Class Notes: Thermodynamics, Hydrophobic Effect, and Protein Purification

Chapter 5: Protein Purification: Column Chromatography

Protein Building Block – Amino Acid

Amino acids at pH 7: They exist as zwitterions, with a net charge of zero.
- At a pH below the pKa, the amino acid is protonated.
- At a pH above the pKa, the amino acid is deprotonated.
- Net charge zero

Amino Acids are Charged

Proteins can be separated and purified based on:
- Size
- Charge
- Binding properties
- Protein solubility (depends on pH, salt concentration, and temperature)

Column Chromatography

Mobile phase:
- A mixture of substances (e.g., protein sample) dissolved in a liquid (usually a buffer).
Stationary phase:
- A column with a porous solid matrix (usually a carbohydrate polymer (e.g., derivatives of cellulose, agarose, or dextran) or synthetic substance (e.g., polyacrylamide or silica).
First step:
- Buffered solution (mobile phase) migrates through the porous solid material (solid phase).
Second step:
- Buffered solution containing the protein to purify migrates through the solid phase.
- The substance/protein interacts with the solid phase with a migration rate that depends on its properties.

1) Ion-Exchange Chromatography

Separates based on the sign and magnitude of the net electric charge of the substance.
pH and concentration of free salt ions are used to alter binding affinity to the column.
Uses bound charged groups:
- Cation exchangers: Bind cations (e.g., Carboxymethyl sephadex).
- Anion exchangers: Bind anions (e.g., Diethylaminoethyl cellulose).
Proteins can bind to both anion and cation exchangers since they have both negative and positive charges.
Affinity to the column depends on the salt concentration and pH, which affects the net charge of the protein.
Steps:
- Apply sample to column.
- Wash column with buffer.
- Collect fractions of things coming off column.
- Elute tight binders by adding (gradually in some cases) eluant (a buffer) with higher salt or different pH to reduce affinity to the column.
- Check for elution (e.g., absorbance or some other assay).

2) Hydrophobic Interaction Chromatography

Purifies nonpolar molecules (nonpolar column).
High salt concentration promotes interaction of nonpolar groups (e.g., from protein) with hydrophobic groups from the column.
The hydrophobic effect is enhanced by increasing ionic strength.
Salt in the buffer reduces the solvation of sample molecules, exposing their hydrophobic regions.
Elute the protein of interest usually with a low salt concentration buffer or by changing the pH.

3) Size-Exclusion Chromatography

Also called gel filtration chromatography.
Separates based on size and shape.
Stationary phase gel beads have pores.
Large proteins emerge from the column before small proteins do because small proteins pass through the pores.

4) Affinity Chromatography

Separates based on binding affinity (noncovalent interactions).
Eluted by high salt concentration, high ligand concentration, or different pH.
Utilizes unique biochemical properties of specific proteins.
Example:
- Nickel column
- His-tagged Protein
- Elute with high concentration of imidazole (looks like Histidine!).

Fast-Protein Liquid Chromatography (FPLC)

Uses high-pressure pumps to move proteins down the column.
Greatly improves resolution.
Can be used for ion exchange (IEX), size-exclusion (SEC), and/or affinity chromatography.
To be used in lab later this semester.

Question 1

Which protein would elute first from a gel filtration column?
- B. protein B, with $M_r = 58,400$
Size-exclusion chromatography, also called gel filtration, separates proteins according to size. Large proteins emerge from the column sooner than small ones do.

Question 2

A new protein resembling myosin was reported. Unlike myosin, it binds calcium. Its isoelectric point and molecular weight are very similar to those of myosin. Which method would BEST separate the new protein from myosin if those two proteins were in the same buffer solution?
- C. affinity chromatography
Attaching calcium to the beads in the column would create an affinity matrix that could help purify the protein. Proteins that do not bind to calcium would flow more rapidly through the column than the new protein, which does bind calcium.

Ionization of Amino Acids

At acidic pH, the carboxyl group is protonated and the amino acid is in the cationic form.
At neutral pH, the carboxyl group is deprotonated, but the amino group is protonated. The net charge is zero; such ions are called Zwitterions.
At basic pH, the amino group is neutral (NH2), and the amino acid is in the anionic form.

Nonionic and Zwitterionic Forms of Amino Acids

The nonionic form does not occur in significant amounts in aqueous solutions.
The Zwitterion predominates at neutral pH.
A Zwitterion can act as either an acid (proton donor) or a base (proton acceptor).

Cation → Zwitterion → Anion

Amino acids with uncharged side chains, such as glycine, have two pKa values:
- The pKa of the α-carboxyl group is 2.34.
- The pKa of the α-amino group is 9.60.
The amino acid can act as a buffer in two pH regions.
Zwitterions predominate at pH values between the pKa values of the amino and carboxyl groups.
For amino acids without ionizable side chains, the Isoelectric Point (pI) is:
- pI = (pK1 + pK2)/(2)
At this pH, the net charge is zero, as a result:
- The amino acid is least soluble in water.
- The amino acid does not migrate in an electric field.
Amino acids have a net charge of zero at a specific pH called the Isoelectric Point (pI).

Salting Out: Proteins are Least Soluble at Their Isoelectric Point

Salting out:
- Adding high salt concentration to decrease protein solubility (e.g., using sulfate salts (NH4)2SO4.
Salts compete for solvent interactions, so the solvent can’t dissolve the protein anymore.

Fractionation by Salting Out (pH = pI)

Adjust salt concentration to just below the precipitation point → eliminate many unwanted proteins.
Remove precipitated proteins by filtration or by centrifugation.
Salt out or precipitate the desired protein (adjust pH to pI value).

Chapter 1: The Hydrophobic Effect

Water as Solvent

$H_2O$ dissolves salts and charged or polar biomolecules by screening electrostatic interactions.
The increase in entropy of the system is largely responsible for the ease of dissolving salts in water.

Water Interacts Electrostatically with Charged or Polar Solutes

Examples of Polar, Nonpolar, and Amphipathic Biomolecules (Shown as Ionic Forms at pH 7)
- Polar: Glucose, Glycine, Aspartate, Lactate, Glycerol
- Nonpolar: Typical wax (hydrocarbons)
- Amphipathic: Phenylalanine, Phosphatidylcholine

Transfer of Hydrocarbons to Nonpolar Solvents is Entropically Driven

$\Delta G = \Delta H - T\Delta S$
Table of Thermodynamic Changes for Transferring Hydrocarbons from Water to Nonpolar Solvents at 25°C (showing changes in enthalpy, entropy, and free energy).

Nonpolar Compounds Force Energetically Unfavorable Changes in the Structure of Water

Nonpolar compounds interfere with the hydrogen bonding among $H_2O$ molecules – increases enthalpy ( $\Delta H$ ) and decreases entropy ( $\Delta S$ ).
The free-energy change ( $\Delta G = \Delta H − T\Delta S$ ) for dissolving a nonpolar solute in water is unfavorable:
- $\Delta H$ has a positive value.
- $\Delta S$ has a negative value.
- $\Delta G$ has a positive value.

Ordering of Water Molecules around Nonpolar Solutes

$H_2O$ molecules form a highly ordered, cage-like shell around each solute molecule.
- This maximizes solvent-solvent hydrogen bonding.
- Minimize contact with $H_2O$ by aggregating together
- less ordered $H_2O$ around nonpolar molecules by aggregation spontaneous

Nonpolar Gases Are Poorly Soluble in Water

Biologically important gases CO2, O2, N2 are nonpolar.
Their movement into aqueous solution decreases entropy by constraining their motion.

Question About Sugar Dissolving

Dissolving table sugar into iced tea is an energetically favorable reaction due to a(n):
- C. increase in entropy as the sugar dissolves.
As table sugar dissolves, individual sugar molecules acquire greater freedom of motion. This increase in entropy is why dissolving table sugar into iced tea is an energetically favorable reaction. In thermodynamic terms, formation of the solution occurs with a favorable free-energy change: $\Delta G = \Delta H − T\Delta S$ , where $\Delta H$ has a small positive value and T $\Delta S$ a large positive value.

Amphipathic Compounds in Aqueous Solutions

Polar, hydrophilic region interacts favorably with $H_2O$ and tends to dissolve.
Nonpolar, hydrophobic region tends to avoid contact with $H_2O$ and cluster together.

The Hydrophobic Effect

The hydrophobic effect refers to the association or folding of nonpolar molecules in an aqueous solution.
- Nonpolar regions cluster together.
- Polar regions arrange to maximize interactions with each other and with the solvent.
It is one of the main factors behind:
- Protein folding
- Protein-protein association
- Formation of lipid micelles and membrane bilayers
- Binding of steroid hormones to their receptors

Amphiphiles Form Micelles & Bilayers

Micelle: Hydrocarbon "tails" cluster inward, while polar "head" groups face outward toward the water.
Bilayer: Two layers of amphiphiles align with tails inward and polar heads facing outward.

Question About the Hydrophobic Effect

The hydrophobic effect:
- C. is driven by an increase in the entropy of water molecules.
When amphipathic compounds are mixed with water, the nonpolar regions of the molecules cluster together to present the smallest hydrophobic area to the aqueous solvent. The smaller the hydrophobic surface area, the fewer water molecules are required in the shell of ordered water. The entropy gained by freeing immobilized water molecules drives the hydrophobic effect.

Release of Ordered Water Favors Formation of an Enzyme-Substrate Complex

Ordered water interacting with the substrate and enzyme is displaced by enzyme-substrate interaction, leading to disordered water.
The enzyme-substrate interaction is stabilized by hydrogen bonding, ionic interactions, and the hydrophobic effect.