March 3

Copyright Notice

  • This lecture presentation and the accompanying PowerPoint slides are the exclusive copyright of Professor Omri.

  • Use is permitted only for students enrolled in Biochemistry I (CHMI-2227 E) in Winter term 2026 at Laurentian University.

  • Unauthorized or commercial use, including uploading to sites off Laurentian University servers, is expressly prohibited.

Proteins: Determination of Amino Acid Residues and Molecular Weight

  • The number of amino acid residues in a simple protein can be calculated as:
    extNumberofResidues=Molecular Weight (MW)110extDaltons(D)ext{Number of Residues} = \frac{\text{Molecular Weight (MW)}}{110 ext{ Daltons (D)}}

  • The average molecular weight of an amino acid in proteins is approximately 110 D.

  • Estimating the molecular weight (MW) of a protein involves multiplying the number of amino acid residues by this average MW:
    MW=Number of Residues×110extD\text{MW} = \text{Number of Residues} \times 110 ext{ D}

Conjugated Proteins and Prosthetic Groups

  • Some proteins are classified as conjugated because they are associated with non-protein components known as prosthetic groups.

  • Examples of Conjugated Proteins: ### Class of Conjugated Protein

    • Lipoproteins: Prosthetic group(s) - Lipids

    • Example: B₁-Lipoprotein of blood

    • Glycoproteins: Prosthetic group(s) - Carbohydrates

    • Example: Immunoglobulin G

    • Phosphoproteins: Prosthetic group(s) - Phosphate groups

    • Example: Casein of milk

    • Hemoproteins: Prosthetic group(s) - Heme (iron porphyrin)

    • Example: Hemoglobin

    • Flavoproteins: Prosthetic group(s) - Flavin nucleotides

    • Example: Succinate dehydrogenase

    • Metalloproteins: Prosthetic group(s) - Iron, Zinc, Calcium, Molybdenum, Copper

    • Examples: Ferritin, Alcohol dehydrogenase, Calmodulin, Dinitrogenase, Plastocyanin

Amino Acid Sequence as Genetic Information

  • The amino acid sequence of a protein is unique to each protein and encoded by the nucleotide sequence of DNA.

  • This sequence represents a form of genetic information.

  • Amino acid sequences are read directionally, from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus).

Architecture of Proteins

Protein Shapes: Globular and Fibrous

  • Globular Proteins:

    • Compact, spherical molecules

    • Typically water-soluble, dissolving in biological fluids

    • Exhibit dynamic and flexible properties; e.g., hemoglobin (Hb) enables specific molecular interactions.

  • Fibrous Proteins:

    • Insoluble in water; e.g., collagen and keratin.

    • Possess high tensile strength, contributing to their structural role.

Example of Keratin Structure

  • The horns of bighorn rams are constructed of α-keratin proteins.

Permanent Waving of Hair (α-keratin)

  • Process:

    • Permanent waving relies on reversible chemical modification of disulfide (–S–S–) bonds between cysteine residues in keratin.

    • The reducing agent (commonly ammonium thioglycolate) cleaves these bonds to form –SH groups, allowing reshaping around curlers.

    • Subsequent oxidation (typically with hydrogen peroxide or sodium bromate) reforms new –S–S– bridges, stabilizing the imposed curl configuration.

    • The hair undergoes treatment with a reducing agent to break disulfide bridges, transforming –S-S- into –SH (HS-) groups, creating curls.

    • To set the shape, an oxidizing agent is applied, converting –SH groups back into –S-S-.

Levels of Protein Structure

Primary Structure

  • Defined as the sequence of amino acid residues joined together by peptide bonds.

  • The primary structure is coded for by DNA.

    • Example Sequence: Leu-Thr-Pro-Glu-Lys-Ser

Secondary Structure

  • As the polypeptide chain undergoes folding, it adopts spatial arrangements of neighboring amino acid residues along the linear sequence.

  • Folding is facilitated by hydrogen bonds formed between carbonyl and N-H groups of the peptide backbone.

Tertiary Structure

  • Represents the overall three-dimensional shape of a protein, involving specific ways in which a protein folds.

Quaternary Structure

  • Refers to the subunit organization of a protein, characterized by proteins containing two or more polypeptide chains.

Forces Determining the Structure of Proteins

Primary Structure

  • Determined by covalent bonds (specifically peptide bonds).

Secondary, Tertiary, and Quaternary Structures

  • All determined by weak forces:

    • Weak Forces Include:

    • Hydrogen bonds

    • Van der Waals interactions

    • Hydrophobic interactions

Purification of Proteins Techniques

  • Proteins typically exist as part of complex mixtures with variable abundance. Some proteins are found in larger quantities while others exist in smaller amounts.

  • The initial step in studying the structure and/or function of a specific protein involves renal purification.

  • Protein Purification Process:

    • Sequential procedures are applied to extract the desired protein progressively from a biological sample until a pure form is obtained.

Factors Aiding Protein Purification

  • Prior Knowledge of Basic Characteristics:

    • Molecular Weight (MW)

    • Isoelectric Point (pI)

    • Solubility

  • Availability of an Assay for Protein Detection:

    • Reagents, enzymatic reactions, physiological effects, etc.

  • Such information significantly facilitates the protein purification process.

Solubility in Purification Techniques

  • Ammonium Sulfate:

    • Commonly utilized for selectively precipitating proteins

    • At low concentrations of ammonium sulfate, protein solubility generally increases slightly (salting-in), attributed to reduced electrostatic attractions between protein molecules.

    • At high concentrations of salt, protein solubility sharply decreases (salting-out), leading to complete precipitation due to diminished water availability to solubilize proteins.

  • Electrostatic Bonds:

    • Proteins can be linked by electrostatic bonds, referred to as "salt bonds".

    • Adding an optimal amount of salt at the isoelectric point (pI) can enhance protein solubility:

    • At pI, proteins carry equal + and - charges.

    • Excess salt addition can lead to proteins precipitation due to water molecules being engaged with salt ions.

Dialysis in Protein Purification

  • Dialysis:

    • Involves semi-permeable membrane bags (e.g., cellophane).

    • Membrane pores allow free passage of molecules below a certain molecular weight (MW).

    • The process is driven by the concentration difference of solutes inside versus outside the membrane.

    • Typically used to remove small molecules (salts) from a protein solution.

Chromatography Techniques in Protein Purification

Ion Exchange Chromatography

  • Allows proteins to be separated according to their charges:

    • Cation-exchange resins (negative) bind positively charged groups.

    • Anion-exchange resins (positive) bind negatively charged groups.

  • The most frequently used anion-exchanger is cellulose and agarose-based resins with attached DEAE (diethylaminoethyl) groups.

  • The most frequently used cation-exchanger is cellulose and agarose-based resin with attached CM (carboxymethyl) groups.

Separation Techniques

  • By increasing the salt concentration or changing the pH of the solvent, specific proteins can be effectively separated.

Solubility versus Salt Concentration

  • Behavior of Proteins:

    • At low salt concentrations, proteins remain soluble due to surface charges promoting electrostatic repulsion and maintaining adequate hydration.

    • As salt concentration increases, solubility decreases due to the salting-out effect:

    • Salt ions compete with proteins for water molecules.

    • Protein hydration shell is reduced.

    • Increased protein–protein interactions lead to aggregation and precipitation.