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Enzymes as Tracers
As naturally occurring components:
Enzyme activities may be monitored to
assess the state of a biological system.
Pure enzymes may also be employed to
modify the condition of analytical
specimens to make analyses simpler. Or,
they may be incorporated into complexes
with ligands or binding components to
produce tags that can be identified by the
generation of unique products.
Endogenous Tracers
Enzymes that are unique to a given cell or
tissue provide a means to locate those cells
or to ascertain the health of the tissue. For
example, alkaline phosphatase is found in
few cells but is present in high levels in
migrating germ cell precursors, allowing
them to be located within the tissues of the
developing embryo. Creatine kinase is
found in muscle & is normally low in serum
unless muscle damage has occurred.
Active caspases are only in apoptotic cells.
Endogenous Tracers (cont.)
Enzymes in tissues may also complicate
analyses that attempt to use pure enzymes
as tracer tags. If samples to be analyzed
contain an enzyme with similar properties
to the tracer, false positives will arise.
Controls lacking tracer enzyme are always
needed & protocols should take care that
assay conditions optimize specificity of the
tracer enzyme activity, e.g., lack of similar
enzymes in samples, pH, salt, temperature,
substrate conditions favoring the tracer.
Pure Tracer Enzymes
Enzymes favored include those with:
High turnover numbers
Low Km for substrate, high Km for product
High Ki
Storage stability
Ease of detection
Low cost/ ease of isolation of pure enzyme
Absent in samples
Compatible with assay conditions
Pure Tracer Enzymes (cont.)
Horseradish peroxidase
Alkaline phosphatase
β-Galactosidase
Urease
Glucose oxidase
Acetylcholinesterase
Lysozyme
Glucose-6-phosphate dehydrogenase
Luciferase
Use of Native HRP as a Neuronal Tracer:
Reaction with Substrates Localizes Enzyme Activity
Horseradish Peroxidase
HRP Models
University of Budapest
Recombinant HRPC1A Complex
with CN & Ferulic Acid
HRP-mediated Chemiluminescence
Tracing for Western Blots
(ECL Amersham)
Oligomer Detection Using Biotin & Streptavidin-HRP
(Chemicon)
Alkaline Phosphatase
University of Oklahoma Health Science Center Clinical Lab
PPT slide #59
Enzyme Structure with Phosphate at Active Site
Alkaline Phosphatase
Chemilumenescence on Nylon
(University of Bologna)
Alkaline
Phosphatase
&
β-Galactosidase
Staining Reactions
There is substantial flexibility available in production of
soluble, insoluble, chromogenic, fluorescent, or
luminescent products from either alkaline phosphatase or
horseradish peroxidase. The choice is largely based on
desired data, limit of detection required, available
instrumentation, ancillary reagents, and prior experience.
Substrates are available from Sigma/Aldrich Chemicals, In
Vitrogen, Anaspec, Pierce Chemical, and a number of other
suppliers.
For some idea of this range see:
SigmaAlkPhos&HRPSubstrates.pdf
Random-Primed Labeling & Detection
via Fluorescein, Anti-fluorescein-AP, &
Fluorescent Product
(Amersham Biosciences)
Western/Southern/Northern Blot Visualization
with Vector DuoLuX chemilumescent &
fluorescent substrates for AP & HRP
Some Cautions: Use of HRP or AP often requires pretreatment of
specimens or solutions to remove endogenous enzyme activity or related
enzyme activities. Be aware that the enzymes can be inhibited by their
products or by inhibitors, e.g., acid pH & phosphate buffers block AP
activity while perservatives such as azide block HRP activity. Enzyme
handling is also crucial: AP solutions can be stored frozen while HRP
solutions cannot; while both enzymes like all proteins are sensitive to
freezing & thawing, this action is hardest on enzymes with prosthetic
groups & those involved in redox reactions; storage with cryoprotectants
such as glycerol, ethylene glycol, or DMSO helps considerably.