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Enzyme linked reactions and kinetics - Coggle Diagram
Enzyme linked reactions and kinetics
Enzymes, substrates and products
Enzyme assays are a common diagnostic tool where the concentration of a substrate/product is measured to determine the activity of an enzyme
Enzyme assays in a lab
spectroscopy is a cheap and easy technique commonly used to follow the progress of an enzyme linked reaction if one of the substrates or products absorbs light in the UV/visible region of the EM spectrum
Beer Lambert Law - recap
The absorbance of an analyte depends on three factors, concentration, path length and the molar extinction coefficient
Assay automation
Enzyme assays used for diagnosis and research are usually highly automated
Effects of pH, temperature and additives can be monitored
Automation is essential to make results timely and variable
Sources of error
pipetting errors
fluctuation of the incident light intensity
calibration errors
Alkaline phosphotase (ALP)
ALP catalyses the dephosphorylation of various compounds in nature
ALP is found in all tissues
Increased levels of ALP can indicate
cholestatic liver disease
osteoblastic activity in childhood
pathological conditions
Pregnancy
ALP assay
The serum sample is mixed with p-nitrophenol phosphate
ALP present in the sample will convert the p-nitrophenol phosphate to p-nitrophenol and phosphate
End point of the reaction is when all the p-nitrophenol phosphate has been converted
the end point does not tell amount of ALP in the sample
The activity of ALP must be calculated from over a particular volume over a set time-period
Substrate concentration
The rate of the enzyme catalysed reaction depends on the concentration of the substrate
The concentration of p-nitrophenol phosphate added to the serum sample must saturate the reaction so that the maximum rate Vmax is measured
This is known as the zero-order reaction where the rate independent of the substrate concentration.
