Measuring the Enzyme Function of Alkaline

PhosphataseWhat is the effect of substrate concentration on

alkaline phosphatase activity?

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)

Background Research

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 L-ALP

Introduction and Background

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

Experimental Design Diagram

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 37-degrees Celsius bath.

Set your spectrophotometer at the optimum wavelength for p-nitrophenol.

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.

Materials and Equipment

Cellular spectrophotometer

6 test tubes Alkaline phosphatase Substrate concentration

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 Table

Data Analysis

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.

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

SpectrophotometerPictures taken by Chelsea Kellar

Photo 1Pictures taken by Chelsea Kellar

Photo 2Pictures taken by Chelsea Kellar

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.

Conclusion

“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.worthington-biochem.com/introbiochem/substrateconc.html

“Alkaline Phosphatase.” 28 November 2011 <http://en.wikipedia.org/wiki/Alkaline_phosphatase>

Works Cited