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Lab6 322 Converted - 1
Lab6 322 Converted - 1
of an enzyme catalyzed
reaction
▪ In this experiment, we will continue to study acid phosphatase kinetics.
Enzyme Substrate
Time Temperature pH Inhibitor
concentration concentration
Objectives
▪ To establish the relationship between pH and the rate of an enzyme catalyzed
reaction.
▪ Place the tubes in a test tube rack situated in 37oC water bath and let stand for 5 min.
• Start the reaction by adding 0.5 ml enzyme and stop it by adding 0.5 ml KOH as
in the following table:
▪ Notes:
• In blank tube add KOH first then the enzyme, to prevent the reaction from happening.
Results
pH Absorbance 405 nm Velocity (µmole of PNP/min)
3
4.5 Plot a graph
5.5 illustrating the
effect of
7 different pHs
9 on the rate of
the reaction.
Calculations:
▪ Velocity (V) = (A x 106) /(E x time)= µmole of PNP/min
▪ A= absorbance
▪ Time = 5 min
The Effect of pH on the Rate of an Enzyme Catalyzed
Reaction.
(µmole of PNP/min)
Velocity
Optimum pH
pH
Discussion
▪ An introductory statement
▪ Principle
▪ From the curve, explain and discuss the shape of the curve and the
relationship between the activity of acid phosphatase and pH.
▪ Define the optimum pH and determine which buffer pH is the best from the
curve.