Chromatography

Non-competitive Inhibition

• Definition: A form of enzyme inhibition where the inhibitor binds to an enzyme away from the active site.

• Key Characteristics:

  • The inhibitor modifies the reaction rate.

  • The substrate still binds with the same affinity, indicating that the Michaelis constant ($K_m$) remains unaltered.

  • The capacity to catalyze the reaction is influenced, leading to a decreased maximum velocity ($V_{max}$).

• Graphical Representation:

  • The Lineweaver-Burk plot demonstrates a decrease in $V<em>{max}$, but the slope indicates that $K</em>m$ remains the same.

Protein Purification

## Learning Objectives

• By the end of this lecture series, you will be able to answer key questions:

  • Why do we study proteins?

  • How do we study proteins?

  • What are proteins?

  • How do proteins work?

Overview of Protein Study

• Interdisciplinary Nature:

  • Protein studies involve biochemistry, chemistry, biology, physics, biophysics, mathematics, and computational biology.

  • This encompasses numerous methodologies to study the properties, structures, distribution, and abundance of proteins.

Purpose of Protein Purification

• To Study Properties: Analyzing the characteristics of proteins requires purifying them from other substances.

• To Study Structure: Understanding the structural biology of proteins necessitates purification to obtain clear insights into their configurations.

• For Distribution and Abundance Analysis: Purification allows researchers to investigate the locations and quantities of proteins in biological contexts.

• Clinical and Commercial Use: Purified proteins can have applications in therapeutics and industrial processes.

Sources of Biomolecules

• Natural Sources:

  • Generally more complex, containing thousands of other proteins and biomolecules, affecting the purification process.

• Synthetic Sources:

  • Often yield incomplete products needing further purification due to similarity with target proteins.

Protein Study Techniques

Types of Protein Sources

• Native Proteins:

  • Extracted from the natural host, providing authentic biochemical characteristics but often in small quantities.

• Heterologous Expression:

  • Involves the use of bacteria, yeast, mammalian, or insect cells to express and produce larger amounts of desired proteins.

  • Note: Might not represent the true native state of proteins.

Biophysical Properties of Proteins

Characteristics of Proteins

• Physical Attributes:

  • Size

  • Mass

  • Shape/Structure, including:

    • Primary (1º) to Quaternary (4º) structures

  • Folding patterns

  • Interactions with:

    • Other proteins

    • Biopolymers

    • Small molecular ligands

    • Metal ions

    • Redox potential

  • Charge properties:

    • Isoelectric point

  • Hydrophobicity

  • Electromagnetic properties:

    • Interaction with UV/visible light

    • Fluorescence characteristics

    • Magnetic traits

Methods of Protein Separation

Utilizing Biophysical Characteristics

• Proteins can be separated based on biochemical properties through two primary methods:

  • Chromatography:

    • Origins in the Greek meaning 'colourful writing'.

    • Involves separation through differential interactions between two phases.

  • Gel Electrophoresis:

    • Derived from Greek: electro (pertaining to amber) and phoresis (meaning 'being carried').

    • This technique allows for the movement of macromolecules through an electric field.

Centrifugation in Protein Isolation

Step 1: Isolating Protein

• Centrifugation allows separation of dense particles from suspension based on density:

  • Isolation Process

    • Direct centrifugation for samples from bacteria and blood.

    • For organ/tissue, samples must be homogenized before centrifugation.

Principles of Centrifugation

• Centrifugal Force: Acts outwardly from the axis of rotation, aiding in separation.

  • Expressed as relative centrifugal force (RCF): $RCF = rac{Centrifugal ext{ force}}{g}$

    • Where $g$ is the acceleration due to gravity, commonly approximated as 10 N/kg.

  • Different samples necessitate varying RCF based on their density and size for effective separation.

Centrifugation Speeds

• Larger objects sediment more quickly at higher speeds.

  • 3,000 g for cell preparations, 20,000 g for organelle isolation, 100,000 g for protein separation.

Steps To Preparing Soluble Proteins

Preparation Methodology

• Utilizing culture flasks containing E. coli growth medium:

  • Transfer to centrifuge tubes.

  • Centrifuge to pellet cells.

  • Break open cells through homogenization or sonication to release soluble proteins.

Protein Purification Steps

Step 3: Isolation and Purification Technique

• Recognizing that centrifugation alone cannot routinely separate proteins, other techniques must be employed:

  • Characteristics for separation: size, mass, shape, interactions, charge, and hydrophobicity.

  • Primarily using various forms of chromatography.

Chromatography Details

Chromatography Principles

• The sample presents unique interactions between the protein and both mobile and stationary phases:

  • Stationary phase typically consists of polymer beads, potentially functionalized for specific interactions.

  • Mobile phase is a compatible buffer system designed for protein interaction during the separation process.

Practical Chromatography

1. Introduce protein mixture in a suitable buffer to chromatography column containing the stationary phase.

2. Mobile phase (buffer) flows through the column:

   • Proteins interact to differing extents, with some moving slower (blue proteins) while others elute quickly (orange proteins).

Detection of Eluate

• Employ UV detectors to quantify eluting proteins which will be plotted as chromatograms, measuring absorbance ($A_{280}$) for protein detection, yielding a visual representation correlating elution volume with absorbance.

Chromatography Varieties

Types of Chromatography

• Thin Layer Chromatography

• Column Chromatography

Size Exclusion Chromatography (SEC)

• Also referred to as gel filtration, separates proteins based on their hydrodynamic radius.

Ion Exchange Chromatography

• Utilizes charge differences among proteins for separation, considering overall charge, charge density, and distribution.

Hydrophobicity and Affinity Chromatography

• Exploits differences in surface hydrophobicity through modification of chromatography media with hydrophobic groups.

• Affinity: Functionalized stationary phases bind specific proteins, e.g., His-tags binding to nickel, Streptactin for biotin, etc.

Summary

• A critical overview: To explore the biochemistry and structure of proteins, efficient separation and purification must be achieved, employing methods such as centrifugation and chromatography based on intrinsic biophysical properties like charge, size, and hydrophobicity.

Protein Quantification

UV/VIS Spectroscopy

• The absorption of light at 280 nm can indicate protein presence due to the absorption characteristics of amino acids such as Tyrosine (Tyr), Tryptophan (Trp), and Disulfides.

• Beer-Lambert-Bouguer Law: $A = au Cl$

  • Where:

    • $A$ = absorbance

    • $au$ = molar absorptivity

    • $C$ = concentration

    • $l$ = light path length

• Molar absorptivity values: Tyrosine = 1490, Tryptophan = 5500, Cystine = 125.

• Extinction Coefficient Calculation:

  • $au(prot) = n<em>Y imes au</em>Y + n<em>W imes au</em>W + n<em>C imes au</em>C$

• Example provided illustrates the extinction coefficient calculation based on protein composition.

Calculation Example

• Calculating protein concentration using absorbance measurement and extinction coefficient:

  • Given: $A = 0.2$, $au(prot) = 47440$

    • From Beer-Lambert Law: $C = rac{A}{ au} = rac{0.2}{47440} <br>ightarrow C = 4.2 μM$