Diagnostic Enzymology Flashcards
How are Enzymes used in analysis?
- As specific bio-reagents reacting with substrate/analyte and as means of producing observable signal
- As removers of interferences
- As signal amplifiers - ‘labels’ in immunoassay
How are enzymes used as specific bio-regeants and an example?
- Examples is Glucose Oxidase is very specific for glucose, but peroxidase can react with many reagents.
[glucose oxidase]
Glucose + O2 → Gluconolactone + H2O2
[peroxidase]
H2O2 + o-toluidine → oxidized o-toluidine (coloured) + H2O
What is an example of enzymes as specific bioreagents in a reversal arrange?
- A converse arrangement can also provide specificity.
- A lack of specificity in the primary enzyme (hexokinase) can be compensated by a high specificity in secondary enzyme(G-6-Pi-DH) linked to detection of the initial product.
[hexokinase]
Glucose + ATP → Glucose-6-Pi + ADP
[G-6-Pi-DH]
Glucose-6-Pi + NADP → 6-Phosphogluconate + NADPH
- The glucose-6-Pi-DH is very specific for its substrate
What are other clinical analytes where enzymes are used as reagents?
- Urate
- Creatinine
- Cholesterol
- Triglycerides
- Lactate
- Urea
- Ammonia
- Total CO2
What are advanatges of he enzymatic method over the jaffe method?
- Enzymatic method for creatinine competes with the traditional ‘Jaffe’ method, which uses alkaline picrate as a colour reagent.
- Jaffe method is long established and cheaper, but is more prone to interferences, particularly from bilirubin.
Modern analysers allow for both methods on the same instrument and can be programmed only to do an enzymatic creatinine analysis if a high bilirubin is detected.
What are types of enzyme mediated analysis?
- End Point Assays
- Kinetic Assays
What are features of End Point Assays?
Many assays using enzymes as reagents are ‘end point’ assays. The reactions are allowed to go to completion.
- Zero order kinetics
- Can demand relatively high amounts of enzyme reagents and they work best with enzymes with a low Km (high affinity)for the substrate which is the subject of the analysis.
- Less sensitive to changes in assay conditions
What can be used when a reaction equilibrium is unfavourable?
Where a reaction equilibrium is unfavourable to the desired direction, a ‘trapping’ reagent may be used to ‘pull’ the equilibrium that way.
- Example – Analysis of lactate
[LDH]
Lactate + NAD ←→→ Pyruvate + NADH
Pyruvate + Hydrazine → Hydrazone
As pyruvate is continually removed from the equilibrium by conversion to its hydrazone, the production of the detection chromophore, NADH, continues until all the lactate is used up.
What are features of Kinetic Assays?
- In kinetic enzyme assays the rate of the reaction is used to assess the concentration of the analyte being measured. This will require several measurements over a period of time, rather than just measuring a change after completion, as in a fixed time/end point methods.l
- Whereas in end point assays the idea is to reach the ‘end’ point as rapidly and completely as possible, in kinetic assays smaller amounts of the primary enzyme may allow a slower process which is then easier to monitor over time.
- Also, the theory behind enzyme kinetics means that an enzyme with a high Km for the substrate being determined may be advantageous as it is desirable to work at substrate/analyte concentrations below 0.2 x Km.
When is the observed reaction velocit proportional to substrate concentration?
- First-order kinetics.
- To achieve a situation where the observed reaction velocity is proportional to the substrate concentration, ideally need to work at substrate concentrations below 0.2 x km.
What are advanatages and disadvantages of Kinetic Assays?
Advantages
- Because there may be less need to wait for a reaction to achieve equilibrium, as in end point assays, kinetic assays may be ‘faster’.
Disadvantages
- Since it is the reaction rate which is being monitored all factors which cause this to vary must be strictly controlled to give precise results.
- Enzyme reagents employed as part of ‘product detection’ (coupled assays) must NOT be rate limiting – use in high concentrations relative to the enzyme reacting with analyte/substrate.
What are examples for Kinetic Assays?
Kinetic Assay example – Ethanol
[ADH]
Ethanol + NAD → Acetaldehyde + NADH
- This example requires only a single enzyme which both reacts with substrate/analyte and also produces an observable product.
- As with examples of end-point analysis, kinetic assays can also use multi-enzyme systems to produce a convenient spectrophotometric change if needed.
How are enzymes used as labels in immunoassays?
- Immunoassay often analyse very low concentrations of analyte. As enzymes can cycle for as long as they have appropriate substrate, they can amplify the signal.
- Enzymes with high turn-over numbers (kcat) are advantageous as they produce more signal per unit time. Peroxidase and ALP are often favoured as such labels.
What are two common techniques for use of labels in immunoassay?
Two common techniques are:
- EMIT – a ‘homogeneous’ immunoassay where no separation step is needed
- ELISA - a ‘heterogeneous’ immunoassay method
How does EMIT take place?
- When the anibody is unbound to the enzyme drug conjugate, the enzyme is active and coverts NAD to NADH and the reaction is spectrometrically read at wavelength of 340nm
- When the antibody binds to the enzyme drug conjuagte, the enzyme is inactive
