Biomolecules - The Isoelectric Point

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Introduction

• Biomolecules are organic molecules found in living organisms
• They play important roles in various biological processes
• The isoelectric point (pI) is a crucial concept in biomolecules

Isoelectric Point (pI)

• The isoelectric point is the pH at which a biomolecule carries no net electrical charge
• At the pI, the molecule is in its zwitterionic form
• It is the average of the pKa values of the ionizable groups

Calculation of pI

• pI can be determined using the Henderson-Hasselbalch equation
• For an amino acid, the pI is given by the average of the pKa values of its ionizable groups

Examples

• Glycine: pKa1 = 2.35, pKa2 = 9.78 (average = 6.07)
• Alanine: pKa1 = 2.35, pKa2 = 9.87 (average = 6.11)
• Serine: pKa1 = 2.21, pKa2 = 9.15, pKa3 = 10.47 (average = 7.28)

Significance of pI

• The behavior of a biomolecule depends on its charge
• At pH values below the pI, the molecule carries a net positive charge
• Above the pI, the molecule carries a net negative charge

Application in Electrophoresis

• Electrophoresis is a technique used to separate biomolecules based on their charge
• It utilizes the pI to determine the direction and speed of migration

Isoelectric Focusing

• Isoelectric focusing is a type of electrophoresis that separates biomolecules based on their pI
• A pH gradient is established, and the molecules migrate to their respective pI values

Analysis of Proteins

• Determining the pI of proteins helps in their identification and characterization
• It provides valuable information about their isoelectric behavior

Summary

• The isoelectric point is the pH at which a biomolecule carries no net electrical charge
• It is calculated using the average of the pKa values of the ionizable groups
• pI plays a significant role in electrophoresis and protein analysis

====== 11. pI Calculation of Amino Acids

• Amino acids have ionizable groups: amino (-NH2) and carboxyl (-COOH)

• For amino acids with one ionizable group, the pI is the average of its pKa and pKb values:

  pI = (pKa + pKb) / 2

• Example: Glycine (pKa = 2.35), (pKb = 9.78)

12. pI Calculation of Amino Acids (cont.)

• For amino acids with two ionizable groups, the pI is calculated using the pKa values of both groups:

  pI = (pKa1 + pKa2) / 2

• Example: Aspartic Acid (pKa1 = 2.09), (pKa2 = 3.86)

13. pI Calculation of Amino Acids (cont.)

• For amino acids with three ionizable groups, the pI is determined using the pKa values of all groups:

  pI = (pKa1 + pKa2 + pKa3) / 3

• Example: Histidine (pKa1 = 2.18), (pKa2 = 6.0), (pKa3 = 9.09)

14. pI Calculation of Peptides and Proteins

• Peptides and proteins are made up of multiple amino acids
• The pI of peptides and proteins can be calculated based on the pI values of the constituent amino acids
• Each amino acid contributes to the overall charge of the peptide/protein at different pH values

15. Application in Protein Separation

• The pI plays a crucial role in protein separation techniques like isoelectric focusing and electrophoresis
• Isoelectric focusing separates proteins based on their pI values
• Proteins migrate towards pH values in the gel that correspond to their pI

16. Determining Protein pI with Electrophoretic Mobility

• The relative electrophoretic mobility of a protein can help determine its pI
• A protein will migrate fastest in an electric field when the pH is significantly different from its pI
• By testing the protein’s mobility at different pH values, the pI can be approximated

17. Calculating pI and Protein Function

• The pI of a protein can provide insights into its function
• Proteins with a pI close to physiological pH are typically water-soluble
• Proteins with extreme pI values may have functions related to their charge, such as ion transport or binding

18. Effects of pH on Protein Structure

• Changes in pH can disrupt protein structure and function
• Extreme pH values can denature proteins, leading to loss of biological activity
• pH changes near the pI can affect protein charge and solubility

19. pI Modifications and Protein Engineering

• Modifying the pI of a protein can be useful in various applications
• Techniques like site-directed mutagenesis can introduce amino acid substitutions to alter the pI
• These modifications can be used to design proteins with specific properties or improve their stability

20. Summary and Review

• The pI is the pH at which a biomolecule carries no net charge
• It is calculated based on the pKa values of ionizable groups in amino acids, peptides, and proteins
• The pI is essential in techniques like isoelectric focusing and electrophoresis for protein separation
• Understanding the pI can provide insights into protein function and structure

Biomolecules - The Isoelectric Point

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Isoelectric Point and Amino Acids

• Amino acids have ionizable groups that determine their charge
• The isoelectric point (pI) is the pH at which an amino acid carries no net charge
• The pI value is calculated based on the pKa values of the ionizable groups

Equation 1: Calculation of pI for Amino Acids

• For amino acids with one ionizable group, the pI is the average of the pKa and pKb values:

  pI = (pKa + pKb) / 2

Equation 2: Calculation of pI for Peptides and Proteins

• For peptides and proteins, the pI can be calculated using the pI values of the constituent amino acids

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Example of pI Calculation

• Let’s calculate the pI value for aspartic acid, which has two ionizable groups (carboxyl and amino)

• The pKa1 value for the carboxyl group is 2.09

• The pKa2 value for the amino group is 9.82

• Using Equation 2, we can find the pI:

  pI = (pKa1 + pKa2) / 2 pI = (2.09 + 9.82) / 2 pI ≈ 5.955

• Therefore, the pI of aspartic acid is approximately 5.955

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Importance of pI in Protein Separation

• The isoelectric point plays a significant role in protein separation techniques such as electrophoresis and isoelectric focusing
• These techniques rely on the differential migration of proteins based on their charge at different pH values

Electrophoresis

• In electrophoresis, proteins migrate in an electric field towards the electrode of opposite charge
• Under different pH conditions, proteins can be separated based on their charge and size

Isoelectric Focusing

• Isoelectric focusing is a technique that separates proteins based on their pI values
• A pH gradient is established in a gel matrix, and proteins migrate to their respective pI values, where they become electrically neutral

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Analysis of Protein Charge

• Determining the pI of a protein is essential for understanding its charge behavior under different pH conditions

• pH < pI:

  • The protein is positively charged
  • It migrates towards the cathode in electrophoresis

• pH > pI:

  • The protein is negatively charged
  • It migrates towards the anode in electrophoresis

• pH ≈ pI:

  • The protein carries no net charge
  • It remains near the position where it was loaded in electrophoresis

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Amino Acid Examples and Their pI Values

1. Glycine:

   • pKa: 2.35 (carboxyl group), 9.78 (amino group)
   • pI = (2.35 + 9.78) / 2 = 6.065

2. Alanine:

   • pKa: 2.35 (carboxyl group), 9.87 (amino group)
   • pI = (2.35 + 9.87) / 2 = 6.11

3. Lysine:

   • pKa: 2.18 (carboxyl group), 9.06 (amino group)
   • pI ≈ 9.12

4. Glutamic Acid:

   • pKa: 2.19 (carboxyl group), 4.07 (additional carboxyl group), 9.47 (amino group)
   • pI = (2.19 + 4.07 + 9.47) / 3 = 5.91

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Effects of pH on Protein Stability

• Extreme pH values can denature proteins and affect their stability

• pH values near the pI can also influence protein solubility

• Above the pI (pH > pI):

  • Protein is negatively charged
  • Repulsion between protein molecules can lead to aggregation and loss of solubility

• Below the pI (pH < pI):

  • Protein is positively charged
  • Attraction between protein molecules can cause aggregation and precipitation

• Near the pI (pH ≈ pI):

  • The protein is least soluble and can precipitate out of solution

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Modifying Protein pI

• The pI of a protein can be modified through various techniques:

1. Chemical Modification:

   • Adding or removing functional groups to alter the ionizable residues
   • May affect protein function

2. Genetic Engineering:

   • Mutagenesis techniques to introduce amino acid substitutions
   • Modifying ionizable residues to change the pI of the protein

3. pH Adjustment:

   • Adjusting the pH of the solution to change the charge state of the protein
   • Can be used to study the effects of different charge states on protein behavior

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Summary and Review

• The isoelectric point (pI) is the pH at which an amino acid, peptide, or protein carries no net charge
• The pI can be calculated based on the pKa values of the ionizable groups
• Understanding the pI is essential in protein separation techniques like electrophoresis and isoelectric focusing
• The pI affects the charge, solubility, and stability of proteins under different pH conditions
• Modifying the pI of proteins can be achieved through chemical or genetic engineering methods and pH adjustment