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Organic Chemistry
Table of Contents
Unit 26 Overview: Biomolecules: Amino Acids & Proteins
26.1 Structures of Amino Acids
26.2 Amino Acids and the Henderson–Hasselbalch Equation: Isoelectric Points
26.3 Synthesis of Amino Acids
26.4 Peptides and Proteins
26.5 Amino Acid Analysis of Peptides
26.6 Peptide Sequencing: The Edman Degradation
26.7 Peptide Synthesis
26.8 Automated Peptide Synthesis: The Merrifield Solid-Phase Method
26.9 Protein Structure
26.10 Enzymes and Coenzymes
26.11 How Do Enzymes Work? Citrate Synthase

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26.2 Amino Acids and the Henderson–Hasselbalch Equation: Isoelectric Points

Citation:

Amino acids are the building blocks of proteins, each with unique properties that affect their behavior in different environments. Their isoelectric point (pI) is the pH at which they have no net charge, crucial for understanding protein interactions and separation techniques.

Electrophoresis uses these charge differences to separate proteins in an electric field. By manipulating pH and creating gradients, we can exploit the unique pI of each protein to achieve precise separation, essential for protein analysis and purification in biochemistry and molecular biology.

Amino Acids and Isoelectric Points

Isoelectric point calculation

  • Isoelectric point (pI) is pH where amino acid has net charge of zero occurs when positive and negative charges are balanced
  • Calculate pI by averaging pKa values of amino and carboxyl groups ($pKa_{COOH}$ and $pKa_{NH3+}$)
    • Non-ionizable side chain amino acids (Ala, Val, Leu, Ile, Met, Phe, Trp, Pro): $pI = \frac{pKa_{COOH} + pKa_{NH3+}}{2}$
    • Ionizable side chain amino acids include side chain pKa in calculation
      • Acidic side chains (Asp, Glu) have lower pKa: $pI = \frac{pKa_{COOH} + pKa_{side chain}}{2}$
      • Basic side chains (Lys, Arg, His) have higher pKa: $pI = \frac{pKa_{side chain} + pKa_{NH3+}}{2}$
  • Example: Alanine (Ala) has $pKa_{COOH} = 2.34$ and $pKa_{NH3+} = 9.69$, so $pI_{Ala} = \frac{2.34 + 9.69}{2} = 6.01$

Amino acid forms at different pH

  • Henderson-Hasselbalch equation relates pH, pKa, and ratio of protonated (HA) to deprotonated (A^-) acid forms: $pH = pKa + log\frac{[A^-]}{[HA]}$
    • pKa is the negative logarithm of the acid dissociation constant
  • pH < pKa: protonated form (HA) predominates more H+ available to protonate the acid
  • pH > pKa: deprotonated form (A^-) predominates more OH- available to deprotonate the acid
  • pH = pKa: equal concentrations of protonated and deprotonated forms [HA] = [A^-]
  • Determine predominant amino acid form at given pH by comparing to pKa values of amino, carboxyl, and side chain groups
    • Group with pKa closest to pH will be partially protonated and deprotonated in equilibrium
    • Groups with pKa significantly lower than pH will be fully deprotonated
    • Groups with pKa significantly higher than pH will be fully protonated
  • Example: Glutamic acid (Glu) has $pKa_{COOH} = 2.19$, $pKa_{side chain} = 4.25$, and $pKa_{NH3+} = 9.67$. At pH 7:
    • Carboxyl group (pKa 2.19) fully deprotonated (-COO^-)
    • Side chain (pKa 4.25) mostly deprotonated (-CH2-CH2-COO^-)
    • Amino group (pKa 9.67) fully protonated (-NH3+)

Amino Acid Properties

  • Amino acids are amphoteric, meaning they can act as both acids and bases
  • The net charge of an amino acid depends on the pH of its environment
  • Polarity of amino acids affects their interactions with water and other molecules
  • Ionic strength of the solution can influence amino acid behavior and interactions

Electrophoresis and Isoelectric Points

Protein separation by electrophoresis

  • Electrophoresis separates proteins based on net charge and size in an electric field
  • Protein net charge depends on buffer solution pH and protein's isoelectric point (pI)
    • pH < pI: protein has net positive charge migrates towards negative cathode
    • pH > pI: protein has net negative charge migrates towards positive anode
    • pH = pI: protein has zero net charge does not migrate
  • Separate proteins by pI using pH gradient in buffer solution
    1. Proteins migrate through pH gradient
    2. Each protein stops migrating when it reaches pH equal to its pI zero net charge
    3. Proteins with different pI values stop at different positions separated
  • Example: mixture of proteins with pI 5, 7, 9 in pH 3-10 gradient
    • pH 5 protein migrates to pH 5 zone and stops
    • pH 7 protein migrates further to pH 7 zone and stops
    • pH 9 protein migrates furthest to pH 9 zone and stops
  • Visualize separated protein bands by staining (Coomassie blue, silver stain)

Key Terms to Review (42)

Photon: A photon is a quantum of electromagnetic energy, essentially a particle of light that carries energy but has no mass. In the context of spectroscopy, photons interact with molecules to cause transitions between energy levels, which is fundamental to understanding molecular structure through techniques like infrared spectroscopy.
Conjugate acid: A conjugate acid is formed when a base gains a proton (H+ ion) during a chemical reaction. It is the species that remains after a base has accepted a proton in the context of the Brønsted–Lowry acid-base theory.
Carboxyl group: A carboxyl group is a functional group consisting of a carbon atom double-bonded to an oxygen atom and single-bonded to a hydroxyl group (-COOH). It is characteristic of carboxylic acids, giving these compounds their acidic properties.
Isoelectric point (pI): The isoelectric point (pI) is the pH at which an amino acid or a peptide has no net electric charge. At this pH, the molecule does not move in an electric field during electrophoresis.
Amphoteric: Amphoteric refers to a substance that can act as both an acid and a base, depending on the conditions. This characteristic allows these substances to neutralize both acids and bases, making them useful in various chemical processes and applications.
Conjugate Acid: A conjugate acid is the species formed when a base accepts a proton (H+) in a Brønsted-Lowry acid-base reaction. It is the acid that results when a base is protonated, and it is a weaker acid than the original acid. Conjugate acids play a crucial role in understanding acid-base chemistry, the strength of acids and bases, and their behavior in various reactions, including SN1 reactions, amine basicity, and the Henderson-Hasselbalch equation for biological amines and amino acids.
PKa: pKa, or the acid dissociation constant, is a measure of the strength of an acid in a solution. It represents the pH at which a particular acid is 50% dissociated into its conjugate base. This value is crucial in understanding the behavior and properties of acids, bases, and their reactions in organic chemistry.
PH: pH, or the potential of hydrogen, is a measure of the acidity or basicity of a solution. It is a scale that ranges from 0 to 14, with 7 being neutral, values less than 7 being acidic, and values greater than 7 being basic or alkaline. The pH of a solution is directly related to the concentration of hydrogen ions (H+) present, and it is a critical factor in many chemical and biological processes.
Acid Dissociation Constant: The acid dissociation constant, denoted as Ka, is a quantitative measure of the strength of an acid in a solution. It represents the equilibrium constant for the dissociation of an acid in water, providing insight into the extent of ionization and the relative acidity of different acids.
Isoelectric Point: The isoelectric point (pI) is the pH at which a particular molecule or surface carries no net electrical charge. This is a crucial concept in understanding the behavior of organic acids, organic bases, and amino acids.
Henderson-Hasselbalch Equation: The Henderson-Hasselbalch equation is a mathematical expression that relates the pH of a solution to the equilibrium concentrations of the conjugate acid-base pair. It is a fundamental tool used to predict and understand acid-base reactions in organic chemistry, biological systems, and various other applications.
Polarity: Polarity is a fundamental concept in chemistry that describes the unequal distribution of electrons within a molecule or between atoms. This unequal distribution creates a separation of electrical charge, resulting in a molecule having a positive and negative end, known as a dipole moment.
Alanine: Alanine is a non-essential amino acid that is commonly found in proteins. It plays a crucial role in various biochemical processes, including protein synthesis, energy production, and amino acid metabolism.
Protonation: Protonation is the process of adding a proton (H+) to a molecule or atom, resulting in the formation of a positively charged species. This fundamental chemical reaction is central to various organic chemistry topics, as it can significantly influence the reactivity and stability of molecules.
Deprotonation: Deprotonation is the process of removing a proton (H+) from a molecule or ion, resulting in the formation of a negatively charged species. This chemical reaction is central to various organic chemistry topics, as it allows for the generation of reactive intermediates and the manipulation of molecular structures.
Carboxyl Group: The carboxyl group is a functional group consisting of a carbon atom double-bonded to an oxygen atom and single-bonded to a hydroxyl group (-COOH). It is a key structural feature in various organic compounds, including carboxylic acids, amino acids, and proteins, and plays a crucial role in their chemical reactivity and properties.
Hasselbalch: Hasselbalch is a key concept in understanding the relationship between pH, the concentration of hydrogen ions, and the equilibrium of biological acids and amino acids. It is central to the Henderson-Hasselbalch equation, which is used to describe the behavior of these systems.
Henderson: Henderson is a term that is closely associated with the Henderson-Hasselbalch equation, which is a fundamental concept in understanding the behavior of biological acids and the isoelectric points of amino acids. The Henderson-Hasselbalch equation provides a way to calculate the pH of a solution based on the acid dissociation constant (Ka) and the concentrations of the acid and its conjugate base.
Glycine: Glycine is the smallest and simplest of the 20 standard amino acids that are the building blocks of proteins. It plays a crucial role in various biochemical processes, including protein synthesis, metabolism, and neurotransmission.
PI: pI, or isoelectric point, is a fundamental concept in biochemistry that describes the pH at which a molecule, such as an amino acid or a protein, has no net electric charge. At the pI, the molecule's positive and negative charges are balanced, making it electrically neutral.
Pro: The prefix 'Pro' is a versatile term that carries several important meanings in the context of amino acids and the Henderson-Hasselbalch equation related to isoelectric points. It often denotes a forward or promoting action, indicating the progression or facilitation of a process or function.
Titration: Titration is an analytical technique used to determine the concentration of an unknown solution by adding a measured volume of a standard solution of known concentration until the reaction is complete, as indicated by a change in color or pH. It is a fundamental tool in organic chemistry for analyzing the properties of amino acids.
Asp: Asp, short for aspartic acid, is a non-essential amino acid that plays a crucial role in various biochemical processes within the body, particularly in the context of amino acids and the Henderson-Hasselbalch equation, which is used to determine the isoelectric points of molecules.
Phe: Phe, or phenylalanine, is an essential amino acid that plays a crucial role in the context of amino acids and the Henderson-Hasselbalch equation, particularly in understanding the concept of isoelectric points. As one of the 20 standard amino acids found in proteins, Phe is involved in various metabolic processes and serves as a building block for many important biomolecules.
Coomassie Blue: Coomassie blue is a dye used to stain and visualize proteins in various biochemical and analytical techniques, particularly in the context of amino acid analysis and the determination of isoelectric points.
Electrophoresis: Electrophoresis is a technique used to separate and analyze charged molecules, such as proteins, nucleic acids, or amino acids, based on their differential migration in an electric field. It is a fundamental tool in biochemistry, molecular biology, and analytical chemistry.
Ionic Strength: Ionic strength is a measure of the concentration of ions in a solution, which affects the behavior and properties of charged species. It is a crucial factor in understanding the equilibrium and reactivity of ionic compounds, particularly in the context of amino acids and the Henderson-Hasselbalch equation used to determine isoelectric points.
Silver Stain: A silver stain is a laboratory technique used to visualize and detect specific biomolecules, such as proteins, in a sample. It is particularly useful in the context of amino acids and the Henderson-Hasselbalch equation, as it can help determine the isoelectric points of proteins.
Net Charge: The net charge of a molecule or ion refers to the overall electric charge that it possesses, which is the sum of the positive and negative charges within the structure. This concept is particularly important in the context of amino acids and the Henderson-Hasselbalch equation when discussing isoelectric points.
Arg: Arg, short for arginine, is a basic amino acid that plays a crucial role in various physiological processes, particularly in the context of amino acids and the Henderson-Hasselbalch equation, which is used to determine the isoelectric points of amino acids.
Amino Group: The amino group is a functional group consisting of a nitrogen atom bonded to two hydrogen atoms. It is a key structural feature of amino acids, which are the building blocks of proteins, and plays a crucial role in the chemistry and properties of these biomolecules.
His: The term 'His' refers to the imidazole side chain of the amino acid histidine, which plays a crucial role in the context of amino acids and the Henderson-Hasselbalch equation, particularly in determining the isoelectric point of a molecule.
Zwitterion: A zwitterion is a neutral molecule that contains both a positive and a negative charge within the same structure. This unique characteristic allows zwitterions to play a crucial role in the structures and properties of amino acids, which are the building blocks of proteins.
Buffer: A buffer is a solution that resists changes in pH upon the addition of small amounts of an acid or base. Buffers play a crucial role in maintaining the optimal pH environment for biological processes, such as those involving amino acids and the Henderson-Hasselbalch equation, which is used to determine isoelectric points.
Lys: Lys, short for lysine, is a proteinogenic amino acid that is essential for human health and development. It is one of the 20 standard amino acids that are the building blocks of proteins in the body. Lys plays a crucial role in the context of amino acids and the Henderson-Hasselbalch equation, particularly in understanding isoelectric points.
Val: Val, short for valine, is an essential amino acid that plays a crucial role in the structure and function of proteins. It is one of the branched-chain amino acids (BCAAs) and is involved in various metabolic processes in the body, including energy production and muscle growth.
Ile: Ile, or isoleucine, is an essential amino acid that plays a crucial role in the structure and function of proteins within the body. As a nonpolar, aliphatic amino acid, Ile is involved in various metabolic processes and is particularly important in the context of amino acids and the Henderson-Hasselbalch equation, which describes the relationship between pH and the isoelectric point of a substance.
Leu: Leu, short for leucine, is an essential amino acid that plays a crucial role in the structure and function of proteins within the body. As one of the 20 standard amino acids, Leu is a key component in the Henderson-Hasselbalch equation and the determination of isoelectric points for amino acids.
Met: Met, or methionine, is an essential amino acid that plays a crucial role in various metabolic processes within the body. As a key component of proteins, Met is involved in the synthesis and regulation of proteins, as well as in the maintenance of cellular structure and function. Additionally, Met is important for the Henderson-Hasselbalch equation and the determination of isoelectric points, which are essential concepts in understanding the behavior of amino acids in different pH environments.
Peptide: A peptide is a short chain of amino acids linked together by peptide bonds. Peptides are fundamental building blocks of proteins and play crucial roles in various biological processes, including the context of amino acids and the Henderson-Hasselbalch equation for determining isoelectric points.
Glu: Glu, short for glutamic acid, is a non-essential amino acid that plays a crucial role in the context of amino acids and the Henderson-Hasselbalch equation, which is used to determine the isoelectric point of a compound. As an amino acid, Glu is a building block of proteins and is involved in various metabolic processes within the body.
Trp: Trp, short for tryptophan, is an essential amino acid that plays a crucial role in the context of amino acids and the Henderson-Hasselbalch equation, particularly in relation to isoelectric points. As an amino acid, Trp is a building block of proteins and has unique chemical properties that influence its behavior in various biological processes.