutral amino acid (Gly, Ala): pI = (pKa₁ + pKa₂)/2
For acidic amino acid (Asp, Glu): pI = (pKa₁ + pKa_R)/2
For basic amino acid (Lys, Arg): pI = (pKa_R + pKa₂)/2
For Cys, Tyr (acidic side chain): pI = (pKa₁ + pKa_R)/2
For His: pI = (pKa_R + pKa₂)/2
The pI is determined by the AVERAGE of the two pKa values FLANKING the neutral form. Below pI: net positive charge. Above pI: net negative charge.

5. The peptide bond

Peptide bonds are amide bonds formed between the α-COOH of one amino acid and the α-NH₂ of the next, releasing water. Peptide bonds have key properties:

Planar: the amide N has \u03c0-character (resonance with C=O), making the C-N bond partial-double-bond and preventing rotation.
Trans-preferred: the carbonyl O and amide H are usually trans (~99.95%); the cis form is energetically disfavoured.
Two rotatable bonds per residue: φ (phi) is N-Cα rotation; ψ (psi) is Cα-C(=O) rotation. The Ramachandran plot shows allowed combinations.
Directional: peptides have an N-terminus (free \u03b1-NH₂) and a C-terminus (free \u03b1-COOH). Sequences are written N to C.

6. Disulfide bonds

Cysteine\'s -SH side chain can oxidise to form a -S-S- disulfide bond between two cysteine residues. Disulfides covalently cross-link protein chains; they are essential for the stability of secreted proteins (insulin, antibodies, ribonuclease). Disulfides form/break depending on redox conditions: reducing environment (cytoplasm) keeps -SH free; oxidising environment (extracellular, ER) promotes disulfide formation. Reagents that disrupt disulfides: dithiothreitol (DTT), \u03b2-mercaptoethanol (BME), tris(2-carboxyethyl)phosphine (TCEP).

7. Secondary structure preview

Amino acids with specific properties favor specific secondary structures:

α-Helix: right-handed coil with H-bonds between residue i and i+4. Favored by Ala, Leu, Met, Glu (helix-formers); destabilised by Pro (no NH for H-bond) and Gly (too flexible).
β-Sheet: extended chain in pleated sheet, H-bonds between strands. Favored by Val, Ile, Tyr, Phe (\u03b2-formers).
Turns and loops: short connections between secondary structures. Pro and Gly common in turns.

8. Detection: ninhydrin reaction

The classic test for amino acids: ninhydrin (2,2-dihydroxy-1,3-indandione) reacts with the α-amino group, releasing CO₂ and an aldehyde, and forming a deep purple-blue product called Ruhemann\'s purple (λmax ~570 nm). The reaction works for primary amines (most amino acids) and gives a distinctive yellow color with proline (which has a secondary amine, not primary). Used for amino acid quantitation and TLC visualization.

Memorise these landmarks α-amino acid backbone: H₂N-C\u03b1H(R)-COOH
Zwitterion at physiological pH: H₃N⁺-C\u03b1H(R)-COO^-
Net charge: positive below pI, negative above pI
Glycine pI = 5.97 (only achiral standard amino acid)
Lysine pI = 9.74 (basic side chain)
Glutamate pI = 3.22 (acidic side chain)
Histidine pKa_R = 6.0 (only one with side-chain pKa near physiological pH \u2192 great pH buffer)
Proline: secondary amine, gives YELLOW with ninhydrin (not purple)
Cysteine: pKa thiol = 8.3; can form disulfide with another Cys
Trp, Tyr, Phe: aromatic; absorb UV at 280 nm \u2192 protein quantitation
Peptide bond is PLANAR (no rotation about C-N). Two rotatable bonds per residue (φ, ψ). Ramachandran plot shows allowed conformations.

9. Systematic amino acid analysis approach

Identify the side chain (R group). Determine: aliphatic, aromatic, polar, acidic, or basic.
Count ionisable groups. Two pKa (no R-group ionisation) or three pKa (R group ionisable).
Calculate pI. Average of two pKa values flanking the neutral form.
Predict net charge at given pH. Below pI \u2192 positive; above pI \u2192 negative.
Predict secondary structure tendency. Helix-former, sheet-former, or turn/loop?
For peptides: identify N-terminus, C-terminus, and any side-chain charges.

Instructions

The Simulation has four parts. Complete in order.

Section I — Amino Acid Library. 8 amino acids. For each: classify (nonpolar, polar, acidic, basic, aromatic); estimate pI range; identify a key chemical feature.

Section II — Titration Bench. 6 unknown amino acid samples. Click "Run Titration", view the pH-vs-equivalents-of-base curve, identify the amino acid by its pKa values and pI.

Section III — Peptide & Property Interpretation. 8 puzzles: pI calculations, peptide bond properties, charge prediction, disulfide bonds, secondary-structure preferences.

Section IV — SDS & Microscale. SDS for 4 lab materials (glycine, NaOH, conc. HCl for protein hydrolysis, ninhydrin) = 16 questions. Then 6 microscale sample-prep scenarios.

Prepare your lab notebook. Use the Example Report as your template.

Prerequisite: Familiarity with acids/bases (Henderson-Hasselbalch equation, buffer pH). The Carbohydrates lab is recommended as a precursor for biomolecule context.

Simulation

Four interactive parts. Use the ↺ Reset Simulation button to clear all answers.

Eight amino acid identification cases. For each: (a) classification; (b) pI range; (c) key feature.

Score: 0 / 24

Six unknown amino acid samples. Select a sample, click Run Titration, observe the pH-vs-base curve, identify the amino acid. Curve labels appear after you answer.

Score: 0 / 6

Eight harder puzzles: peptide bonds, pI calculations, charge prediction, disulfides, secondary structure.

Score: 0 / 8

Round 1 — SDS interpretation

Four amino acid lab materials. Each has 4 questions.

SDS score: 0 / 16

Round 2 — Microscale sample prep

Six sample-prep scenarios.

Microscale score: 0 / 6

Team Questions

Discuss with your team before answering.

Question 1 — Zwitterion. Draw the zwitterion form of glycine and label the charged groups.

Question 2 — pI calculation. Aspartate has pKa values 1.88, 3.65, 9.60. Calculate its pI.

Question 3 — Peptide bond geometry. Why is the peptide bond planar, and what bonds in the backbone CAN rotate?

Question 4 — Charge at pH 7. What is the net charge of lysine (pKas: 2.18, 8.95, 10.53) at pH 7.0?

Question 5 — Histidine in physiology. Why is histidine especially important as a buffering amino acid in proteins at physiological pH?

Question 6 — Disulfide bonds. What amino acid forms disulfide bonds, and what is the chemistry?

Example Lab Notebook Entry

Use the format below as a template.

Amino Acids & Peptides — Lab Notebook Entry

Submitted by: [Student Name]

Course: Organic Chemistry II · Section: 202-A · Date: May 12, 2026

Objective

To learn the systematic identification of amino acids from titration curves; understand the zwitterion form and isoelectric point (pI) calculation; appreciate how side-chain pKa values determine charge distribution at any pH; understand the geometric and chemical properties of the peptide bond; and connect amino acid properties to protein structure and function.

Bench results (Section II) — titration curves

Sample	Amino acid	pKa₁	pKa₂	pKa_R	pI	Class
1	Glycine	2.34	9.60	\u2014	5.97	Nonpolar
2	Alanine	2.34	9.69	\u2014	6.01	Nonpolar
3	Lysine	2.18	8.95	10.53	9.74	Basic
4	Glutamate	2.19	9.67	4.25	3.22	Acidic
5	Histidine	1.82	9.17	6.00	7.59	Basic (His)
6	Cysteine	1.96	10.28	8.18	5.07	Polar (thiol)

Discussion

Amino acids are the monomeric building blocks of proteins. Their distinctive structural feature \u2014 a chiral α-carbon bearing both an amino group and a carboxyl group \u2014 makes them amphoteric: they can act as acids (donating H⁺ from the α-NH₃⁺) and as bases (accepting H⁺ at the α-COO^-). At physiological pH (~7.4), the α-amino is protonated (-NH₃⁺) and the α-carboxyl is deprotonated (-COO^-); the amino acid exists as a zwitterion with two charges but a net charge of zero.

The pI is the pH at which the amino acid carries no NET charge. Below pI, the molecule has a net positive charge (the carboxyl is partially protonated, the amine fully protonated). Above pI, the molecule has a net negative charge (carboxyl fully deprotonated, amine partially deprotonated). At the pI specifically, the charge balance is exact. For amino acids without ionisable side chains, pI = (pKa₁ + pKa₂)/2. For acidic amino acids (Asp, Glu, Cys, Tyr), pI is the average of pKa₁ and pKa_R. For basic amino acids (Lys, Arg, His), pI is the average of pKa_R and pKa₂.

The titration bench (Section II) revealed amino acid identity through diagnostic curve shapes. Glycine and alanine show TWO inflection points (around pH 2.3 and 9.6), characteristic of simple amino acids without side-chain ionisation. Lysine shows THREE inflections (2.2, 9.0, 10.5), with the high-pH plateau showing the additional buffering capacity of the -NH₃⁺ side chain. Glutamate shows three inflections (2.2, 4.3, 9.7), with the ADDITIONAL low-pH plateau from the side-chain carboxyl. Histidine\'s side-chain imidazole has pKa = 6.0, giving a buffering region near physiological pH \u2014 critically important biologically (hemoglobin uses His to buffer blood pH; many enzyme active sites use His for proton transfers). Cysteine has a thiol side chain (pKa = 8.3) that can be deprotonated under mildly basic conditions; this is important because the deprotonated -S^- is a strong nucleophile (catalytic in many enzymes) and can be oxidised to form disulfide bonds.

The peptide bond is one of the most important structures in biochemistry. Although nominally a "simple" amide, the peptide bond has special properties that determine protein architecture: (1) PLANAR \u2014 the C-N bond has partial double-bond character from resonance with the C=O, restricting rotation; this means that 6 atoms (Cα, C, =O, N, H, Cα) lie in a plane; (2) trans-preferred (~99.95%) \u2014 the cis form would create steric clash between the two Cα groups; the only common exception is X-Pro, where cis is more frequent (~5-10%); (3) two rotatable bonds remain per residue: \u03c6 (phi) is N-Cα, and \u03c8 (psi) is Cα-C(=O); the Ramachandran plot maps allowed combinations and predicts secondary structure preferences.

The connection to higher-order protein structure: the 20 amino acids\' diverse side chains (hydrophobic, polar, acidic, basic, aromatic, sulfur-containing) provide the chemistry needed for: hydrophobic core formation (Leu, Val, Ile, Phe, Trp); H-bond networks (Ser, Thr, Asn, Gln); ionic interactions (Asp/Glu vs. Lys/Arg); covalent cross-links (Cys-Cys disulfides); catalysis (Ser, Cys, His, Asp, Glu in enzyme active sites); and recognition (specific side-chain combinations for substrate binding). The next biomolecule labs (lipids/nucleic acids, then pericyclic) will continue to show how chemistry constrains and enables biology.

Conclusion

Amino acid chemistry is the foundation of protein chemistry. The 20 standard amino acids combine in nearly infinite sequences (20ⁿ for an n-residue protein) to give the structural and catalytic diversity of all proteins. Understanding individual amino acid properties \u2014 chirality, zwitterion behavior, pI, side-chain ionisation, hydrophobicity \u2014 is essential for predicting and explaining protein structure, function, and behavior in solution.

References

1. Solomons, T. W. G.; Fryhle, C. B.; Snyder, S. A. Organic Chemistry, 12th ed., Wiley, 2016, Ch 24.
2. Lehninger, A. L.; Nelson, D. L.; Cox, M. M. Principles of Biochemistry, 7th ed., W.H. Freeman, 2017, Ch 3.
3. McMurry, J. Organic Chemistry, 9th ed., Cengage, 2016, Ch 26.
4. Berg, J. M.; Tymoczko, J. L.; Stryer, L. Biochemistry, 9th ed., W.H. Freeman, 2018, Ch 2-3.

Practice Questions

Work through each before peeking at the hint.

Practice 1 — Glycine pI

Glycine has pKa₁ = 2.34 and pKa₂ = 9.60. Calculate its pI.

Hint: For an amino acid without ionisable side chain, pI = (pKa₁ + pKa₂)/2 = (2.34 + 9.60)/2 = 5.97. The amino acid has zero net charge at this pH.

Practice 2 — Lysine pI

Lysine has pKas: 2.18 (\u03b1-COOH), 8.95 (\u03b1-NH₃⁺), 10.53 (side-chain NH₃⁺). What is its pI?

Hint: For basic amino acids, pI is the average of the two pKa values flanking the neutral form: pI = (pKa₂ + pKa_R)/2 = (8.95 + 10.53)/2 = 9.74. Above pH 9.74, lysine has a net negative charge; below, net positive. At pH 7 (physiological), lysine has +1 net charge (the side-chain amine is protonated).

Practice 3 — Hemoglobin & His

Hemoglobin has 38 histidine residues. Why is histidine\'s pKa_R ~6.0 critically important in oxygen transport?

Hint: The pH change between lungs (slightly basic, ~7.5) and tissues (slightly acidic, ~7.0-7.2 due to metabolism) is small but biologically important. His pKa = 6.0 is right in this range, so His can shift between protonated/deprotonated forms in this small pH window. Hemoglobin uses this property: in tissues, the lower pH partially protonates His, stabilizing deoxy-Hb and releasing O₂; in lungs, higher pH deprotonates His, allowing oxy-Hb. This is the basis of the Bohr effect.

Practice 4 — Hydrophobic core

Of the following amino acids, which would you expect to find in the HYDROPHOBIC INTERIOR of a globular protein: Lys, Val, Asp, Phe, Ser?

Hint: Hydrophobic = nonpolar amino acids = those that prefer to avoid water. Val and Phe are nonpolar (Val: aliphatic; Phe: aromatic) \u2014 found in the hydrophobic core. Lys (basic), Asp (acidic), Ser (polar) are charged or polar \u2014 found on the protein surface where they can interact with water. Other hydrophobic amino acids: Leu, Ile, Met, Trp, Ala (and Cys when not in disulfide bonds).

Practice 5 — Disulfide bond

Insulin contains three disulfide bonds. Why does insulin (an extracellular hormone) have so many disulfides while most cytoplasmic proteins have none?

Hint: Disulfide bonds form by oxidation of two thiol -SH groups. The cellular environment determines redox state: cytoplasm is REDUCING (high glutathione, NADPH \u2192 keeps -SH free). Extracellular environment is OXIDISING (oxygen, no glutathione \u2192 promotes -S-S- formation). Secreted/extracellular proteins (insulin, antibodies, ribonuclease, lysozyme, blood proteins) have many disulfides for stability. Intracellular proteins have few or no disulfides because they\'d be reduced as fast as they form.

Practice 6 — Proline & secondary structure

Proline is rare in α-helices but common in turns. Why?

Hint: Proline is the only amino acid with a SECONDARY amine (its side chain ring connects back to the α-amino N). This means: (1) the N has NO H to donate \u2014 disrupts the α-helix H-bond pattern (the H of residue i+4 normally bonds to the C=O of residue i; without H on Pro, the helix kinks); (2) the rigid five-membered ring restricts the \u03c6 angle to ~-60\u00b0, an angle compatible with helix initiation but not with helix continuation. Result: Pro is a "helix-breaker" but ideal for tight turns and the start of helices.

Practice 7 — Net charge calculation

For glutamate at pH 5.0 (pKas: 2.19, 4.25, 9.67), what is the approximate net charge?

Hint: At pH 5.0, the \u03b1-COOH (pKa 2.19) is deprotonated (-COO^-, charge -1). The side-chain COOH (pKa 4.25) is also mostly deprotonated (pH 5 > pKa 4.25 by ~0.75 units, so ~85% deprotonated, charge ~-0.85). The \u03b1-NH₃⁺ (pKa 9.67) is fully protonated (charge +1). Total: -1 - 0.85 + 1 = -0.85. So the net charge at pH 5 is approximately -1 (mostly fully deprotonated). At pH = pI = 3.22, both the \u03b1-COOH and side-chain COOH are partially deprotonated and the molecule is neutral overall.

Practice 8 — Trp absorption

A protein has ε₂₈₀ = 22,000 M^-1 cm^-1. The protein contains 4 tryptophan, 6 tyrosine, and 1 cystine (disulfide). Are these consistent? Use \u03b5_Trp\u2248 5500, \u03b5_Tyr \u2248 1490, \u03b5_cystine \u2248 125 M^-1 cm^-1.

Hint: Sum: 4 \u00d7 5500 + 6 \u00d7 1490 + 1 \u00d7 125 = 22,000 + 8940 + 125 = 31,065 M^-1 cm^-1. The measured 22,000 is LESS than the predicted 31,065 \u2014 not consistent. Either the protein has fewer aromatic residues than stated, or the Trp/Tyr are buried in a way that reduces \u03b5, or there\'s a folding effect. Note: the rough rule for protein \u03b5₂₈₀ assumes all aromatics are exposed; folded proteins may have slightly lower \u03b5 due to environment effects on the chromophores.

Practice 9 — Ninhydrin

A ninhydrin test gives a YELLOW color (not the usual purple). What amino acid is most likely present?

Hint: PROLINE. Ninhydrin reacts with primary amines (-NH₂) to give Ruhemann\'s purple. Proline has a SECONDARY amine (the side chain ring connects to the \u03b1-amino N), so it cannot form Ruhemann\'s purple; instead it gives a yellow product. This selective response is used to identify Pro in TLC and amino acid analysis.

Practice 10 — Combining techniques

A small peptide is hydrolyzed (6 M HCl, 110\u00b0C, 24h) and the products are run on an amino acid analyzer. Equal amounts of Gly, Ala, and Lys are detected. The original peptide had MW ~290 and a positive Tollens\' test for the C-terminus. Suggest the structure.

Hint: The peptide is a tripeptide of Gly + Ala + Lys (sum of MW: 75 + 89 + 146 = 310; minus 2 \u00d7 18 for two peptide bonds = 274, close to MW 290 with rounding for charges/water). The positive Tollens\' suggests an aldehyde at the C-terminus \u2014 this is unusual for a normal peptide. Wait: Tollens\' positive at C-terminus could mean the C-terminus is an aldehyde (rare); more likely the test indicates a free amino group reactive with Tollens\' (which is unusual). The acid hydrolysis breaks all peptide bonds. The order of residues in the original peptide cannot be determined without sequencing techniques (Edman, MS-MS).