BIO LAB REPORT - Misfolded AP is investigated to determine...

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Misfolded AP is investigated to determine if it is the cause of a severe and rare form of osterporosis. Our hypothesis states that if the protein has abnormal folding it will have a different pH optimum than normally functioning AP. Preliminary information needed to be gathered in order to determine the abnormal AP’s optimum pH. First an Absorbance Spectrum was determined for p-nitrophenol, a substrate of AP. After gathering the absorbencies over varying wavelengths, the data is plotted on a graph to show the wavelength that maintained optimum absorbance. Next, a standard curve is constructed by measuring absorbencies of different concentrations of a p-nitrophenol solution. This standard curve will allow the extinction coefficient to be determined and unknown concentrations of p-nitrophenol solutions to be measured using the known absorbance. Next, an Optimal Timecourse is created to discover the optimum incubation time of an enzyme solution consisting of PNP, AP, and a pH 10 buffer. Absorbance is then determined and plotted on a graph to show where the peak is. Finally, the optimal pH level of our AP can be tested by mixing different pH buffers with PNP and AP. After the reactions occur, absorbencies are again determined and graphed to show the peak. Results concluded that the tested AP is in fact abnormally folded because it has optimum functionality of pH 6. This finding allows further testing to reveal if this misfolded protein is the cause of this rare life-threatening disease. If it is found to be true, a treatment or cure could be developed to fight the disease. Introduction Enzymes are defined as a protein catalyst used by living organisms to speed up and control biological reactions (Freeman, 2005). Environmental conditions such as pH
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and temperature can denature the proteins, or otherwise cause protein misfolding (Freeman, 2005). When proteins are misfolded, function of the enzyme is compromised (Lipton 2007) and can be the underlying cause of many highly debilitating disorders (Leila, 2008). Alkaline phosphatase (ALP) is glycoprotein structured metalophosphatase with several defined functions (Handan et. al. 2007). It is present in many tissues of all living beings from bacteria to mammals (Handan et. al. 2007), found in high concentrations in the liver, bile, and bones of humans and has an optimum functionality at pH 10 (Landon, 2008). Misfolded AP will have a different optimum pH level than normal human AP. The goal of the experiment is to determine if mutant AP affects function, and causes a rare osteoporosis is young women. The pH of the protein’s environment will affect the folding of that particular protein, so the misfolded protein will
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This note was uploaded on 04/07/2008 for the course BSCI 105S taught by Professor Nelson during the Spring '08 term at Maryland.

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BIO LAB REPORT - Misfolded AP is investigated to determine...

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