Lecture 1-4: Introduction to Amino Acids

Small Molecules and Macromolecules

• Small molecules

  • Sugars, amino acids, nucleotides, carboxylic acid derivatives

  • Act as building blocks for macromolecules

• Macromolecules

  • Proteins – chains of amino acids

  • Polysaccharides – chains of simple sugars

  • Nucleic acids – chains of nucleotides

Macromolecule Example

Myoglobin is a protein that stores O2 in muscle tissue

How large is a protein molecule?

• Most proteins: 10,000 to 100,000 g mol-1

• Protein size is expressed in kiloDaltons (kDa)

  • 1 Dalton (Da) = 1 g mol-1 (mass of H atom)

  • 1 kDa = 1000 g mol-1

• Myoglobin is 16.5 kDa – small protein

• P-glycoprotein is 170 kDa – large protein

Macromolecule Building Block Principle

What are Proteins Made Up Of?

• Linear chains of amino acids

• Linked by peptide bonds (type of amide bond)

• Each protein has:

  • Unique sequence of different amino acids

  • A well defined size and structure

• Proteins have diverse functions including:

  • Catalyzing reactions (enzymes)

  • Forming complex subcellular structures

Describe the Basic Amino Acid Structure

• Each amino acid has an amino group and a carboxylate group

• Each amino acid has a different side chain R

• 20 different amino acids are found in proteins

• Note the central alpha carbon!

What form of bond links two amino acids together?

Peptide Bonds!

• Condensation (Dehydration Synthesis): To join two amino acids, the carboxyl group (COO- /COOH) of the first amino acid reacts with the amino group (NH3+ /NH2-) of the second. A molecule of water (H2O) is removed (an -OH from the carboxyl end and an -H from the amino end). The resulting covalent linkage (CO-NH) is called a peptide bond (or an amide bond).

• Hydrolysis (Breaking the Bond): The reverse reaction breaks the peptide bond. Adding a water molecule splits the bond and regenerates the free carboxylic acid and amino groups

• NOTE: At physiological pH (~7):

  • Amino group (NH₃⁺): Protonated (gains/retains H⁺).

  • Carboxyl group (COO⁻): Deprotonated (loses H⁺).

Where is the point of weakness in the newly formed molecule?

C=O! Oxygen is significantly more electronegative than carbon:

• Oxygen pulls the shared electrons strongly toward itself, giving the oxygen atom a partial negative charge

• This drains electron density from the carbon atom, leaving it with a partial positive charge

• Because that carbon is starved for electrons, it acts as an electrophile—essentially a target waiting for any electron-rich molecule to come along

• H2O has two unshared lone pairs of electrons on its oxygen atom:

  • Because opposite charges attract, the electron-rich oxygen of water attacks the partially positive carbonyl carbon

Where does the unique combinations occur in a protein?

The "R" groups (or side chains) are precisely where all the diversity, individuality, and unique chemical combinations occur in a protein

• The Main Chain (Backbone): The repeating -N-Cα-C-(=O)- unit highlighted across the slide is identical in every amino acid. It simply acts as the structural scaffold holding the chain together.

• The Side Chains (R1, R2, R3): These are the chemical groups attached to each central alpha-carbon (Cα).

• The combination of different side chains R1, R2, R3, etc. gives each protein its unique properties.

Side Chain Structure

• Carbon atoms of the amino acid core are identified by Greek letters

• The α-carbon is the central backbone atom

• The β- carbon is the first atom of the side chain, the y-carbon is the second, etc

• Functional groups may be linked to different core atoms:

  • α-amino

  • e-amino

The 20 natural amino acids

• Amino acids share a common backbone, but differ in the side chain

• Amino acids can be grouped according to structures or by similar properties

How many “categories” of amino acids are there? How many belong in each group?

• 5 Based on Polarity Properties

  • 6 with very non-polar side chains

  • 5 with moderately non-polar side chains

  • 4 with polar but uncharged side chains

  • 3 with positively charged side chains (very polar)

  • 2 with negatively charged side chains (very polar)

What Side Chains do the very non-polar amino acids tend to have?

• The side chains are dominated by hydrocarbon, and consist only of C-C and C-H bonds

• Hydrocarbon is non-polar and hydrophobic (or water avoiding)

• Ala, Val, Leu, Ile, Met, Phe

Recap: Polar and Non-Polar Properties

• Polarity is a consequence of atoms having different electronegativity or tendency to hold bonding electrons

• Electronegativity: Electronegativity is a measure of an atom's tendency to attract shared electrons toward itself in a chemical bond

• O > N > S > C = (Roughly) H

• Atoms with similar electronegativity share bonding electrons equally, e.g. C-C, C-H, and are non-polar

• Pairs of atoms with different electronegativity distribute bonding electrons unequally – more electronegative atoms such as O or N get greater than 50% share, and this leads to unbalanced charges and polar bonds

Moderately Non-Polar Amino Acids

• Glycine has single H atom as side chain, not enough to be very non-polar

  • Hydrophobicity is related to the number of CH, CH2 or CH3 groups present

• Cysteine contains the slightly polar SH group

• Proline is unique because the side chain links to α-N as well as to α-C. The polar
  N moderates the non-polar hydrocarbon.

• Tyrosine has a single polar group that partly offsets the very non-polar benzene ring.

• Tryptophan behaves similarly.

Amino acids with polar uncharged side chains

• Ser, Thr, Asn, Gln

• Serine and threonine have side chains that include the polar hydroxyl group -OH (simple alcohol)

• Asparagine (Asn) and Glutamine (Gln) both contain the polar amide group

• These side chain groups do not gain or lose H+ in aqueous solution at pH 7, so they are uncharged

• All four side chains act as good hydrogen bond donors or acceptors

Recap: Hydrogen Bonds

• Hydrogen bonds are electrostatic attractions between a H-bond donor and an acceptor

• An acceptor = electronegative atom with an available lone pair of electrons

  • It "accepts" the partially positive hydrogen by attracting it toward its lone pair.

• A donor = hydrogen atom covalently bonded to another electronegative atom.

  • It "donates" its hydrogen's positive partial charge to the interaction because it pulls electron density away from that hydrogen.

• The hydrogen bond is about 5-10% as strong as a covalent bond, enough to make the two molecules stick loosely to each other but not to form a permanent link

• H-bonds are directional - stronger if donor and acceptor line up with one another

The Amino Acids with Positively charged side chains (very polar)

• These side chains contain weak bases that gain H+ (become protonated) and so are positively charged in aqueous solution at neutral pH

• Charge make them very polar, overriding the non-polar hydrocarbon chain

The Amino Acids with Negatively charged side chains (very polar)

• Side chains have carboxylic acid groups R-COOH that lose H+ (become deprotonated) at neutral pH

• When deprotonated these are described as carboxylate groups R- COO-

• Carboxylate groups are negative and also very polar

• Asp side chain: -CH2-COO-

• Glu side chain: -CH2-CH2-COO-

What can amino acids act as?

• As both acids and bases!

• Recall: HA → A- + H+

• The amino and carboxylate groups of amino acids and the side chains of some amino acids can act as acids and bases

• These groups will gain (protonate) or lose (deprotonate) H+ depending on availability of H+ in solution

• Normal biochemical processes occur close to pH 7 (physiological pH is 7.0-7.4)

Worked Example 1

Worked Example 2

Important! What is the correct way to represent an Amino Acid at a Neutral pH?

• The correct structure to represent an individual amino acid at neutral pH is NH3+ and COO-

• This is also known as a Zwitterion

• But when the amino acid is part of a peptide chain, the α-amino groups and α-carboxylate groups are linked as uncharged amide bonds (except for the ones at the N and C termini, which are also NH3+ and COO- at pH 7)

How many amino acids have ionizable side chains?

• Seven!

• Note: The polar but uncharged amino acids go from 0 to -1 charge

• Note: The very polar and charged amino acids go from +1 to 0 charge

What does the value of pKa tell you in regards to deprotonation?

• The value of pKa tells you where in the pH scale a group undergoes deprotonation

• A molecule can have several ionizable groups

• Each group has its own pKa value

• Value of a pKa depends on its chemical context

• An amino acid will have a slightly different pKa when it is part of a peptide chain

Example/Explanation of Deprotonation’s correlation with pH with Glutamic Acid

• Starting with glutamic acid at very low pH, all three functional groups
  are fully protonated.

• As we raise the pH, [H+] becomes less available, so deprotonation is more likely to occur.

• Each group undergoes a transition as pH shifts from 0 to 14, starting ~1 unit below its pKa and almost complete by ~1 unit above its pKa.

• When pH = pKa, 50% of the group is in the protonated form and 50% is in the deprotonated form

• Note that once a group deprotonates, it remains that way for the rest of the pH range

• REMEMBER: The ionization state of a group at a given pH depends on its pKa value

Important: How to assess the state of ionization of a functional group

• If pH is one unit or more below the pKa, the group is fully protonated

• If pH is one unit or more higher than pKa, the group is fully deprotonated

• If pH is equal to pKa, the group is 50% deprotonated and 50% protonated

• If pH is less than one unit away from pKa, a calculation may be needed to determine the exact state

Does being protonated mean a functional group is always positively charged? How does the ionizing atom determine charge?

• Charge of an ionized group depends on the functional group

• The relationship of pH and pKa tells you whether a group is protonated or deprotonated, NOT whether it is positive or negative → Make sure to look at those R groups!

• Adding H+ always shifts the charge by +1 (more positive), but the net charge depends on the atom involved

• Groups that ionize on O or S atoms are neutral when protonated, and negative when deprotonated (e.g., side chains of Asp, Glu)

• Groups that ionize on N are positive when protonated, and neutral when deprotonated (e.g., side chains of Arg, Lys, His)

• There is no group that goes from positive to negative when it becomes deprotonated!!

REVIEW SLIDES ON HOW TO DO CALCULATIONS 😃 😃😃

What are the two processes of amino acid analysis? Also what three things can it be?

• Amino acid analysis helps to determine protein structure

  • Separation of a mixture into components

  • Detection of the components of interest

• Can be qualitative (tells you what is present)

• Can be quantitative (tells you how much is present)

• Can be preparative (separated components can be recovered for further experiments)

What method is important for separating different parts of a mixture? What are the two phases involved?

• Partition Chromatography

• Particles of solid are chosen with a specific property, e.g. silica gel has HO-Si-OH groups that can hydrogen-bond to polar amino acids

  • Stationary phase

• Liquid solvent or buffer flows past the particles and is non-polar

  • Mobile phase

• Amino acids exchange (partition) between phases

• Polar amino acids P spend more of their time hydrogen bonded to silica and move slowly

• Non-polar amino acids N spend more time in solvent, and move almost as fast as solvent

The Stationary and Mobile Phase

• Stationary Phase (The "Stick-Around" Phase):

  • Solid beads packed into a vertical glass column.

  • Here, silica gel is used, which is covered in exposed hydroxyl groups (HO-SI-OH). Because of these (-OH) groups, the stationary phase is highly polar and readily forms hydrogen bonds.

• Mobile Phase (The "Moving" Phase):

  • A liquid solvent or buffer poured into the top that percolates downward through the packed beads.

  • In this setup, the solvent chosen is non-polar.

• Polar Amino Acids: They bind and unbind repeatedly, spending most of their time stuck to the stationary phase. As a result, they move down the column slowly and come out (elute) last

• Non-Polar Amino Acids: They prefer dissolving in the non-polar mobile phase. They travel along with the flowing liquid and exit the bottom first.

Based on this graph and test tubes, how are the Amino Acids Identified?

• Concentration of amino acids is measured in each test tube and the results are graphed

• To elute more polar amino acids, progressively polar mobile phase is run through the column

• Compounds can be identified by their characteristic elution volume

• N and A,B,C: Non-Polar (interact weakly with the polar silica stationary phase, so they wash through rapidly with minimal elution volume)

• P and D,E: Polar: Amino acids stick tightly to the polar stationary phase via hydrogen bonding and require larger elution volumes

What is another method to identify amino acid properties?

• Thin layer chromatography

• Silica gel is spread in a thin layer on a plastic sheet

• Samples are applied near the lower edge

• The lower edge is placed in solvent

• As solvent soaks up the sheet, different components of sample move with the solvent at different rates

• The highest point reached by solvent is the solvent front

• Each amino acid can be identified by its characteristic relative mobility RF

• Very polar amino acids have low RF, non-polar amino acids have high RF

How are amino acids detected?

• Amino acids are colourless, and samples may be 10-6 to 10-10 moles

• They can be detected by adding ninhydrin which reacts with primary and secondary amines

• Gives intense purple colour (10-8 moles detectable), or yellow colour for proline

• Spray ninhydrin onto TLC plates, or add to amino acid solution, and heat

• Colour intensity is proportional to quantity of amino acid, and can be measure

• Alternative is fluorescamine, giving yellow fluorescence under UV light (10-10 moles detectable)

Ion Exchange Chromatography

• Separates on the basis of charge

• Uses charged resins as stationary phase

• Cation exchange Chromatography- resins contain negative groups, which bind positive molecules (cations)

• Anion exchange Chromatography - resins contain positive groups, which bind negative molecules (anions)

How do you break that ionic attraction to wash them out (elute them?)

• Salt Competition (e.g., increasing (NaCl):

  • Floods the column with counterions (Na+ and Cl-) that physically outcompete the amino acids for the charged binding sites on the resin.

  • Molecules with lower net charges are displaced first at low salt, while highly charged molecules require higher salt concentrations to release.

• Altering pH (Charge Neutralization / Reversal):

  • Shifting buffer pH changes the protonation state of the amino acid's ionizable groups based on their pKa

  • This eliminates (or reverses) the molecule's net charge—e.g., raising pH deprotonates positive groups, causing them to lose electrostatic attraction and elute from a cation resin

How does low pH (e.g., pH 2.5) allow amino acids to bind to a cation exchange resin?

• Resin charge: Cation exchange resin is negatively charged.

• Amino acid charge at pH 2.5:

  • a-amino group is fully protonated as NH3+ (+1).

  • a-carboxylate group (pKa approx 2.5) is 50% COO- (-1) and 50% -COOH (0).

  • Net core charge is overall positive (+0.5), enabling electrostatic binding to the negative beads.

• Binding strength: Determined by the magnitude of the amino acid's total net positive charge (including side chains).

What are the two methods used to elute bound amino acids from a cation exchange column?

• Na+ Salt Competition (left):

  • High Na+ counterions compete for and displace amino acids from negative resin binding sites.

  • Weakly bound (lower positive charge) amino acids displace first at low Na+ tightly bound ones elute as Na+ increases.

• Increasing {pH} (right):

  • Raising pH (e.g., to 6.5) causes amino acids to lose protons (H+).

  • Neutralizes or reverses the net positive charge, eliminating attraction to the negative resin so they wash out.

Summary of Ion Exchange Slide

Where are these proteins extracted from?

• Proteins are derived from natural sources such as microbial cultures, plants, or animal tissues such as liver

• The cells are broken open to release the proteins into a solution – crude extract

• Extracts may contain thousands of different proteins

• Separation by ion exchange is based on charge differences among proteins (Net Charge)

• Depends on the relative number of Asp + Glu (negative) versus His + Lys + Arg (positive) in each protein, and on pH

• ~65% of all proteins are negatively charged at pH 7

Charge Differences Between Proteins and Amino Acids

Example Question

Affinity Chromatography

• Beads in the column – has covalently attached ligand

• Any molecule or ion that binds specifically and reversibly to a target protein or macromolecule to form a complex.

• Proteins with affinity towards ligand - bind to it

  • The ligand doesn't just slow the target protein down—it usually stops it completely (remains anchored in place at the top of the column.)

• All other unwanted proteins lack affinity for the ligand and flow straight through the column without sticking (the "flow-through").

• The target protein must be detached from the ligand so it can be collected. This is typically done by:

• Salt (weakens binding between ligand and proteins) or

• Ligand (compete with attached ligands)

Affinity Chromatography - Use of Tagged Proteins

Tag = A peptide or protein that binds a ligand that is fused to the gene encoding target protein

Ligand vs Tag Protein Table

Immobilized Metal Affinity Chromatography (IMAC)

• Principle: Target proteins are genetically engineered with a 6–8 His-tag at the N- or C-terminus, which binds tightly to chelating resin charged with {Ni}^{2+} or {Co}^{2+} ions.

• Binding/Washing: Column is made up of chelating resin containing
  Ni2+. Only the His-tagged protein coordinates with the immobilized metal ions, while non-tagged background proteins wash straight through.

• Elution: Released by adding imidazole to the buffer. Free imidazole mimics the histidine side chain and out-competes the His-tag for {Ni}^{2+} binding sites.

• Pros/Cons: High degree of purification in one step; however, the tag can affect native protein activity and may need to be enzymatically cleaved post-isolation.

Separating proteins on the basis of size

• Gel filtration or molecular exclusion chromatography allows separation of proteins on the basis of size

• Beads of polymeric gel - a loose network of polymer with many water-filled pores.

• Protein molecules can enter the pores if they fit

• Larger proteins are excluded from the pores

• Proteins of intermediate size may enter some of the pores

• Proteins separate by size; larger proteins elute first, smaller proteins elute later

Gel filtration can be used to measure the molar mass of proteins -Sample is compared to proteins of known size

• Measure elution volume of proteins of known mass

• Elution volume Ve is the volume of buffer needed to move a protein from top to bottom of column

• Elution volume is a linear function of log molar mass (negative slope)

• Then measure elution volume of unknown protein and project back to the log mass axis

• Find antilog to determine the molar mass of unknown

• Alternately, find Mr by using coordinates to derive equation for straight line y=mx+b

Proteins can be separated and characterized by electrophoresis

• Electrophoresis - movement of charged molecules in an electric field

• Does not contribute to purification as structure is commonly affected by electrophoresis

• Can visualize and characterize purified proteins

• Can be used to estimate:

• Number of different proteins in a mixture

• Degree of purity of a mixture

• Isoelectric point

• Approximate molecular weight

• Rate of movement depends on size, shape and charge