Download What is the effect of substrate concentration on - jehs

Measuring the Enzyme Function
of Alkaline Phosphatase
What is the effect of substrate concentration on alkaline
phosphatase activity?
Background Research
 Alkaline phosphatase is pertinent in recycling phosphate within living
cells
 Alkaline phosphatase catalyzes the cleavage of a phosphate group from
many types of molecules, including nucleotides, protein, and alkaloids
 In humans alkaline phosphatase is particularly concentrated in the liver,
bile duct, kidney, bone, and the placenta.
 Human phosphatase enzymes
 ALPI-intestinal
 ALPL-tissue non-specific (liver/bone/kidney)
 ALPP- placental (Regan isozyme)
Introduction and Background
The purpose of this experiment is to determine the effects of
substrate concentration on the functioning of the enzyme,
alkaline phosphatase. The serum ALP measurement’s most
important clinical attribute is its sensitivity in distingushing liver
disease. The measurement of corticosteroid-induced ALP can
also be used as a screening test for hyperadrenocorticosim in
dogs and has also been shown to be a prognostic indicator for
canine osteosarcoma.
The Table shows condition or disorders resulting in increased LALP
Experimental Design Diagram
Hypothesis: What is the effect of substrate concentration on
alkaline phosphatase activity?
Independent Variable: Artificial substrate concentration
Levels of
IV
Buff.
PNP-p
DW
PNP
Enz
AB
Trials
1
2
3
4
5
6
Dependent Variable: Alkaline phosphatase
Controlled Variables (constants)
•N/A
Materials and Equipment




Cellular spectrophotometer
6 test tubes
Alkaline phosphatase
Substrate concentration
Procedure
 Set up (6) glass test tubes as
follows. Volumes are given in
microliters, not milliliters.
 Add only the first 4 ingredients,
then place the test tubes into a 37degrees Celsius bath.
 Set your spectrophotometer at the
optimum wavelength for pnitrophenol.
 Without touching the knobs on the
spectrophotometer, read the
absorbance to 3 decimal places.
 Read the absorbance in the
spectrophotometer.
 Record your data in notebook.
Data Table
DV: Alkaline phosphatase
Tube:
B
Trial 1
Trial 2
Trial 3
Trial 4
Trial 5
Trial 6
Buff.
500
500
500
500
500
500
500
PNP-p
0
0
0
25
25
50
50
DW
400
300
300
375
375
350
350
PNP
0
100
100
0
0
0
0
Enz
100
100
100
100
100
100
100
AB
Data Analysis
Tube:
B
Trial 1
Trial 2
Trial 3
Trial 4
Trial 5
Trial 6
Buff.
500
500
500
500
500
500
500
PNP-p
0
0
0
25
25
50
50
DW
400
300
300
375
375
350
350
PNP
0
100
100
0
0
0
0
Enz
100
100
100
100
100
100
100
AB
0.042
0.062
0.082
0.103
0.103
0.124
0.143
Statement of Analysis:
The graph shows that the activity of alkaline phosphatase is measured
under the conditions defined by the experiment. In the substrate
experiment, you will need to calculate the actual concentration of
substrate in moles per liter for each incubation.
Spectrophotometer
Pictures taken by Chelsea Kellar
Photo 1
Pictures taken by Chelsea Kellar
Photo 2
Pictures taken by Chelsea Kellar
Conclusion
 My hypothesis was that substrate concentration
catalyzes alkaline phosphatase activity.
 The results I obtained tell about the properties of
alkaline phosphatase and about the functioning of
the tissue from which it was prepared by varying the
substrate concentration and measuring the rate of
the enzyme catalyzed reaction. The appearance and
intensity of yellow color thus indicated the degree to
which the substrate had been acted upon by the
enzyme.
Works Cited
 “Enzyme Kinetic Analysis of Alkaline Phosphatase.” 11 November 2011
<http://www.public.asu.edu/~laserweb/woodbury/classes/chm467
/Enzkin/alkphosphat.html>
 Kaslow, Jeremy E., M.D. “Alkaline Phosphatase.” 11 November 2011
<http://www.drkaslow.com/html/alkaline_phosphatase.html>
 Sonoma State University. “Enzyme Function: Effects of Substrate
Concentration on Alkaline
 Phosphatase Activity.” 11 November 2011
<http://www.sonoma.edu/users/h/hanesda/b324/alk_phos.html>
 “Substrate Concentration (Introduction to Enzymes)”. 28 November
2011 http://www.worthingtonbiochem.com/introbiochem/substrateconc.html
 “Alkaline Phosphatase.” 28 November 2011
<http://en.wikipedia.org/wiki/Alkaline_phosphatase>