Enzyme activity assay, applications, automation Flashcards
What are some CONDITIONS to consider when conducting an enzyme activity assay?
You have to consider the optimal pH, ionic strength, temperature, cofactors, and substrate concentration. And make sure it is free from INHIBITORS.
What detection techniques can be used for enzyme activity?
Spectrophotometry (light absorption), calorimetry (heat released/absorbed), spectrofluorimetry (formation of product), manometry (gas consumption/production), electrochemical methods (electrical potential/change in potential), enthalpimetry (energy change), radiochemical methods (picomolar concentration of reactants & products with radioisotopes), and dry-reagent methods (immobilise on solid).
How does spectrofluorimetry work to analyse enzyme activity?
It measures the PRODUCTION of the product or the reduction of REAGENT CONCENTRATIONS by attaching a fluorophore. Lipase activity is measured with fluorescein.
What does manometry do?
Manometry measures the enzyme activity when one or more COMPONENTS ARE GASEOUS. Oxygen consumption or production.
How does enthalpimetry work?
Enthalpimetry measures the enthalpy change THROUGHOUT the reaction.
What are the 2 electrochemical methods that can be used to detect enzyme activity?
Potentiometry and polarography. Potentiometry measures the electrical potential GENERATED by the enzyme undergoing an ELECTROCHEMICAL REACTION. Polarography measures the CHANGE IN POTENTIAL of the test sample when INCREASED VOLTAGE is applied.
What radioisotopes can be used in radiochemical methods for enzyme activity assay?
3H tritium, 32P, 35S, 131iodine.
What can industrial enzymes be applied in?
Industrial enzymes can be used in genetic diseases (Fabry/Gaucher/SCID/PKU), coagulation diseases, cancer, and infectious diseases.
What methods can be applied to check for enzyme purity?
Enzyme purity can be assayed with ELECTROPHORESIS (SDS-PAGE), ANTIBODY-MEDIATED identification, CHROMATOGRAPHY (ion-exchange, gel filtration, affinity)
What are examples of enzyme markers for diagnosis?
Enzyme markers include PLASMA SPECIFIC enzymes (adenylate cyclase, kinases, coagulation factors, GAPDH), SECRETED enzymes (AST/ALT), and CELLULAR enzymes (creatine kinase).
When are plasma enzymes raised in the body?
Plasma enzymes are raised when there is cell DAMAGE, increased cell TURNOVER/PROLIFERATION/enzyme SYNTHESIS, and decreased CLEARANCE.
How does enzyme measurement help diagnosis?
Enzyme measurement indicates the extent of DAMAGE, ORIGINAL enzyme LEVEL, and amount of TISSUE affected.
How can we identify where the enzyme originated from?
By measuring ORGAN-SPECIFIC enzymes or ISOenzymes. And by analysing PATTERNS OF SEVERAL ENZYMES.
What enzyme markers are elevated in myocardial infarction within 8 days?
LDH, BHBD, then CPK. CPK reachest peak earliest in myocardial infarction.
What enzyme markers are elevated in acute VIRAL hepatitis within 7 weeks?
ALT (highest), AST, ALP (constant), GGT
