1. Measuring the Enzyme Function of Alkaline Phosphatase What is the effect of substrate concentration on alkaline phosphatase activity?

2.  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

3. 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

4. Hypothesis: What is the effect of substrate concentration on alkaline phosphatase activity? Independent Variable: Artificial substrate concentration Levels of IV Buff.PNP-pDWPNPEnzAB Trials123456 Dependent Variable: Alkaline phosphatase Controlled Variables (constants) N/A Experimental Design Diagram

5. 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

6. DV: Alkaline phosphatase Tube:BTrial 1Trial 2Trial 3Trial 4Trial 5Trial 6 Buff.500 PNP-p00025 50 DW400300 375 350 PNP0100 0000 Enz100 AB Data Table

7. 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:BTrial 1Trial 2Trial 3Trial 4Trial 5Trial 6 Buff.500 PNP-p00025 50 DW400300 375 350 PNP0100 0000 Enz100 AB0.0420.0620.0820.103 0.1240.143

8. Spectrophotometer Pictures taken by Chelsea Kellar

9. Photo 1 Pictures taken by Chelsea Kellar

10. Photo 2 Pictures taken by Chelsea Kellar

11.  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

12.  “Enzyme Kinetic Analysis of Alkaline Phosphatase.” 11 November 2011  Kaslow, Jeremy E., M.D. “Alkaline Phosphatase.” 11 November 2011  Sonoma State University. “Enzyme Function: Effects of Substrate Concentration on Alkaline  Phosphatase Activity.” 11 November 2011  “Substrate Concentration (Introduction to Enzymes)”. 28 November 2011 http://www.worthington- biochem.com/introbiochem/substrateconc.htmlhttp://www.worthington-  “Alkaline Phosphatase.” 28 November 2011 Works Cited