Hemoglobin

SDS-PAGE

  • Definition: SDS polyacrylamide gel electrophoresis is a laboratory technique used to separate proteins based on their molecular weight.

  • Standard Curve: A standard curve is plotted using the degree of migration of known proteins against the log of their known molecular weights.

  • Estimation of Molecular Weight: This allows for the estimation of the molecular weight of unknown proteins through their migration patterns.

Affinity Chromatography

  • Concept: This method uses resin that has a specific ligand (affinity reagent) that interacts strongly with the target molecule.

  • Process:

    • The target molecule binds to a column resin.

    • Non-target molecules flow through without binding.

    • The target molecule is eluted using a specific solution that breaks the binding.

  • Example: Histidine-tag (H6) binds to nickel ions to retain target proteins; elution can be performed using imidazole.

Native Purification of Enzymes

  • Measurement: Purity is defined by specific activity measured as units of enzymatic activity per mg of protein (units/mg).

Purification Techniques

  • Quiz Question - Which cannot purify active proteins?

    • a) Ion exchange chromatography

    • b) Gel filtration

    • c) SDS-PAGE

Conjugated Proteins

  • Definition: Proteins with permanently attached chemical groups are termed conjugated proteins.

  • Prosthetic Groups: The non-amino acid components are called prosthetic groups; examples include the heme group in myoglobin and hemoglobin which contains iron.

Importance of Heme

  • Function: Heme is crucial for oxygen binding, important since oxygen poorly dissolves and must be sequestered due to its reactivity.

  • Iron Sequestering: Free iron can produce reactive oxygen species; heme allows iron to bind oxygen safely.

Hemoglobin Structure

  • Composition: Hemoglobin is a multisubunit protein composed of four polypeptide chains (2 α and 2 β chains) and binds four heme groups. Each chain binds one oxygen molecule.

  • Tertiary Structure: Hemoglobin's tertiary structure facilitates effective heme binding.

Heme Structure

  • Composition: Heme consists of an iron atom within a porphyrin ring, with a planar geometry.

  • Co-ordination Bonds: Iron forms six coordination bonds, four in the plane of the ring and two others, including one to a histidine side chain in hemoglobin.

Hemoglobin Function

  • Oxygen Binding: Each heme can bind one O2, thus hemoglobin can bind four O2.

  • Roles of Hemoglobin and Myoglobin: Hemoglobin transports oxygen in the blood, while myoglobin stores it in muscle tissues.

Oxygen Partial Pressure

  • O2 Levels:

    • Lungs: 15 kPa

    • Resting Muscle: 5 kPa

    • Working Muscle: 1 kPa

  • Molecule Functionality: Hemoglobin picks up O2 at high pressures (lungs) and releases it at low pressures (muscle). Myoglobin retains O2 at low pressures.

Binding Curves

  • Comparison: Hemoglobin and myoglobin binding curves illustrate their distinct oxygen binding properties.

    • Hemoglobin exhibits a sigmoidal curve due to cooperative binding, meaning the binding of one O2 makes it easier for subsequent O2s to bind.

Conformational Change of Hemoglobin

  • Mechanism: Oxygen binding changes hemoglobin from a tense (T) state to a relaxed (R) state, enhancing O2 affinity.

Transmission of Conformational Change

  • Oxygen Effect: Oxygen binding alters the porphyrin ring’s plane, affecting histidine and adjacent helix, facilitating the switch to a relaxed state.

Positive Cooperativity Theory

  • Ligand binding at one site positively influences the likelihood of binding at another, enhancing efficiency in binding O2.

Interaction with H+ and CO2

  • Mechanism: Hemoglobin binds H+ and CO2, which inversely affects O2 binding capacity. Low pH reduces O2 affinity as H+ serves as a negative allosteric effector.

H+ Influence on Binding

  • Specific Interaction: Protonation of His HC3 (H146) stabilizes the T state through ionic interactions with D94, lowering O2 binding.

2,3-BPG Interaction

  • Modulator Role: 2,3-bisphosphoglycerate (BPG) functions as a negative allosteric modulator, regulating O2 binding in red blood cells.

BPG Mechanism

  • Function: BPG binds to basic residues in the T state, maintaining that conformation, while the R form cannot accommodate BPG due to its smaller cavity.

Mutant Hemoglobin Effects

  • E6V Substitution: A single amino acid change in the β-chain results in poorly soluble hemoglobin causing red blood cell deformations but provides malaria resistance.

  • Thalassemia Types:

    • α-thalassemia: Loss of α-subunits.

    • β-thalassemia: Aggregation of α-subunits.

Binding States of Hemoglobin

  • Higher Affinity for Oxygen: The R state displays higher affinity for oxygen than the T state.

Enzymes & Enzyme Kinetics

  • Reaction Equation:

    • Sucrose + O2 -> CO2 + H2O + Energy - highly exergonic.

    • Enzymes lower activation energy to increase reaction rates.

Enzyme Mechanism

  • Catalysts: Enzymes effectively increase reaction speed by lowering activation energy, e.g., catalyzing H2O2 breakdown.

Enzyme Requirements

  • Cofactors & Coenzymes: Some enzymes require inorganic cofactors or tightly bound coenzymes (prosthetic groups), with an absence resulting in apoenzymes.

  • Characteristics: Enzymes are efficient, highly specific, and operate optimally under physiological conditions.

Enzyme Kinetics Development

  • Early Studies: Began in 1902, focusing on enzymatic reactions' initial rates as substrate concentrations increase until saturation occurs.

Vmax in Kinetics

  • Saturation Point: At high substrate concentrations, the initial reaction rate reaches a maximum (Vmax) independent of substrate concentration.

Michaelis-Menten Theory

  • Complex Formation: Michaelis and Menten established that E + S forms an enzyme-substrate complex (ES), leading to product formation.

  • Rate Constants: k1 (binding rate) is dependent on enzyme and substrate concentration, while k-1 (dissociation rate) depends solely on ES.

Saturation Explanation

  • Formation Breakdown: At high substrate concentrations, the formation and breakdown of the ES complex explains saturation in reactions.

Rate-Limiting Step

  • Catalytic Process: The breakdown of ES into products (k2) is typically the slowest, determining the rate of the overall reaction.

Michaelis-Menten Equation Derivation

  • Basic Equation: Vo = Vmax[S] / (Km + [S]), where Vo is initial velocity, Vmax is maximum rate, Km indicates substrate concentration for half-max velocity.

Understanding Km

  • Units of Km: Expressed in concentration units (M). It equals the substrate concentration when Vo is half of Vmax.