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Enzyme Assays + Automation - Coggle Diagram
Enzyme Assays + Automation
Why perform enzyme assays?
Clinical applications
What goes wrong in disease
Staging of disease
Biomarkers
Drug targets
Enzymes as drug molecules
Biotechnology tool kits
Tools to synthesise other molecules
Analytical tools to measure other molecules
Considerations for assays
pH
Optimal temp
Ionic strength
Optimal reactions states
Substrate conc.
Coenzymes/cofactors
Free from inhibitors
Detection techniques
Photospectrometry
Calorimetry
Spectrofluorimetry
Detection is measured by attaching a moiety that fluoresces at defined wavelengths
Accurate measurements when fluorescent samples are at low concentrations
Common fluorophore is fluorescin
Used when products formed are non-fluorescing
Manometry
One component is in gaseous form
Samples + reagents in different compartments
Mixed at set time periods
End point + kinetic assays can be performed
e.g. oxygen consumption in measured in glucose oxidase
e.g. CO\(_2\) production in carboxylases
Electrochemical methods
Potentiometric techniques
Electrical potential gradient is generated dependent on the concentration or property of the substance in solution that is udergoing electrochemical reaction
Polarography/voltammetry
Fixed voltage applied between 2 electrodes
Potential difference generated can be measured
Composition of test solution determines the current which flows at each instance
Enthalmipetry
Measures enthalpy change during the course of a reaction
Very senstive
Easikly adaptable
Measures changes in heat
Freedom from interference
Accurate thermostatting + excellent insulation are required
Radiochemical
Radiolabelled substrate follows the enzymatic reaction
Highly sensitive where picomolar conc. can be measured
Enzymatic assay is perfomred for a time period + quenched with using a reagent
Substrate is sepaarted using chromatography or electrophoresis + radiactive fraction of product is used to estimate the activity of the enzyme
Solid phase/dry reagent
Immobilise components of assay on a solid phase
Methods of immobilisation
Polystyrene
Physical adsorption
VdW's
Hydrogen bonding
Hydrophobic interaction
Covalent binding
Acute pancreatitis
Fluorescent enzyme assay
In healthy individuals, fluorescence does not occur
TPE used to diagnose
Bioluminescence
Andibody-dependent cell-mediated cytotoxicity (ADCC)
Complement action releases endogenous GAPDH
ATP fomation measured with luciferin + luciferase mix
Light signal produced