James' Unwanted Blood Clots
The antithrombin III test measures how well James' plasma inhibits thrombin.
This part of the tutorial illustrates aspects of enzyme kinetics.
The antithrombin III assay measures how well a patient's serum inhibits thrombin. Thrombin has several physiological substrates. The principle one is fibrinogen, but thrombin also cleaves several other clotting factors.
A better thrombin substrate for studying enzyme kinetics:
For convenience, people have developed several artificial thrombin substrates that change color when cleaved. One of these substrates is hexahydrotyrosyl-L-alanyl-L-arginine-p-nitroanilide (H-D-HHT-L-Ala-L-Arg-pNA).
Thrombin catalyzes the reaction:
H-D-HHT-L-Ala-L-Arg-pNA (colorless) → H-D-HHT-L-Ala-L-Arg + pNA (colored)
For this substrate, thrombin has Km=3.24 μM
Question:
The amount of thrombin that is present in a sample is measured by adding a bit of the sample to a solution containing the substrate in a buffer.
Use your knowledge of enzymes to figure out what the laboratory should measure in order to determine the concentration of thrombin in the sample.
- color of the solution vs time
- rate of change of color vs amount of enzyme added
- rate of change of color vs amount of substrate added
- all of the above
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