Enzyme Activity and GST Importance

Sep 11, 2024

Determination of Enzyme Specific Activity

Objectives

  • Determine GST enzyme activity.
  • Determine protein concentration accurately.
  • Determine specific activity of GST enzyme.

Fundamentals

  • GST enzyme activity in protein mixtures depends on protein amount.
  • Protein content in mixtures is crucial.

Monomers and Polymers

  • Monomer: Small molecule (e.g., amino acids).
  • Polymer: Long chain of repeated monomers (e.g., proteins).

Amino Acids

  • Building blocks of proteins.
  • 20 different kinds, differentiated by R chain.
  • Can form linear chains (e.g., dipeptides).

Quantifying Proteins

  • Methods:
    • Volumetric
    • Gasometry
    • Spectrophotometry
      • UV Light: For proteins with aromatic groups.
      • Visible Light: Biuret, Lowry methods.

Biuret Method

  • Measures protein-peptide complex with
  • Advantages: Simple, fast.
  • Disadvantages: Less sensitive, color instability.

Importance of Measuring Enzyme Activity

  • Critical for understanding biological processes.
  • GST enzymes participate in detoxification processes.

Experiment Overview

  1. Preparation of Cytosolic Fraction

    • Prepare GST enzyme from mice.
    • Centrifuge to separate enzyme.
    • Store cytosolic fraction.

  2. Determination of GST Activity

    • Use CDNB method.
    • Measure absorbance at 340 nm.

  3. BSA Standard Curve

    • Create standard concentrations (100-700 µg/mL).
    • Use for determining GST concentration.

  4. Protein Concentration Determination

    • Mix cytosol fraction, buffer, biuret reagent.
    • Measure at 550 nm.

  5. Enzyme Specific Activity Calculation

    • Calculate specific activity (units/mg protein).

Enzyme Activity and Pharmaceutical Relevance

  • GST enzymes are involved in detoxification and drug metabolism.
  • Potential targets for enhancing chemotherapy efficacy by inhibiting GST.

Conclusion

  • Understanding GST activity helps in drug development and toxicity studies.

These notes summarize the lecture on determining enzyme specific activity, emphasizing the importance of GST in detoxification and pharmaceutical applications. The process involves preparation, activity measurement, and analysis to understand enzyme functions.