Enzymes Flashcards
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
How do we standardise assays in the lab?
We ensure that:
- assay protocols are followed
- control material is assayed
- instrumentation is maintained and in proper working order
Why do we need enzyme units?
(3)
Enzymes are not always available in pure form: they are often purified from tissues
e.g. trypsin from the intestine; the greater the activity per unit of protein the more pure the enzyme preparation
As the preparation may not be 100% pure you need to know what the activity of your preparation is i.e. how active the enzyme is
What is the enzyme unit of measurement?
The katal
What is the katal?
(3)
The SI unit for catalytic activity
Based on two other SI units: the mole (amount) and the second (unit of time)
The katal is the amount of enzyme which will catalyse the transformation of one mole of substrate to product per second or moles/sec
What do we usually use instead of the katal and why?
The katal is usually very small numbers e.g. nanokatals or microkatals
Instead we use international units which are more practical
What is the international unit
The amount of enzyme which will catalyse the transformations of 1 micromole of the substrate to product per minute per litre, under standard conditions: umol/L/min
What is functional serum enzyme?
Responsible for reaction taking place in blood e.g. clotting of blood
What are non functional serum enzymes?
Do not have their function in blood but they are found in low concentrations in serum due to wear and tear of tissues and normal cell turnover
What are the three types of enzymes in serum?
Functional serum enzyme
Non functional serum enzymes
Obstruction to secretory pathway
What causes enzymes in serum due to obstruction to secretory pathway?
Reflux back into blood because of blockage
Comment on the clinical significance of enzymes
(2)
Enzymes are normally intracellular and injury or death of tissues can cause the release of tissue-specific enzymes into the bloodstream
Serum enzyme level may be increased by disease that cause increase in its rate of release or decrease in their rate of excretion
Comment on the clinical significance of enzymes
(2)
Enzymes are normally intracellular and injury or death of tissues can cause the release of tissue-specific enzymes into the bloodstream
Serum enzyme level may be increased by disease that cause increase in its rate of release or decrease in their rate of excretion
What are 9 commonly assayed enzymes
Transaminases - ALT and AST
Alkaline phosphate
Acid phosphatase
Gamma-glutamyl transferase
Amylase
Lipase
Creatinine kinase
Lactic dehydrogenase
Prostate specific antigen
What are isoenzymes?
(5)
Enzymes that occur in different molecular forms which differ in their physiochemical properties but catalyse the same chemical reaction
They vary with respect to their kinetic parameters, electrophoretic mobility and localisation
They all have independent action
They can be used to identify the specific affected tissues
They can be differentiated from each other and can be clinically quantified in the lab
What are the three main problems with measuring enzyme activity
Sometimes can be common to more than one tissue
Timing of sample collection is critical
Macroenzymes -> rarely pathological but can cause confusion
How can enzymes being more common to more than one tissue cause problems?
An increase in the activity of the enzyme could reflect damage to one of several different tissues
Two ways of overcoming this problem
- measuring test profiles (more than one analyte associated with the disease)
- measuring specific isoforms or isoenzymes
Why might timing of sample collection cause problems?
Enzymes will rise and then fall as they are cleared
Why might macroenzymes cause problems
Enzymes can form complexes causing reduced clearance
How is greater specificity is achieved in what three ways?
(3)
Interpreting investigations in the light of clinical features e.g. jaundice patient with high ALT in hepatitis and therefore not muscle injury
Combining measurement with other analytes
- ALP up in cholestasis and bone diseases
- confirmed by increased GGT in cholestasis
Isoenzyme determination
- CK up in muscle injury
- CK-MB up in cardiac muscle injury
What enzyme is defective in albinism
Tyrokinase
What enzyme is defective in Glucose 6 phosphate dehydrogenase
G6PD deficiency
What three areas can enzymes be used in therapy?
Substitution of missing production of digestive enzymes (e.g. digestive enzymes) e.g. pepsin, trypsin
Removal of deposits of death tissue or fibrin (e.g. in lungs or eyes) treatment of skin defects -> proteinases, nucleases, collagenase
Clot lysis in myocardial infarction
- streptokinase, urokinase
