Protein Electrophoresis and Ionization Notes

Protein Electrophoresis and Ionization

Introduction

Proteins are essential cellular components, constituting about 50% of a cell's dry weight. Their diverse functions, including enzymatic activity, transport, and hormonal signaling, stem from their unique structures, which are determined by the sequence and types of amino acids linked by peptide bonds. With 20 different amino acids and an average protein length of around 300 amino acids, the variety in protein composition is vast. Electrophoresis exploits this diversity to separate and identify proteins based on their net electrical charge, size, and shape.

Electrophoretic Separation

Electrophoresis separates proteins by applying an electric field to a protein mixture. Proteins migrate according to their net charge: negatively charged proteins move toward the positive pole, positively charged proteins move toward the negative pole, and neutral proteins remain stationary. At physiological pH, proteins possess carboxyl groups (at the carboxy terminus) and amino groups (at the amino terminus), which can be negatively and positively charged, respectively. The R groups of amino acids further contribute to a protein's overall charge. These R groups are categorized as non-polar, polar, or ionic, with the ionic category being most relevant to electrophoresis.

Ionic R Groups

Five amino acids—aspartic acid, glutamic acid, lysine, arginine, and histidine—have ionic R groups. Aspartic acid and glutamic acid have carboxyl groups that become negatively charged at high pH as they donate a proton to hydroxide ions:

(\text{protein})-C-OH + OH^- \xrightarrow{\text{high pH}} (\text{protein})-C-O^- + H_2O

At low pH, these carboxyl groups can accept a proton and become neutral:

(\text{protein})-C-O^- + H^+ \xrightarrow{\text{low pH}} (\text{protein})-C-OH

Lysine, arginine, and histidine have amino or imino groups that can carry a positive charge. At low pH, an amino group can gain a proton:

(\text{protein})-N-H + H^+ \xrightarrow{\text{low pH}} (\text{protein})-NH^+

At high pH, a positively charged amino group can react with hydroxyl ions to become neutral:

(\text{protein})-NH^+ + OH^- \xrightarrow{\text{high pH}} (\text{protein})-N-H + H_2O

Net Charge and Isoelectric Point

The net charge of a protein varies with pH. Lower pH generally results in a more positively charged protein, while higher pH leads to a more negatively charged protein. The isoelectric point is the pH at which a protein has no net charge.

Experimental Variables

In electrophoresis experiments, pH is the independent variable systematically changed to observe its effect on protein charge. Net electrical charge is the dependent variable, determined by observing the direction of protein migration in an agarose gel under an electric field. The pH influences the unique electrical charge that each protein acquires.

Experimental Procedure

The electrophoresis procedure involves:

  1. Preparing an agarose gel at different pH levels.

  2. Loading protein samples into the gel.

  3. Applying an electric field across the gel.

  4. Analyzing the gel to determine protein migration distances.

Agarose Gel Preparation

Gels are prepared at different pH levels. Agarose is mixed with a buffer solution and heated until translucent. A small amount is poured to seal the edges of the gel tray, followed by the remaining solution. A well comb is inserted to create wells for the protein samples, and the gel is allowed to solidify.

Loading Samples

After the gel solidifies, the comb and casting tray are removed. Protein samples (15 microliters each) are added to separate wells, noting the protein and well positions using provided diagrams.

The protein solutions are:

  • Serum Albumen (blue dye added)

  • Cytochrome C (orange)

  • Myoglobin (brown)

Applying Electric Field

The electrophoresis box lid is placed, and the power supply is connected with correct polarity. The apparatus is run for 30-45 minutes to allow for adequate protein separation.

Analyzing the Gel

After the run, the power supply is turned off, and the gel tray is removed. The distance each protein migrates from the well is measured and recorded. If no movement is observed, the distance is recorded as zero.

Electrophoresis Overview

  1. Proteins are amino acid chains, varying in number and type of amino acids.

  2. Amino acids share a common structure: an amino group and a carboxyl group, with differing R groups.

  3. Aspartic acid has two carboxyl groups, while lysine has two amino groups.

  4. In a protein chain, amino acids are linked, and the net electric charge depends on the pH of the environment.

Reaction Examples

  • Aspartic acid in a high pH environment:
    CH2-C-OH + OH^- \rightarrow CH2-C-O^- + H_2O

  • Aspartic acid in a low pH environment:
    CH2-C-O^- + H^+ \rightarrow CH2-C-OH

  • Lysine in a low pH environment:
    (CH2)4-N-H + H^+ \rightarrow (CH2)4-NH^+

  • Lysine in a high pH environment:
    (CH2)4-NH^+ + OH^- \rightarrow (CH2)4-N-H + H_2O

Protein Charge and Migration

  • In an aspartic acid-rich protein at low pH, the net charge is +1 and it migrates toward the negative field.

  • In an aspartic acid-rich protein at high pH, the net charge is -3 and it migrates toward the positive field.

  • In a lysine-rich protein at low pH, the net charge is +3 and it migrates toward the negative field.

  • In a lysine-rich protein at high pH, the net charge is +4, so it will move towards the negative field.

Isoelectric Point

The isoelectric point is the environmental pH at which a protein has a net zero charge.

Electrophoresis and Ionization Questions

  1. Major property influencing migration: Net charge, determined by charged amino acid R-groups (acidic and basic).

  2. Distinction between aspartic/glutamic acid and lysine/arginine/histidine: Aspartic and glutamic acids are negatively charged (acidic), while lysine, arginine, and histidine are positively charged (basic) at physiological pH.

  3. Charge changes with pH: Acidic amino acids are neutral at low pH and negatively charged at high pH. Basic amino acids are positively charged at low pH and neutral at high pH.

  4. Different net charges: Proteins vary in acidic and basic amino acid content. Net charge changes with pH due to proton gain or loss. The isoelectric point (pI) is the pH at which a protein has no net charge.

  5. Protein movement explanation:

    • Serum albumin (pI = 4.9):

      • pH < 4.9: Positive charge, moves to the cathode.

      • pH > 4.9: Negative charge, moves to the anode.

    • Myoglobin (pI = 7.2):

      • pH < 7.2: Positive charge, moves to the cathode.

      • pH > 7.2: Negative charge, moves to the anode.

    • Cytochrome C (pI = 10.7):

      • pH < 10.7: Positive charge, moves to the cathode.

      • pH > 10.7: Negative charge, moves to the anode.

Proteins move toward the cathode at pH lower than their pI and toward the anode at pH higher than their pI.

Diversity of Life on Earth I

Introduction

Earth's diversity of life is immense, with estimates ranging from 10 to 100 million species, of which only about 1.4 million have been described. Classification involves identifying major features and relationships among organisms. A phylogenetic system is used, organized by three domains: Eubacteria, Archaea, and Eukarya.

Phylogenetic Tree

The phylogenetic tree illustrates the ancestry of organisms, starting from a single ancestral population that split into various groups over time. Each split is defined by new characteristics or synapomorphies. For example, Fungi and Amoebozoa share a common ancestor, with chitin being a synapomorphy for fungi and blunt pseudopods for Amoebozoa. A nucleus is a synapomorphy for all Eukarya.

Classification Hierarchy

Organisms are classified into a hierarchy, with each species belonging to multiple groups

Purpose

The purpose is to learn the names, appearances, and properties of 14 groups of organisms representing prokaryotes, protists, and fungi. This involves grouping similar organisms and differentiating diverse organisms, using the provided