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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

Amino acid analysis is crucial for understanding peptide structure and composition. It involves preparing peptides through disulfide bond reduction and amide bond hydrolysis, then separating amino acids using chromatography techniques like ion-exchange and RP-HPLC.

Interpreting chromatograms and quantifying results reveal amino acid ratios in peptides. Advanced techniques like mass spectrometry and Edman degradation provide deeper insights into peptide sequences, while proteolytic enzymes help break down complex peptides for easier analysis.

Amino Acid Analysis of Peptides

Peptide preparation for analysis

  • Disulfide bond reduction
    • Cysteine residues form disulfide bonds stabilize peptide structure
    • Reduce disulfide bonds to free thiols using reducing agents (dithiothreitol or 2-mercaptoethanol)
    • Alkylate free thiols prevent re-formation of disulfide bonds using alkylating agents (iodoacetamide)
  • Amide bond hydrolysis
    • Peptide bonds (amide bonds) link amino acids in peptide
    • Hydrolyze peptide bonds using strong acids (6M HCl) at high temperatures (110-165°C)
    • Complete hydrolysis yields individual amino acids for analysis
    • Partial hydrolysis may occur for certain amino acids (serine, threonine, tyrosine) due to sensitivity to acid hydrolysis conditions

Chromatography in amino acid separation

  • Ion-exchange chromatography
    • Separates amino acids based on net charge at given pH
    • Uses cation-exchange resins (sulfonated polystyrene) for separation
    • Elutes amino acids using buffers with increasing ionic strength or pH gradient
  • Reverse-phase high-performance liquid chromatography (RP-HPLC)
    • Separates amino acids based on hydrophobicity
    • Derivatizes amino acids with chromophore or fluorophore for detection
    • Elutes amino acids using gradient of increasing organic solvent (acetonitrile) in mobile phase
  • Post-column derivatization
    • Eluted amino acids react with derivatizing agent (ninhydrin) to enhance detection
    • Forms colored or fluorescent compounds for quantification

Interpretation of analyzer results

  • Chromatogram interpretation
    • Each peak represents individual amino acid
    • Compare retention time of each peak to known amino acid standards for identification
    • Peak area or height proportional to quantity of each amino acid
  • Quantification methods
    • External standard method: compares peak areas to calibration curve constructed using known concentrations of amino acid standards
    • Internal standard method: adds known amount of non-naturally occurring amino acid (norleucine) to sample before hydrolysis for normalization
  • Determining molar ratios
    • Calculate relative amounts of each amino acid in peptide
    • Divide quantified amount of each amino acid by amount of least abundant amino acid
    • Round molar ratios to nearest integer determine stoichiometry of amino acids in peptide
    • Provides information about amino acid composition of the peptide

Advanced Peptide Analysis Techniques

  • Mass spectrometry
    • Determines the mass-to-charge ratio of peptide fragments
    • Useful for identifying the primary structure of peptides
  • Edman degradation
    • Sequential removal and identification of N-terminal amino acids
    • Helps in determining the peptide sequence
  • Proteolytic enzymes
    • Used to cleave peptides at specific sites
    • Aids in peptide sequencing by creating smaller, more manageable fragments

Key Terms to Review (37)

Elution: Elution in the context of organic chemistry and specifically in analyzing amino acids in peptides, is the process of extracting one material from another by washing with a solvent. It is a critical step in separating and identifying the components of peptides during amino acid analysis.
Peptide bond: A peptide bond is a covalent chemical bond formed between two amino acid molecules when the carboxyl group of one molecule reacts with the amino group of another molecule, releasing a molecule of water (a condensation reaction). This bond is integral in forming the primary structure of peptides and proteins.
Edman degradation: Edman Degradation is a method used in organic chemistry for sequencing amino acids in a peptide by selectively and sequentially removing one residue at a time from the amino end of the peptide. This process utilizes phenyl isothiocyanate in an alkaline environment to label and cleave the terminal amino acid, which can then be identified.
Primary structure: The primary structure of a protein is the specific sequence of amino acids in a polypeptide chain, determined by covalent peptide bonds. It dictates the protein's overall shape and function.
Mass spectrometry (MS): Mass spectrometry is an analytical technique used in organic chemistry to determine the mass-to-charge ratio of ions. It helps identify the composition of a sample by generating ions and measuring their mass and charge.
Hydrolysis: Hydrolysis is a chemical reaction in which a compound is cleaved into smaller molecules by the addition of water. This process involves the breaking of chemical bonds through the insertion of water molecules, often resulting in the formation of new functional groups or the decomposition of larger molecules.
Serine: Serine is a non-essential amino acid that plays a crucial role in various biological processes, including protein synthesis, cell signaling, and metabolism. It is a key component in the understanding of chirality, amino acid structures, peptide analysis, and phospholipid composition.
Acetonitrile: Acetonitrile, also known as methyl cyanide, is a colorless, volatile, and flammable organic compound with the chemical formula CH3CN. It is widely used as a solvent in various chemical reactions and analyses, particularly in the context of organic chemistry and biochemistry.
Mass Spectrometry: Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of ions to identify and quantify the chemical composition of a sample. It provides detailed information about the molecular structure and fragmentation patterns of compounds, making it a powerful tool in organic chemistry and various other fields.
Chromophore: A chromophore is a functional group or conjugated system within a molecule that is responsible for the molecule's color. It is the part of a molecule that absorbs specific wavelengths of light, leading to the observed color of the molecule.
Peptide Bond: A peptide bond is a covalent chemical bond formed between the carboxyl group of one amino acid and the amino group of another amino acid, resulting in the creation of a peptide chain. This bond is crucial in the formation and structure of proteins, which are essential macromolecules for life.
Internal Standard Method: The internal standard method is an analytical technique used to quantify the concentration of a specific analyte in a sample. It involves adding a known amount of a reference compound, called an internal standard, to the sample and comparing the response of the analyte to the response of the internal standard to determine the analyte's concentration.
Threonine: Threonine is an essential amino acid that plays a crucial role in protein synthesis and various metabolic processes within the body. It is one of the 20 standard amino acids found in proteins and is particularly important in the context of understanding the structures of amino acids and analyzing peptides through amino acid analysis.
Chromatogram: A chromatogram is a graphical representation of the separation and detection of the components of a mixture as they elute from a chromatographic column or plate. It is a fundamental tool used in analytical chemistry, particularly in the analysis of amino acids in peptides.
Proteolytic Enzymes: Proteolytic enzymes are a class of enzymes that catalyze the breakdown of proteins into smaller peptides or amino acids. They play a crucial role in various biological processes, including digestion, protein turnover, and regulation of cellular functions.
Norleucine: Norleucine is a non-proteinogenic amino acid that is structurally similar to the essential amino acid leucine. It is commonly used as an internal standard in amino acid analysis of peptides and proteins due to its unique properties and behavior during chromatographic separation.
Amino Acid Composition: Amino acid composition refers to the specific arrangement and relative amounts of the different amino acids that make up a peptide or protein. It is a crucial aspect of understanding the structure and function of these biomolecules.
Amide Bond: An amide bond is a covalent chemical bond formed between the carbonyl carbon of one molecule and the nitrogen atom of another molecule. This type of bond is crucial in the structure and function of peptides and proteins, as it serves as the linkage between individual amino acid units.
Molar Ratios: Molar ratios are the relative amounts of substances involved in a chemical reaction, expressed in terms of moles. They describe the proportions of reactants and products in a balanced chemical equation and are used to predict the quantities of substances produced or consumed during a reaction.
Dithiothreitol: Dithiothreitol (DTT) is a reducing agent commonly used in biochemical applications to maintain proteins in their reduced state by preventing the formation of disulfide bonds. It is particularly relevant in the context of peptides and proteins, as well as amino acid analysis of peptides.
Ion-Exchange Chromatography: Ion-exchange chromatography is a separation technique that relies on the reversible interaction between charged analytes and oppositely charged functional groups immobilized on a stationary phase. This method is particularly useful for the analysis and purification of charged biomolecules, such as amino acids and peptides.
Fluorophore: A fluorophore is a molecule or a part of a molecule that can absorb light energy and re-emit that energy as light of a different wavelength. Fluorophores are an essential component in various analytical techniques, including amino acid analysis of peptides, where they are used to detect and quantify the presence of specific amino acids.
Retention Time: Retention time is a fundamental concept in chromatography, which is a technique used to separate and analyze complex mixtures of chemical compounds. It refers to the time it takes for a specific compound to travel through a chromatographic column and reach the detector, providing valuable information about the identity and behavior of the analytes in the sample.
External Standard Method: The external standard method is an analytical technique used in quantitative analysis to determine the concentration of an unknown analyte in a sample by comparing its response to that of a known standard solution. This method is commonly employed in various analytical techniques, including amino acid analysis of peptides.
Disulfide Bond: A disulfide bond is a covalent chemical bond formed between two sulfur atoms, typically found in the side chains of cysteine residues in proteins. These bonds play a crucial role in the stabilization of protein structure and function.
Primary Structure: The primary structure of a protein refers to the linear sequence of amino acids that make up the polypeptide chain. It is the most fundamental level of protein structure and forms the foundation for the higher-order structures that give proteins their unique functional properties.
2-mercaptoethanol: 2-mercaptoethanol is a small organic compound containing a sulfhydryl (thiol) group. It is commonly used in the context of amino acid analysis of peptides as a reducing agent to break disulfide bonds and maintain proteins in a reduced state.
Iodoacetamide: Iodoacetamide is a chemical reagent commonly used in the analysis of peptides and proteins. It is a reactive compound that selectively modifies the thiol (sulfhydryl) groups of cysteine residues, which are important structural and functional components of many proteins.
Cation-Exchange Resins: Cation-exchange resins are solid, insoluble materials that can selectively remove positively charged ions (cations) from a solution through an ion-exchange process. These resins are commonly used in various applications, including amino acid analysis of peptides.
Reverse-Phase High-Performance Liquid Chromatography: Reverse-phase high-performance liquid chromatography (RP-HPLC) is a widely used analytical technique for the separation, identification, and quantification of various chemical compounds, including peptides and amino acids. It involves the use of a non-polar stationary phase and a polar mobile phase to separate analytes based on their hydrophobicity.
Cysteine: Cysteine is a sulfur-containing amino acid that plays a crucial role in the structure and function of proteins. It is one of the 20 standard amino acids found in proteins and is known for its unique chemical properties that contribute to various aspects of biochemistry covered in this chapter.
RP-HPLC: RP-HPLC, or Reverse Phase High-Performance Liquid Chromatography, is a widely used analytical technique in the field of amino acid analysis of peptides. It is a separation method that utilizes a non-polar stationary phase and a polar mobile phase to effectively separate and identify the individual amino acids present in a peptide sample.
Post-column Derivatization: Post-column derivatization is a technique used in analytical chemistry, particularly in the analysis of amino acids, where a chemical reaction is performed on the sample after it has been separated by a chromatographic method, such as high-performance liquid chromatography (HPLC). This process helps to enhance the detection and quantification of the analytes of interest.
Tyrosine: Tyrosine is a non-essential amino acid that plays a crucial role in the structure and function of proteins. It is one of the twenty standard amino acids found in proteins and is involved in various metabolic processes within the body.
Ninhydrin: Ninhydrin is a chemical reagent commonly used in the analysis of amino acids and peptides. It is a versatile tool that plays a crucial role in the identification and quantification of these biomolecules, particularly in the context of amino acid analysis of peptides.
Edman Degradation: Edman degradation is a method used to determine the amino acid sequence of a peptide or protein by sequentially removing and identifying the N-terminal amino acid residue. It is a crucial technique in the field of protein analysis and peptide sequencing.
Peptide sequencing: Peptide sequencing is the process of determining the precise order of amino acids in a peptide chain. This technique is crucial for understanding protein structure and function, as the sequence of amino acids dictates how a protein folds and operates. Knowledge of peptide sequences can also assist in identifying proteins, studying enzymatic activity, and developing therapeutic drugs.