Biochemistry Notes: Amino Acids, Proteins, and Separation Techniques (BIOC*2580)

Biochemistry Notes: Amino Acids, Proteins, and Separation Techniques (BIOC*2580)

• Source material is a slide deck for Introduction to Biochemistry (Lectures 1-4, Fall 2025).

• Purpose: comprehensive study notes with key concepts, definitions, examples, and equations (LaTeX).

• Use these notes to replace or review the provided transcript content.

Macromolecules in Biochemistry

• Biochemistry studies molecules that build life: small molecules and macromolecules.

• Macromolecules include proteins, polysaccharides , nucleic acids; they are built from smaller building blocks.

• Small molecules vs macromolecules distinction:

  • Small molecules: simple, low molecular weight components.

  • Macromolecules: large, polymeric assemblies (e.g., proteins, nucleic acids).

Protein Building Blocks: Amino Acids

• Proteins are polymers of amino acids linked by peptide bonds (a type of amide bond).

• Each protein has:

  • A unique sequence of amino acids (primary structure).

  • A well-defined size and three-dimensional structure (secondary, tertiary, quaternary levels).

  • Diverse functions: catalysis (enzymes), structural roles, subcellular organization, signaling, etc.

• Amino acids share a common backbone but differ in the side chain (R group).

Protein Size and Mass Units

• Most proteins: about

  • $10{,}000 ext{ to } 100{,}000 ext{ g mol}^{-1}$

• Protein size is expressed in Daltons (Da) or kilodaltons (kDa):

  • $1 ext{ Da} = 1 rac{ ext{g}}{ ext{mol}}$

  • $1 ext{ kDa} = 1000 ext{ g mol}^{-1}$

• Example proteins:

  • Myoglobin: approximately $16.5 ext{ kDa}$ (a small protein).

  • P-glycoprotein: approximately $170 ext{ kDa}$ (a large protein).

Building Block Principle of Macromolecular Structure

• Macromolecules are built from repeating, simple bonds between building blocks.

• Understanding: building blocks (monomers) + their linkages explain macromolecule properties.

• For proteins, the building blocks are amino acids connected by peptide (amide) bonds.

Amino Acids: Structure and Nomenclature

• Each amino acid has:

  • An amino group (–NH2 or –NH3+ at physiological pH).

  • A carboxylate group (–COO− or –COOH at different pH).

  • A unique side chain (R group) that defines identity and properties.

  • A central alpha carbon (Cα) bearing the amino and carboxylate groups and the side chain.

• Generic amino acid formula: $ext{H}_2 ext{N}- ext{CH}( ext{R})- ext{COOH}$ (at low pH; zwitterion form at neutral pH).

Peptide Bonds and Protein Chains

• Peptide bond formation (condensation/dehydration) involves removal of water when linking amino acids.

  • Condensation: removal of H₂O from linking units.

  • Hydrolysis: reformation of carboxylate and amino groups by addition of water.

• The C=O of the amide bond is a site of weakness where water attack can occur.

• Large numbers of amino acids linked form a peptide chain; side chains (R groups) confer properties.

• Terms:

  • Polypeptide: a long chain of amino acids (often a full protein).

  • Oligopeptide: a short chain (a fragment).

• Myoglobin example: 153 amino acids → ~16.5 kDa polypeptide.

Terminology: Residues, Polypeptides, and Oligopeptides

• Amino acids in proteins are linked via peptide bonds; water is released during bond formation.

• The amino acid components that remain in a protein chain after bond formation are called amino acid “residues.”

• Mass relationships:

  • Average molecular weight of amino acids in proteins ≈ $128$ Da.

  • Water molecule removed per peptide bond = $18$ Da.

  • Therefore, average amino acid residue mass in a protein ≈ $128 - 18 = 110$ Da.

• Protein length estimation: number of residues ≈ protein mass / 110.

Backbone and Side-Chain Nomenclature in Amino Acids

• Backbone atoms are identified with Greek lettering; the central backbone atom is the α-carbon (Cα).

• The first atom of the side chain is the β-carbon (Cβ); the second is the γ-carbon (Cγ); etc.

• Functional groups may be attached to different core atoms (examples include α-amino, α-carboxylate, and side-chain functions such as ε-amino in Lysine).

The 20 Natural Amino Acids and Grouping by Side-Chain Properties

• The 20 standard amino acids can be grouped by side-chain properties:

  • Non-polar (very hydrophobic): 6 residues (examples include Ala, Val, Leu, Ile, Met, Phe).

  • Moderately non-polar: Gly, Cys, Pro, Tyr, Trp (with varying contributions to hydrophobicity).

  • Polar uncharged: Ser, Thr, Asn, Gln.

  • Positively charged (basic): Histidine, Lysine, Arginine.

  • Negatively charged (acidic): Aspartate, Glutamate.

• Each amino acid is denoted by a 1-letter code (e.g., A for Alanine, I for Isoleucine) and a 3-letter code (e.g., Ala, Ile).

• Notable notes:

  • Methionine (Met, M) initiates translation in protein synthesis.

  • Glycine (Gly, G) is the smallest amino acid with no side-chain stereochemistry.

  • Cysteine (Cys, C) can form disulfide bridges.

  • Tyrosine (Tyr, Y) and Tryptophan (Trp, W) contain aromatic rings; Tyr has a polar –OH group.

  • Proline (Pro, P) has a cyclic side chain that can constrain backbone conformation.

Non-Polar Side Chains: Very Non-Polar Grouping (examples and notes)

• Very non-polar side chains are dominated by hydrocarbon content (C–C and C–H).

• Common examples: Ala (A), Val (V), Leu (L), Ile (I), Met (M), Phe (F).

• Hydrocarbon side chains are non-polar and hydrophobic.

Polar and Non-Polar Properties; Electronegativities

• Polarity arises from differences in electronegativity (O > N > S > C ≈ H).

• Non-polar bonds occur when atoms share electrons equally (e.g., C–C, C–H).

• Polar bonds arise when electrons are unequally shared, creating partial charges (δ− and δ+).

• Hydrogen bonds form between a donor (–OH, –NH) and an acceptor (O, N with lone pairs).

• Hydrogen bond strength is about 5–10% of a covalent bond, providing directional interactions.

• Hydrogen bonds contribute to secondary structure and protein folding.

Polar Uncharged Side Chains and Hydrogen Bonding

• Serine (Ser, S) and Threonine (Thr, T) have –OH groups capable of hydrogen bonding and phosphorylation.

• Asparagine (Asn, N) and Glutamine (Gln, Q) contain amide groups that serve as hydrogen bond donors and acceptors.

• These four side chains act as good hydrogen bond donors and acceptors.

Hydrogen Bonding in Proteins

• Hydrogen bonds are electrostatic attractions between a hydrogen atom bonded to an electronegative atom (donor) and an electronegative atom with a lone pair (acceptor).

• Donor examples: –O–H, –N–H.

• Acceptors: electronegative atoms with lone pairs (O, N).

• Directionality: aligned donor-acceptor pairs strengthen H-bonds.

Charged Side Chains: Positive and Negative at Physiological pH

• Positively charged side chains (basic) at neutral pH: Lysine (Lys, K), Arginine (Arg, R), Histidine (His, H).

  • These residues gain protons (protonated form) and become positively charged.

  • Observed in proteomics and protein purification as basic groups.

• Negatively charged side chains (acidic) at neutral pH: Aspartate (Asp, D), Glutamate (Glu, E).

  • Side chains deprotonate to form carboxylate groups (–COO−).

• Note: protonation states determine charge and influence protein solubility and interactions.

Ionizable Groups and pKa Values (Overview)

• Ionizable groups include:

  • α-Amino group and α-Carboxylate group of the backbone.

  • Side chains of certain amino acids (Asp, Glu, His, Cys, Tyr, Lys, Arg) can ionize.

• General idea: Each ionizable group has a characteristic pKa, which depends on the chemical context (peptide vs free amino acid).

• Physiological pH is approximately 7.0–7.4.

• The pH-pKa relationship is governed by Henderson–Hasselbalch equation:

  • $ext{pH} = ext{p}K_a + \log\left(\frac{[ ext{A}^-]}{[ ext{HA}]}\right)$

• The ionization state (protonated vs deprotonated) determines the overall charge of the amino acid or residue.

Ionization States at Neutral pH and pKa Context

• At pH 7, the α-carboxylate group typically exists as COO− (deprotonated) with pKa ≈ 2.4–2.5 in free amino acids; in polypeptides, this can shift slightly.

• At pH 7, the α-amino group is typically NH3+ (protonated) with pKa ≈ 9.5 in free amino acids; in polypeptides, shifts occur.

• Many side chains have pKa values in the 3–13 range (depending on residue):

  • Aspartate (Asp) pKa ≈ 4.0

  • Glutamate (Glu) pKa ≈ 5.0

  • Histidine (His) pKa ≈ 6.5

  • Cysteine (Cys) pKa ≈ 8.5

  • Tyrosine (Tyr) pKa ≈ 10.0

  • Lysine (Lys) pKa ≈ 10.2

  • Arginine (Arg) pKa ≈ 12.5

• Amide groups in backbone do not ionize like side chains; their charge state is not typically considered ionizable at physiological pH.

• Terminal groups:

  • N-terminus (α-amino) pKa ≈ 9.5 (free amino acid) or ~9.0–9.6 in peptides.

  • C-terminus (α-carboxylate) pKa ≈ 2.4–2.5 (free) or ~2.5 in peptides.

• The ionization state of a group at a given pH depends on its pKa value and environment.

Assessing Ionization State: Practical Rules of Thumb

• If pH is one unit or more below the pKa, the group is fully protonated (HA).

• If pH is one unit or more above the pKa, the group is fully deprotonated (A−).

• If pH equals pKa, the group is 50% protonated and 50% deprotonated.

• If pH is within one unit of pKa, exact state requires calculation (ratio of deprotonated to protonated).

• We distinguish between groups that ionize on O or S atoms (neutral when protonated and negative when deprotonated) and groups that ionize on N (positive when protonated and neutral when deprotonated).

• Important note: there is no group that goes from positive to negative upon deprotonation.

Calculating Partial Charges for Ionizable Groups

• Three-step method to find partial charges for ionizable groups:
  1) Determine ratio of deprotonated to protonated species from the Henderson–Hasselbalch equation.
  2) Compute the fractions of protonated and deprotonated forms.
  3) Compute the overall (average) charge by weighting each fraction by its respective charge.

• Example: Histidine at pH 7.0 with pKa ≈ 6.5 for the side chain:

  • Ratio: $rac{[ ext{Dep}]}{[ ext{Pro}]} = 10^{ ext{pH} - ext{p}K_a} = 10^{7.0 - 6.5} = 3.2$

  • Solve for fractions: α = fraction deprotonated; (1 − α) = fraction protonated; α/(1−α) = 3.2; α ≈ 0.76; (1−α) ≈ 0.24

  • Partial charge calculation: charge = (fraction protonated × +1) + (fraction deprotonated × 0) = 0.24

  • Therefore, at pH 7, histidine side chain contributes approximately +0.24 charge on average per molecule.

• The same approach applies to other ionizable groups (Asp, Glu, Cys, Tyr, Lys, Arg, etc.). For groups with multiple ionizable sites, perform the calculation for each site and sum contributions.

• Practical takeaway: at pH 7, many side chains exist as mixtures of protonation states; the average charge is a weighted sum of fractions.

A Dipolar Ion (Zwitterion) at Neutral pH

• In physiological pH, most amino acids exist as zwitterions: the amino group is positively charged and the carboxylate is negatively charged, resulting in a net dipolar molecule.

• Zwitterion state is characteristic of amino acids in solution near neutral pH.

Amino Acid Analysis and Separation Techniques

• Analyzing amino acids helps determine protein structure and composition.

Analysis processes:

• Separation of a mixture into components.

• Detection or identification of the components.

• Types of analysis:

  • Qualitative (what is present).

  • Quantitative (how much is present).

  • Preparative (recover components for further experiments).

Chromatography for Amino Acids

• Partition chromatography is used to separate amino acids based on polarity.

• Principles:

  • Stationary phase: solid beads with functional groups (e.g., silica gel with –OH groups that can hydrogen-bond to polar amino acids).

  • Mobile phase: liquid solvent/buffer that flows past stationary phase; generally non-polar relative to the amino acids.

  • Polar amino acids spend more time hydrogen-bonded to the stationary phase and move more slowly; non-polar amino acids spend more time in the mobile phase and move faster.

• Elution volume (Ve) measurements identify amino acids: higher Ve often corresponds to more polar amino acids.

• Detection: elution volumes are correlated with amino acid identity; concentration is measured in test tubes, often plotted vs. elution volume to identify components.

• In TLC/TLC-like formats: RF values are used to describe relative mobility on a plate.

Elution Volume, RF, and Identification in Chromatography

• Elution Volume (Ve): the buffer volume required to move a given amino acid from the top of the column to the bottom.

• Relative mobility (RF) in TLC-type formats: RF = (distance moved by compound) / (distance moved by solvent front).

• Polar amino acids typically have low RF (move slowly) in silica-based systems; very non-polar amino acids have high RF (move with solvent).

Detecting Amino Acids in Chromatography

• Amino acids are colorless and present in very low quantities; detection methods include:

  • Ninhydrin: reacts with primary/secondary amines to give purple color (color intensity proportional to quantity).

  • Proline gives a yellow color with ninhydrin.

  • Fluorescamine: yields yellow fluorescence under UV.

• These detection methods enable qualitative and quantitative analysis of amino acids.

Types of Chromatography for Protein/Peptide Separation

• Ion-exchange chromatography separates based on charge differences using charged resins:

  • Cation exchange: resins with negative groups bind positively charged molecules (cations).

  • Anion exchange: resins with positive groups bind negatively charged molecules (anions).

  • Elution strategies:

  • Compete with high salt (NaCl) to displace bound analyte.

  • Change pH to alter charge so the molecule no longer binds.

• The net charge and elution behavior depend on the pH and the pKa values of all ionizable groups.

Protein Charge and Ionization at pH 2.5 (Example of Ion Exchange)

• At pH 2.5, α-amino groups exist as NH3+ and α-carboxylate groups exist as 50% COO− and 50% COOH, giving a net positive charge for amino acids with side chains that can contribute to charge.

• Side chains can contribute to the overall net charge.

• The exact net charge depends on the specific amino acid and its pKa values.

• The size of the net charge determines how tightly an amino acid binds to ion-exchange resins. Higher charge → stronger binding.

• Elution can be achieved by increasing Na+ concentration or adjusting pH to reduce binding.

Affinity Chromatography and Protein Tags

• Affinity chromatography uses a ligand covalently attached to beads that specifically binds the target protein.

• Proteins without affinity pass through; bound proteins are eluted by high salt or by competing ligand.

• Genetic tagging: fusion of a tag (peptide or protein) to the target protein enables purification by affinity chromatography.

• Common tags and ligands include:

  • His-tag (polyhistidine) with Ni2+ affinity.

  • GST tag with glutathione.

  • MBP tag with maltose.

  • FLAG, Chitin-binding domain, Protein A, etc.

• Tags can be cleaved after purification if needed.

Metal Affinity Chromatography (His-tag)

• His residues bind tightly to Ni2+ or Co2+ on a chelating resin.

• A target protein with a His-tag is captured on Ni2+ resin.

• Elution achieved by introducing imidazole (or other competing chelators) to out-compete His-tag binding.

• Pros: high purification with a single step; cons: the tag may affect protein properties; tags can be removed after purification.

Size-Exclusion (Gel Filtration) Chromatography

• Separation based on size (molecular weight): larger proteins elute first; smaller proteins enter pores and elute later.

• Beads are a polymeric gel with water-filled pores; larger molecules are excluded from entering the pores.

• Elution volume (Ve) is measured; Ve is linearly related to log(molar mass):

  • ext{Ve} = m \, ext{log}(M_r) + b \, , \ m < 0

• Procedure to determine unknown molar mass: compare Ve to standards of known mass, then read off log mass and compute M_r (antilog).

Electrophoresis of Proteins (SDS-PAGE)

• Proteins are separated by movement in an electric field; primarily used for analysis, not purification (can alter structure).

• SDS-PAGE specifics:

  • Proteins are treated with SDS, which denatures and coats them with a uniform negative charge proportional to length.

  • Separation is based mainly on size (molar mass) rather than charge or shape.

  • Molecular weight estimation is done by comparing to standards.

• Gel type: polyacrylamide gels (typical 5–15% acrylamide).

• Visualization: Coomassie blue stain binds to proteins; more staining indicates higher protein content.

Isoelectric Focusing and 2D Gels

• Isoelectric focusing (IEF) separates proteins by isoelectric point (pI): the pH at which net charge is zero.

• In IEF, proteins migrate in a pH gradient until they reach the pH where their net charge is zero, then stop.

• Two-dimensional (2D) gels combine IEF with SDS-PAGE to separate complex mixtures into individual proteins:

  • First dimension: separation by pI (IEF).

  • Second dimension: separation by size (SDS-PAGE).

Mass Spectrometry for Protein Identification

• Mass spectrometry identifies proteins by measuring the mass of ionized protein fragments.

• Process:

  • Protein is ionized and fragmented; ions travel to detector; velocity inversely related to mass.

  • The mass-to-charge ratio (m/z) is measured to determine precise mass.

  • Measured mass is compared to a database of known proteins to identify the sample.

Protein Purification: Enzyme Activity and Specific Activity

• Purification often targets enzymes (proteins that catalyze reactions).

• After each purification step, monitor enzyme activity to assess purification progress:

  • Enzyme Activity (per time) = μmol of substrate converted to product per minute (μmol/min) at 25°C under assay conditions.

  • Definition: Enzyme Activity = rate of substrate turnover by the enzyme in solution.

• Specific Activity: enzyme activity per milligram of total protein:

  • $ext{Specific Activity} = rac{ ext{Enzyme Activity}}{ ext{Total Protein}} ext{ (μmol min}^{-1} ext{ mg}^{-1})$

• Purification goal: increase Specific Activity toward a maximum corresponding to pure enzyme.

• Example: If a pure enzyme has a specific activity of 10 μmol min−1 mg−1 and a sample has 2 μmol min−1 mg−1, the latter is 20% pure.

Purification Metrics During Stepwise Purification

• After each purification step:

  • Enzyme Activity may drop due to losses during purification.

  • Total Protein decreases as contaminants are removed.

  • Specific Activity increases if the target enzyme is enriched relative to contaminants.

• When a single protein species is obtained (e.g., by SDS-PAGE), purification has achieved homogeneity.

Quick Reference: Key Equations and Concepts

• Protein mass units:

  • $1 ext{ Da} = 1 rac{g}{mol}$

  • $1 ext{ kDa} = 1000 rac{g}{mol}$

• Peptide bond mass accounting:

  • Residue mass ≈ $128 - 18 = 110 ext{ Da}$

• Number of residues in a protein ≈ mass / 110

• Henderson–Hasselbalch (acid–base equilibrium):

  • $ext{pH} = ext{p}K_a + \log rac{[ ext{A}^-]}{[ ext{HA}]}$

• Ion-exchange elution strategies:

  • Elute with high salt concentration (e.g., NaCl) or by changing pH to alter charge.

• Gel filtration (size-exclusion): Ve vs log(Mr) relationship:

  • ext{Ve} = m \, ext{log}(M_r) + b, ext{ with } m<0

• RF in thin-layer chromatography:

  • $RF = rac{X<em>B}{X</em>F}$

• Enzyme activity and specific activity:

  • $ext{Activity} = rac{ ext{μmol of substrate converted}}{ ext{min}}$

  • $ext{Specific Activity} = rac{ ext{Activity}}{ ext{Total Protein}}$

• Mass spectrometry: identification by matching measured mass to known protein masses in databases.

Quiz and Practice Resources (from Transcript)

• Q1: Organic chemistry primer (functional groups) – quiz link provided in slide.

• Q2–Q9: Online quizzes and worked solutions referenced throughout the deck (various topics: amino acid identification, single-letter codes, charge calculations, purification techniques).

• Use Q2–Q9 links to test knowledge and review calculations (not included here).

Additional Notes and Common Points to Remember

• The 20 standard amino acids: memorize 1-letter and 3-letter codes and general properties by group (non-polar, polar, charged).

• pKa context matters: amino and carboxylate groups shift in peptides vs free amino acids; environment influences ionization.

• The concept of zwitterions at physiological pH is central to understanding protein solubility and interactions.

• Purification strategies often combine multiple separation methods; success is judged by enzyme activity and specific activity.

• Mass spectrometry and two-dimensional gel electrophoresis are powerful modern tools for protein identification and separation in complex mixtures.

Closing Reminders

• Be comfortable with the core formulas and the logic behind ionization states, charge calculations, and separation techniques.

• Practice with the online quizzes to reinforce understanding of amino acids, charges, and purification metrics.

• Review the definitions of key terms: residue, polypeptide, oligopeptide, zwitterion, isoelectric point (pI), and affinity tags.