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Lecture

Chemical and Biochemical Engineering 2290A/B Lecture Notes - Alkaline Phosphatase, Reaction Rate, Sodium Hydroxide


Department
Chemical and Biochemical Engineering
Course Code
CBE 2290A/B
Professor
Mita Ray

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WEEK 3
Aim: To study the effect of temperature on enzyme reaction rate
Protocol: End point Assay method
Assay mixture:
3.9ml Tris HCl pH 9.0
1.0ml substrate solution
At T=0 , add 0.1ml alkaline phosphatase (enzyme)
At T=4min, add 2ml 2N NaOH
Read absorbance at end of 4min at 410nm.
Repeat 3 times at each temperature
Observation (End point assay method)
Sr. No
TEMPERATURE (˚C)
Observation (Absorbance @ 410nm) t= 4 min
1
2
3
1
0
2
25
3
35
4
45
5
55
6
65
Calculate:
a. For each treatment, plot product concentration vs. time.
b. For each treatment, determine reaction rate using the graphical method and put the
appropriate information on the figure as a text box.
c. For each treatment’s figure legend, in addition to the standard information, add the
resulting reaction rate and product concentration at equilibrium (based on absorbance at
the plateau; if available).
d. Plot temperature vs. reaction rate.
Discuss:
i) Effect of temperature on enzyme activity
ii) Graphical representation of Temperature vs Reaction rate
iii) Based on your knowledge of enzyme structure, function and means of regulation, what
effect(s) do/does temperature have on enzyme function (i.e. rate and saturation, if you
recorded saturation) and how do your data demonstrate that/those effect(s)?
Bonus Question:
Using your knowledge of enzyme structure and function, explain why a prolonged period of high fever is
dangerous to humans.
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