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AP Kinetic Parameters Spring 2011

AP Kinetic Parameters Spring 2011 - BCHM464 Spring 2011 AP...

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BCHM464, Spring 2011 AP Kinetic Parameters 1 Alkaline Phosphatase Activity: Determination of Michaelis-Menten Parameters ( K M , k cat , V max ) A. Introduction In this experiment, you will use the Shimadzu UV-1800 spectrophotometer to assay alkaline phosphatase activity and determine its Michaelis-Menten kinetic parameters ( K M , k cat , V max ). In the first part, you will become familiar with running the assay and check the accuracy of your pipetting. In the second part of this laboratory, you will examine the activity of alkaline phosphatase in buffers of different pH, in order to determine the optimum pH range in which alkaline phosphatase functions most efficiently. Finally, you will determine the kinetic parameters of wild-type alkaline phosphatase. Alkaline phosphatase (AP) nonspecifically hydrolyzes many types of phosphate esters. The activity of AP can be conveniently measured by using substrates that give rise to colored products. p-Nitrophenyl phosphate (pNPP) is a common substrate used to measure the enzymatic activity of phosphatases. Upon hydrolysis by AP, pNPP yields p-nitrophenol (pNP, pKa = 7.1), which forms an intensely yellow phenolate ion (pNP - ) under the slightly basic assay conditions. Other colorimetric substrates of alkaline phosphatase are frequently used in molecular biology and biotechnology applications, such as detection of immunoblots and ELISAs. One of the most common of these is 5-bromo-4-chloro-3-indolyl phosphate (BCIP or XP). After hydrolysis of the phosphoester bond, the product (5-bromo-4-chloro-3-oxindole) is oxidized. The oxidation product exhibits an intense blue color that is easily visible on agar plates and hybridization membranes.
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BCHM464, Spring 2011 AP Kinetic Parameters 2 Using the extinction coefficient determined in the first exercise, you will be able to determine the rate of the AP reaction, by quantitating the amount of nitrophenolate product released over time. Typically, photometric enzyme assays are carried out by first preparing a solution that contains a buffer (to maintain a constant pH), any other salts necessary to maintain optimal enzyme activity, and the desired substrate (e.g. pNPP). The reaction is initiated by adding a solution containing the enzyme to the sample buffer. The change in absorbance caused by increasing concentration of the colored product is then measured over time. B. Procedures PART I: Enzyme Activity Assay Prepare the following materials: 1 mM pNPP in AP assay buffer Alkaline phosphatase (be sure to record the [AP] in your notebook) Spectrophotometer settings for kinetics mode (do NOT use “kinetics rate”) : 400 nm 75 s total time 10 s lag time 0.1 cycle time 65 s end time Obtain AP assay buffer (1.0 mM pNPP in 1.0 M Tris, pH 8.0) containing 1 mM p-nitrophenyl phosphate (pNPP) and a sample of alkaline phosphatase. The pNPP substrate is sensitive to light and is unstable at room temperature. pNPP should always be stored on ice and protected from room light with a cover of aluminum foil. The alkaline phosphatase should also be stored on ice.
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AP Kinetic Parameters Spring 2011 - BCHM464 Spring 2011 AP...

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