Enzyme Assays Flashcards
Why are enzyme assays so important?
- Use in biotechnology.
- Discovery requires reliable functional testing as well as high throughput testing - Clinical applications.
- Diagnostics of disease progression, research of the effectiveness of enzyme inhibitors, using enzymes as drugs
Define an assay
An investigative procedure for qualitative assessment or quantitatively measuring the Prescence, amount or functionality of a target entity
Enzymes can be both the analyte and the analytical tool, define both.
- Analyte - the entity you are interested in
- Analytical tool - the tool you use to assess something else
What do you measure in an enzyme assay?
In all enzyme assays you measure a change in the level of either the reactant/substrate (decrease) or the reaction product (increase)
Is an enzyme assay more sensitive measuring product or substrate?
All else being equal the enzyme assay would be more sensitive measuring the product as you start with nothing compared to a substrate way you may start with a lot therefore the relative error is small.
Describe UV/VIS optical method
Typically the more amendable to direct continuous assays that can be performed fast and at a high throughput
Describe the principle of fluorescence in enzyme assays
Excitation occurs in a single direction. Emission occurs in all directions and is typically measured under an angle different from the incoming light.
Describe chromatography in terms of enzyme assays.
Use when various compounds in the reaction mixture. The products are separated. This takes time and therefore these assays are always discontinuous
Describe manometry in terms of enzyme assays
Change in pressure as the result of the formation/depletion of gaseous compounds
Describe calorimetric assay
Measurement of changes in release/consumption of energy/temperature. Near universal method and always ‘direct’.
Describe radiochemical assay
- Radioactively labelled substrate is used to follow the enzymatic reaction
- The reaction occurs over a defined period and quenched using a reagent.
- The substrate is then separated using electrophoresis and the radioactive fraction of the substrate is used to estimate the activity of the enzyme.
What is a continuous assay?
- Signal you are measuring is detected in real time
-the signal change is directly proportional to the activity you are interested in - Can be both direct or indirect
What is a discontinuous assay?
- Reaction is stopped to allow for follow up procedure for detection
What is a direct assay?
When the formation immediately gives a detectable signal
What are the 2 types of indirect assay?
- The product itself has no detectable signal but its effect on the physiochemical characteristics of the reaction mixture can be monitored by another compound.
- Detection of the product via a follow-up reaction, which are sometimes enzyme catalyzed as well.
Describe the sulphatase activity assay
- Direct Continuous
- Product formation is detected via UV/VIS spectroscopy
- Product is visible under the reaction circumstances, no follow up reaction or change in reaction condition needed
Describe D-analyl-D alanine dipeptidase assay
- Indirect Continuous
- Detection via UV/VIS spectroscopy
- Reaction product is not easily detectable and spontaneously decomposes. Instead thiophenol is detected by reaction with Ellmans reagent that is fast and irreversible under the same reaction conditions
Describe Alanine Aminotransferase assay
- Indirect assay
- ALT converts to L-alanine to pyruvate. The latter can be detected in two different ways
Describe Epoxide Hydralase assay
- Direct discontinuous
- Extraction of the reaction mixture with diethyl ether removes both substrate and product from the aqueous phase
- Diethyl ether with extracted compounds is injected into a GC column for separation of substrate and product
agar plate assay - Production of an acid can be detected by…
A halo
- A pH indicator is included in the groth medium
- Local accumulation of protons causes the pH indicator to turn yellow
In Agar plate assays - polymer degradation can be detected by…
appearance of a halo.
- The insoluble polymer is degraded to its soluble smaller monomers ad oligomonomers, causing the direct surrounding of the bacteria producing the enzyme to become transparent
Agar plate assay - describe hydrolysis of chromogenic x-sulfate
Production of a blue precipitating dye as a result of hydrolysis of chromogenic x-sulfate that is included in the medium.
Doesnt form a halo because it is insoluble
What is an advantage of using a chromogenic substrate
An advantage of using a chromogenic substrate over one the relies on the formation of a halo is that you can fit more colonies on a single plate without increasing the chance of a false positive
Describe BVMOs
Selectively catalyze the introduction of an oxygen atom next to a keto- group to generate a carboxyl ester using O2 and NADPH. Regeneration of expensive cofactor also done enzymatically
Describe how enzymes can be used as drugs.
Enzyme replacement therapy in treatment of genetic diseases. Typically used for treatment rather than curative.
Breakdown, clean up, removal of harmful substances
What is Fabrys disease and how are enzymes used?
- Genetic disorder causing deficiency in a-galactosidase A
- Cause build up of globtriaosylceramide
- Enzyme replacement therapy - IV administrations of a-galactosidase A
What is CSID and how are enzymes used
- Congenital Sucrose-Isomaltase Deficiency - genetic disorder causing deficiency in sucrase
- Treatment - Sacrosidase taken orally
- Enzyme enable sucrose hydrolysis, allowing a normal diet
What is cystic fibrosis and how are enzymes used?
- Genetic disease causing extreme mucus build up in the lungs
- Dornase a reduces viscosity by hydrolysing extracellular DNA relieving some of the worst symptoms
Give some reasons why enzymes could be raised in plasma
- Cell damage
- Increased cell turnover
- Proliferation of cells
- Increased enzyme synthesis
- Decreased clearance
Why are isoenzymes useful?
Particular isoforms are particular to certain organs.
Determine levels of the different isoenzymes to determine which organ is effected
How are isoenzymes different from eachother
Isoenzymes catalyse the same reaction, however…
- different primary structure
- Different physical and chemical properties
- Differentiated - e.g. cofactor use
How would you separate different LDH isoenzymes to determine organ damage
electrophoresis
What are the 3 creatine kinase isoenzymes and where are they found
- CK1 - abundant in brain and smooth muscle
- CK2 - abundant in cardiac muscle and some skeletal muscle
- CK3 - abundant in skeletal and cardiac muscle and is approx 100% of serum CK
Why is direct preferred over indirect in high throughput scenarios
Minimisation of number of liquid handling steps
What detection method would be preferred in a high throughput scenario?
Fast and quantitative methods such as optical methods over chromatography as chromatography required separation which take more time and cost more
Describe the 4 steps of high throughput for sulfatase activity…. Model system to illustrate trade-off between accuracy and throughput
- Fluorescent Activated Droplet Sorting (FADS)
- Agar plate screening - qualitative
- Microtitreplate screening - complicated but quantitative
- Whole cell MM kinetics
What are the benefits of enzyme assay automation?
- Reproducibility
- Reduction in running cost - continuous operation - parallel runs - reduced manual labor
- reliable results
Describe the process of continuous flow analysis
There is one tube and you add the sample containing the enzyme and gravity mixes the sample due to the coiled affect in the tube. You leave the reaction to take place. The contents travels through one spec, where a reading is taken, then it travels through an incubator for a set amount of time at a set temperature and then travel through a second spec where a second reading is taken and these readings are then plotted on a graph
Is continuous flow cost effective?
Only when you are continuously analyzing the enzymes as it cant just be switched off
Benefits of automated analysers
- Ideal for kinetic studies
- Computer controlled
- Fast
What are the benefits of microfluidic chips?
- Effectively miniaturized versions of continuous flow analysers
- Save on reagent costs - can be significant
- Amount of patient sample is greatly reduced
- Settings can be changed
