Enzymology Flashcards
How are enzymes used as reagents and why are they useful?
As specific bio-reagents
- reacting with substrate/analyte - as means of producing observable signal
As removers of interferences (Enable us to detect an observable signal without interference)
As signal amplifiers - ‘labels’ in immunoassay (enzymes can be conjugated to increase signal generation)
As specific bio-reagents, eg glucose oxidase.
Glucose oxidase generates H2O2. Can’t measure oxygen depletion or any of the products, but by linking to a peroxidase coupled assay (secondary reaction), where the H2O2 is converted to oxidized o-toluidine, catalysed by peroxidase. This is a chromagen so it produces a coloured product proportional to the amount of glucose in the sample. Hence is a very specific assay coupled to a non-specific assay.
A converse arrangement can also provide specificity.l
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. Very specific assay can be measured as NADPH increase can be recorded by spectrophotometry.
Other examples of clinical analytes that are commonly determined using enzyme reagents include: Urate (uricase). Creatinine(whole blood enzymes) Cholesterol (cholesterol esterases) Triglycerides (esterse) Lactate(LDH) Urea (urease) Ammonia Total CO2
Using creatinine as an example, explain how enzymes are useful as reagents.
Another Example – Creatinine – Using Creatininase et al
creatininase Creatinine + H2O -> Creatine
creatinase Creatine + H20 -> Sarcosine + urea
sarcosine oxidase Sarcosine + O2 + H2O -> Glycine + HCHO + H2O2
peroxidase H2O2 + dye reagent -> colour
A fifth enzyme is also present – Ascorbate oxidase. This enzyme is there only to prevent interference from ascorbic acid.
The enzymatic method for creatinine competes with the traditional ‘Jaffe’ method, which uses alkaline picrate as a colour reagent. This 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.
How are end point assays useful as a type of enzyme mediated analysis?
Many assays using enzymes as reagents are ‘end point’ assays. The
reactions are allowed to go to completion.
Zero order kinetics - low Km
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.
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.
How are kinetic assays useful as a type of enzyme mediated analysis?
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 end point methods.
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 (Enzymes with high km and low affinity to mimic first order kinetics. Involves using a substrate with a concentration that is 20% of the enzyme).
Because you’re using low conc of substrate it is more prone to changes in conditions. 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.
Because there may be less need to wait for a reaction to achieve equilibrium, as in end point assays, kinetic assays may be ‘faster’.
As with examples of end-point analysis, kinetic assays can also use multi-enzyme systems to produce a convenient spectrophotometric change if needed.
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.
How are enzymes used as labels in immunoassays?
In immunoassay it is often the case that analysis is taking place for very low concentrations of analyte. Because enzymes can cycle for as long as they have appropriate substrates they can amplify many fold what would otherwise be a very small signal.
Enzymes with high turn-over numbers (kcat - constant between enzyme forming the complex and the product) are advantageous as they produce more signal per unit time. Peroxidase and ALP are often favoured as such labels.
Two common techniques are:
EMIT – a ‘homogeneous’ immunoassay where no separation step is needed
ELISA - a ‘heterogeneous’ immunoassay method
Describe how Enzyme Mediated Immunoassay Technique (EMIT) works.
Enzyme is bound as a conjugate, when the drug is in the blood stream it binds to the antibody leaving the active site free so the substrate can bind. So when the drug isn’t present the reagent it bonds to the conjugate hapten (similar structure to the drug), sterically hindering the active site, and preventing formation of the product. Concentration of drug in the blood stream is proportional to the activity of the enzyme (product turnover), which is proportional to generation of product, which you can measure (eg generation of NADH).
Describe how Enzyme Linked Immunosorbant Assays work?
ELISA’s are usually two stage assays: A ‘capture’ antibody is used to bind analyte in the sample to a solid phase followed by a wash step and then addition of a second antibody for the analyte which is linked to an enzyme label.
96 well plate format using a capture antibody immobilised to the plate, binds to antigen of interest and the uses a washing step. capture antibody added, that is conjugated to an enzyme for signal generation. Enzyme with higher Kcat can induce more signals.
Describe how dry reagent systems work.
Enzyme reagents can be immobilised onto a solid support, e.g. cellulose and used in test ‘strips’. Urine testing for glucose generally uses this method – the glucose oxidase, the peroxidase and dye (potassium iodide chomogen) are incorporated into the reagent pad.
‘Dry’ reagent approaches are particularly prominent in Point of Care Testing.
An example:
VITROS dry slides are multilayered slides. Patient sample is deposited onto the slide and the spreading layer contains the appropriate substrate and reagents needed for the reaction. The reaction products diffuse into the underlying layer, and are monitored by reflectance spectrophotometry.
The test types are colorimetric, enzymatic end point, or multi-point rate, or potentiometric. The rate of change in reflection density in converted to enzymatic activity or the amount of colored complex formed is proportional to the analyte concentration in the sample.
Why is diagnostic enzymology important?
Because of their potential ability to produce a large ‘a’ despite being present at a low concentration, enzyme detection can be relatively easy compared to purely structural proteins (not enzymes), which usually require immunoassay.
As we shall see, sometimes we actually prefer to use immunoassays or other methods, e.g. MS based methods, to measure a specific type of an enzyme, or an enzyme whose activity is not easy to monitor.
Why are enzymes measured?
Detection of suspected disease at pre-clinical stage
Confirmation of suspected disease and assessing severity
Localisation of disease to organs
Characterisation of organ pathology
Assessing the response to therapy
Organ function assessment
Assessing genetic susceptibility to drug side effects
What factors determine enzyme activities in serum/plasma?
age gender pregnancy genetics drugs disease process, treatment - e.g. surgery
Generally, serum enzyme concentrations are low and rise only when there is damage to cells and release of contents. The release of enzymes from cells may be triggered by a number of
processes:
Cellular damage due to chemicals, drugs
Physical damage due to trauma, surgery, burns, etc.
Immune disorders – anaphylaxis, autoimmune disease, etc
Microbiological agents – bacteria, viruses, etc
Genetic defects – many, e.g. Duchenne’s Muscular Dystrophy
Nutritional disorders – protein-calorie, vitamin status - enzymes often require cofactors that are vitamins.
The localization of enzyme within the cell/extent of cell damage. Enzymes in the cytosol are the most prone to leakage. Enzymes within organelles are less likely to leak and elevated concentrations in the circulation may indicate more serious damage.
What are the tissue sources of the following enzymes?
Heart Muscle [AST], [LDH], [CKMB]
Liver/biliary tract [AST], [ALT], [γGT], [ALP]
Skeletal muscle [AST], [CK] (total)
Pancreas [Amylase], [lipase], [trypsin], [chymotrypsin]
Prostate gland [Acid phosphatase], [PSA]
Bone [ALP]
RBCs [LDH]
When is isoenzyme analysis useful?
Few enzymes have a ‘unique’ tissue source.
For some enzymes, e.g. amylase from the pancreas, there is generally little concern about other sources (but parotid gland is also a source of amylase).
For many serum enzymes there is often a concern about source and this has led to a ‘panel’ approach.
- For example if a patient has a raised ALP and a raised γGT then it is likely that the excess ALP has arisen from the liver and not from bone.
What is the difference between an isoenzyme and an isoform?
Isoenzyme: Multiple forms of an enzyme that possess the ability to catalyse the enzyme’s characteristic reaction but that differ in structure because they are encoded by distinct structural genes.
Isoform: Multiple forms of an enzyme that differ as a result of post-translational modification.
How does studying creatinine kinase isoenzymes help in diagnosis?
CK has a dimeric structure. Two types of subunit are found the ‘M’ (muscle) subunit and the ‘B’ (brain) subunit.
Muscle tissue CK is predominantly ‘M-M’
Brain tissue CK is predominantly ‘B-B’
Cardiac muscle tissue has a substantial fraction of structure ‘M-B’
All the forms have the same enzymatic activity, immunoassays were developed to measure CK-MB to help diagnose and monitor a heart attack, but troponin has replaced CK-MB in this role.
Total CK useful in diagnosis of Rhabdomyolysis
Also raised by: fitting, exercise, prolonged coma, myositis, hypothyroidism, muscular dystrophies, statins.
