The Effects of varying p-Nitrophenyl Phosphate concentrations on Alkaline Phosphatase Activity Nathan Overholt Louis Settembrino March 5, 2002 Abstract: The enzyme Alkaline Phosphatase was set up in solutions of varying p-nitrophenyl phosphate concentrations. A fixed time assay was used to find the Michaelis constant and Vmax. All solutions were incubated at 30 C° for 30 mins. Absorbances were measured at 405 nm, and the Km was found to be 0.002 M (pNPP) and the Vmax to be 0.433 A/min.
Introduction: Alkaline Phosphatase: Alkaline Phosphatase is an enzyme that comes from liver, bone, placenta, and intestine. This enzyme hydrolyses phosphate esters, is secreted into the serum by osteoblasts and is used as a diagnostic marker for increased metabolic activity. Growing bones need alkaline phosphatase. Any condition of bone growth will cause alkaline phosphatase levels to rise. The condition may be normal, such as a childhood growth spurt or the healing of a broken bone; or the condition may be a disease, such as bone cancer, Paget’s disease, or rickets. During pregnancy, alkaline phosphatase is made by the placenta and leaks into the mother’s bloodstream. This is normal. Some tumors, however, start production of the same kind of alkaline phosphatase produced by the placenta.
These tumors are called germ cell tumors and include testicular cancer and certain brain tumors. (Nordensen, 2002) Healthy livers usually carry alkaline phosphatase away along with other harmful substances out through the bile duct. If the liver becomes diseased in some sort, then the bile duct may become blocked producing large amounts of built up alkaline phosphatase and eventually this leaks out into the bloodstream. There are different forms of alkaline phosphatase called isoenzymes. The determination of the particular isoenzyme will determine the target problem area. (Sorensen, 2002) Enzymes: Enzymes are catalysts, meaning, they help a reaction proceed without being consumed.
The substrate binds to a specific site on the enzyme, and is converted to the product(s) and then released. The reaction would occur in the absence of the enzyme, but a significantly lower rate. As the substrate is converted to product, the concentration of substrate decreases and the concentration of product increases. The actual rate of reaction is the rate constant times either the substrate, in the case of the forward reaction, or rate constant times the product for the reverse reaction. As the substrate is converted to product, the rate constant stays the same, but the rate of reaction decreases as substrate is used up and product concentration increases.
Reaction: E + S ??? ES ??? P Enzymes have two factors when measuring activity, the maximum velocity (Vmax), and the Michaelis constant,(Km). The Vmax can be defined as the maximum rate at a given enzyme concentration that it can work or rate of product formation when active site is saturated.
The Michaelis constant, Km, is a measure of the affinity of the enzyme for the substrate, and has the same units of concentration. The substrate used with alkaline phosphatase for this lab was p-nitrophenyl phosphate. If the enzyme has a high Km, than it has a low affinity for the substrate and if it has a low Km than it has a high affinity. Km is also the substrate concentration at which the enzyme has half the Vmax, or the substrate concentration when the active site is filled half the time. Conversely, if the substrate concentration required for half maximal velocity is high, then the affinity of the enzyme for the substrate is low.
These measures of activity for the enzyme can be determined mathematically through the Michaelis Menten equation (equation 1): Vo= [S]*Vmax equation 1 [S] + Km This equation when plotting velocity vs. substrate concentration will result in a hyperbola approaching Vmax and by taking Vmax/2 one can estimate Km. However, to get a more efficient Km value for substrate one should perform the Lineweaver-Burk plot which linearizes data giving a definite value. This is done by taking the inverse of the Michaelis Menten equation (equation 2) such that: 1/Vo= Km (1/[S]) + 1 equation 2 Vmax Vmax Examples of two of these graphs are given by figures 1 and 2.
Fig 1- Michaelis Menten Plot Fig 2- Lineweaver-Burk Plot By interpreting the Lineweaver-Burk plot one can easily deduce the Km and Vmax by using the x and y intercepts. The x-intercept is equivalent to -1/Km and the y-intercept is equivalent to 1/Vmax.
Materials And Methods: The PLHC-1 cells were grown for a period of 7 days prior to the experiment. On the 7th day the cell extract was prepared by rinsing the cells the cells with 2 mL of PBS (Phosphate buffered saline), than adding 2 mL of 3.5 mM SDS until the cells were disrupted. The cells were checked under the phase contrast microscope to verify disruption. Twelve test tubes were set up with varying volumes of Buffer (0.1 M 2-amino-2-methyl-1-propanol, 0.001 MgSO4, pH 10), Substrate (0.01 M p-nitrophenyl-phosphate ), and Deionized Water. The cell extract was added last and in numerical order, for time consistency. (Table 1) 1 2 3 4 5 6 7 8 9 10 11 12 buffer 500 500 500 500 500 500 500 500 500 500 500 500 substrate 0 25 25 50 50 100 100 200 200 400 400 400 deionized water 400 375 375 350 350 300 300 200 200 0 0 0 extract 100 100 100 100 100 100 100 100 100 100 100 100 Table 1 – Volumes (µL) added sequentially to each test tube.
Tube 1 was the blank, and tube 12 was used as a correction. (Sorenson, 2002) Each tube was immediately vortexed following the addition of the extract. After tube 12 was vortexed 0.04 M NaOH was immediately added and it was revortexed to stop the reaction before incubation. All 12 tubes were than incubated for a 30-minute period at 30 C°. Than 0.04 M NaOH was added to each tube in the same numerical order to keep the reaction times consistent for all the tubes. The absorbances were measured at 405 nm using a Spectronic 20 colorimeter. (Sorenson, 2002) Test tube 1 was used to set the blank, and tube 12 was used to proportionately subtract out the spontaneous hydrolysis of the substrate before the reaction begins. For example tubes 10 and 11 subtract the value, and for tubes 8 and 9 subtract half the value because the substrate concentration is 200/400.
Results: Based upon the values from Lineweaver-Burk the Vmax for p-nitrophenyl-phosphate in the presence of alkaline phosphatase is 0.433 M and the Km is 0.002 M. This was determined from the x and y-intercept of the Lineweaver-Burk plot in figure 3.
Fig 3- Lineweaver-Burk Plot for enzyme alkaline phosphatase and substrate p-nitrophenyl-phosphate. Km and Vmax are determined from x and y-intercept respectively. Units for 1/V are in min/absorbance and units for 1/[S] are L/mol.
Discussion: Based upon our results one can determine accurately the Michaelis constant (Km) and Vmax by a Lineweaver-Burk plot. Vmax is defined as the maximum rate at a given enzyme concentration that it can work or rate of product formation when active site is saturated. This means that at a rate of 0.433 A/min the enzyme alkaline phosphatase was forming product. By determining Vmax, the affinity of the enzyme for substrate or the sum of all the forces causing a strong steric fit can be calculated.
This may also be known as the concentration of substrate when the active site is occupied half the time. This p-nitrophenyl phosphate concentration was .002 M (pNPP). Results of similar Michaelis constants were investigated and in research done by Ozegowski and Muller it was determined that the same substrate had a Michaelis constant of 1.25 X 10(-3) M. In a second study by Delaunay, Fischer, et al the Michaelis constant using p-nitrophenyl phosphate as substrate was 2.5 X 10(-3) M.
The Michaelis constant could have some error due to the correction factor. If NaOH was not immediately added to stop the reaction this may have changed the absorbance of the correction factor for tube 12 and changed all tube correction factors proceeding.
Literature Cited: Delaunay J, Fischer S, Piau JP, Tortolero M, Schapira G. Apr. 2, 1993, Properties of a membrane-bound phosphatase activity in normal and abnormal red blood cells. (Abstract) Nordenson, Nancy J. 2002. Alkaline Phosphatase Test.
http://www.ahealthyme.com/topic/topic100586408 Ozegowski JH, Muller PJ. Dec. 25, 1984, Metabolism of phosphate-limited Streptomyces cultures. I. Purification and characterization of alkaline phosphatase produced by Streptomyces hygroscopicus. (Abstract) Sorensen, Ralph A. Spring 2002, Alkaline Phosphatase Activity, http://www.gettysburg.edu/~rsorense/Celllab02/ap.htm