Enzymes Flashcards
(46 cards)
How can the activity of an enzyme be monitored?
(2)
By following the concentration of substrate or product change with time
Following the progress of the reaction the substrate will decrease and the product will build up
What do we often use to measure enzymes?
Artificial substrates
Why do we use artificial substrates
(2)
As enzymes work so fast we use artificial substrates
This slows the reaction down so we can easily measure the rate
Why do we use artificial substrates
(2)
As enzymes work so fast we use artificial substrates
This slows the reaction down so we can easily measure the rate
Comment on the initiation of an enzyme reaction
(3)
The reaction may not start immediately when the reagents are mixed
There may be a lag time required to permit formation of a measurable product
Typically, the most accurate value is obtained when the reaction just gets going i.e. initial velocity
What are we measuring in a kinetic enzyme assay?
We are measuring the change of absorbance over time or the rate at which the absorbance changes
It is critical to measure the activity of the enzyme when the reaction rate depends only on the amount of enzyme present (first order of kinetics)
This is the linear phase of the progress curve
What is the first order kinetics
It is critical to measure the activity of the enzyme when the reaction rate depends only on the amount of enzyme present
Define velocity
Reaction progress per unit time
What are the three different types of progress curves?
A = rate is constant
B = rapidly becomes non-linear
C = there is a bigger lag phase
What are coupled enzyme assays?
(2)
Reactions linked together such that a second reaction generates a compound which can be measured spectrophotometrically
The indicator reaction needs to be directly proportional to the rate of reaction (product formation) in the primary reaction
Give an example of a coupled enzyme assay
Measurement of AST
What are the two reactions in a coupled enzyme assay
Primary assay reaction
Indicator reaction
Give some examples of enzymes used
Urease
Uricase
Glucose Oxidase
Perioxidase
Hexokinase
Cholesterol Oxidase
Lipase
Alkaline phosphatase
Reverse transcriptase
Write about measuring enzyme substrates
(3)
Can also exploit the specificity of an enzyme to measure the concentration of a substance
Typically the enzymes substrate
e.g. a common method to measure glucose based on glucose oxidase - peroxidase (GOD-PAP)
Write about the GOD-PAP method
(6)
An end point method/equilibrium method
Using a coupled system to produce a colour
The glucose oxidase gives the specificity
The peroxidase generates the colour
A trapping reaction is necessary to ensure that the enzyme will quantitatively convert substrate to product
This traps the product and makes the reaction go to completion
Give three examples of indicator reactions in coupled assays
The production of H2O2 by an oxidase is followed by production of the red compound quinoneimine (cholesterol, TAGs, glucose)
The consumption of NADH is followed by a decrease in absorbanace at 340nm (ALT, AST)
Release of the yellow coloured nitroanilide (GGT, ALP)
What are the five advantages of enzyme assays?
Specificity -> contrast with chemical methods
Sensitivity -> nmol and pmol amounts easy to do
Versatility -> using coupled systems we can assay virtually anything
Reproductibility -> as long as the same amount of enzyme is added to the tube the reaction rate will be the same
Ease of use -> enzymes like mild conditions: most chemical methods need harsh ones
Write about immobilised enzymes
Enzymes can also be immobilised onto surfaces
Allow for the measurement of the enzymes substrate (specific analytes)
What are some clinical applications of immobilised enzymes?
Immobilised enzyme: used for the detection of abnormal substances in urine
e.g. Paper coated with GOD-PAP for glucose in urine
What 7 factors affect enzyme reactions?
Enzyme concentration
Substrate concentration
pH
Temperature
Inhibitors and activators
Coenzymes and prosthetic groups
Timing
Write about enzyme assay standardisation
(3)
One analyte may have a large number of different methods or different assay conditions used to measure it
This makes it difficult to compare results from different labs
This also makes it more difficult for clinicians to interpret results
Why is standardisation of assay conditions critical?
(4)
Comparable results
Common units
Common reference ranges
Indication of trueness (accuracy)
How is enzyme assay standardisation done?
(4)
Enzyme preparation is generated with known catalytic activity
Used as a standard (reduces variability)
Leaves a path of ‘traceability’ patient results can be traced back to the original standard
Requires co-operation between international bodies, manufacturers, labs
How do manufacturers standardise assay conditions?
By optimising:
-pH
- Temperature
- Substrate conditions
- Method parameters (kinetic versus endpoint)
- Units
