Effects Of Varying P-Nitrophenyl Phosphatase Activity

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
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
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