Enzyme Assays

Question 1

Why are enzyme assays so important?

Answer 1

1. Use in biotechnology.
   - Discovery requires reliable functional testing as well as high throughput testing
2. Clinical applications.
   - Diagnostics of disease progression, research of the effectiveness of enzyme inhibitors, using enzymes as drugs

Question 2

Define an assay

Answer 2

An investigative procedure for qualitative assessment or quantitatively measuring the Prescence, amount or functionality of a target entity

Question 3

Enzymes can be both the analyte and the analytical tool, define both.

Answer 3

• Analyte - the entity you are interested in
• Analytical tool - the tool you use to assess something else

Question 4

What do you measure in an enzyme assay?

Answer 4

In all enzyme assays you measure a change in the level of either the reactant/substrate (decrease) or the reaction product (increase)

Question 5

Is an enzyme assay more sensitive measuring product or substrate?

Answer 5

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.

Question 6

Describe UV/VIS optical method

Answer 6

Typically the more amendable to direct continuous assays that can be performed fast and at a high throughput

Question 7

Describe the principle of fluorescence in enzyme assays

Answer 7

Excitation occurs in a single direction. Emission occurs in all directions and is typically measured under an angle different from the incoming light.

Question 8

Describe chromatography in terms of enzyme assays.

Answer 8

Use when various compounds in the reaction mixture. The products are separated. This takes time and therefore these assays are always discontinuous

Question 9

Describe manometry in terms of enzyme assays

Answer 9

Change in pressure as the result of the formation/depletion of gaseous compounds

Question 10

Describe calorimetric assay

Answer 10

Measurement of changes in release/consumption of energy/temperature. Near universal method and always ‘direct’.

Question 11

Describe radiochemical assay

Answer 11

• 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.

Question 12

What is a continuous assay?

Answer 12

• 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

Question 13

What is a discontinuous assay?

Answer 13

• Reaction is stopped to allow for follow up procedure for detection

Question 14

What is a direct assay?

Answer 14

When the formation immediately gives a detectable signal

Question 15

What are the 2 types of indirect assay?

Answer 15

1. The product itself has no detectable signal but its effect on the physiochemical characteristics of the reaction mixture can be monitored by another compound.
2. Detection of the product via a follow-up reaction, which are sometimes enzyme catalyzed as well.

Question 16

Describe the sulphatase activity assay

Answer 16

• 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

Question 17

Describe D-analyl-D alanine dipeptidase assay

Answer 17

• 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

Question 18

Describe Alanine Aminotransferase assay

Answer 18

• Indirect assay
• ALT converts to L-alanine to pyruvate. The latter can be detected in two different ways

Question 19

Describe Epoxide Hydralase assay

Answer 19

• 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

Question 20

agar plate assay - Production of an acid can be detected by…

Answer 20

A halo
- A pH indicator is included in the groth medium
- Local accumulation of protons causes the pH indicator to turn yellow

Question 21

In Agar plate assays - polymer degradation can be detected by…

Answer 21

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

Question 22

Agar plate assay - describe hydrolysis of chromogenic x-sulfate

Answer 22

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

Question 23

What is an advantage of using a chromogenic substrate

Answer 23

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

Question 24

Describe BVMOs

Answer 24

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

Question 25

Describe how enzymes can be used as drugs.

Answer 25

Enzyme replacement therapy in treatment of genetic diseases. Typically used for treatment rather than curative. Breakdown, clean up, removal of harmful substances

Question 26

What is Fabrys disease and how are enzymes used?

Answer 26

- Genetic disorder causing deficiency in a-galactosidase A - Cause build up of globtriaosylceramide - Enzyme replacement therapy - IV administrations of a-galactosidase A

Question 27

What is CSID and how are enzymes used

Answer 27

- Congenital Sucrose-Isomaltase Deficiency - genetic disorder causing deficiency in sucrase - Treatment - Sacrosidase taken orally - Enzyme enable sucrose hydrolysis, allowing a normal diet

Question 28

What is cystic fibrosis and how are enzymes used?

Answer 28

- Genetic disease causing extreme mucus build up in the lungs - Dornase a reduces viscosity by hydrolysing extracellular DNA relieving some of the worst symptoms

Question 29

Give some reasons why enzymes could be raised in plasma

Answer 29

- Cell damage - Increased cell turnover - Proliferation of cells - Increased enzyme synthesis - Decreased clearance

Question 30

Why are isoenzymes useful?

Answer 30

Particular isoforms are particular to certain organs. Determine levels of the different isoenzymes to determine which organ is effected

Question 31

How are isoenzymes different from eachother

Answer 31

Isoenzymes catalyse the same reaction, however... - different primary structure - Different physical and chemical properties - Differentiated - e.g. cofactor use

Question 32

How would you separate different LDH isoenzymes to determine organ damage

Answer 32

electrophoresis

Question 33

What are the 3 creatine kinase isoenzymes and where are they found

Answer 33

1. CK1 - abundant in brain and smooth muscle 2. CK2 - abundant in cardiac muscle and some skeletal muscle 3. CK3 - abundant in skeletal and cardiac muscle and is approx 100% of serum CK

Question 34

Why is direct preferred over indirect in high throughput scenarios

Answer 34

Minimisation of number of liquid handling steps

Question 35

What detection method would be preferred in a high throughput scenario?

Answer 35

Fast and quantitative methods such as optical methods over chromatography as chromatography required separation which take more time and cost more

Question 36

Describe the 4 steps of high throughput for sulfatase activity.... Model system to illustrate trade-off between accuracy and throughput

Answer 36

1. Fluorescent Activated Droplet Sorting (FADS) 2. Agar plate screening - qualitative 3. Microtitreplate screening - complicated but quantitative 4. Whole cell MM kinetics

Question 37

What are the benefits of enzyme assay automation?

Answer 37

- Reproducibility - Reduction in running cost - continuous operation - parallel runs - reduced manual labor - reliable results

Question 38

Describe the process of continuous flow analysis

Answer 38

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

Question 39

Is continuous flow cost effective?

Answer 39

Only when you are continuously analyzing the enzymes as it cant just be switched off

Question 39

Benefits of automated analysers

Answer 39

- Ideal for kinetic studies - Computer controlled - Fast

Question 40

What are the benefits of microfluidic chips?

Answer 40

- 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