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P-Nitrophenol Enzyme Lab Report

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The influence of changing amino acids sequence to the Alkaline Phosphatase (AP) enzyme optimal functions

Abstract
Hydroxyapatite in the bone is regulated by an enzyme called alkaline phosphatase. If a mutation in the AP enzyme exists, a possible outcome can be a decreased level of bone density. Through various experimentations, the functions of normal and mutant AP enzymes were observed. The variations in function of the enzyme were determined through different pH levels. In order to accomplish this, the activity of alkaline phosphatase needed to be examined by assessing how fast the product p-nitrophenol was forming. By incorporating the spectrophotometer, the optimal wavelength of light to use in the AP experiment had to be determined by …show more content…

P-nitrophenol was measured at various wavelengths through a spectrophotometer to find the optimal absorption. Once determined, p-nitrophenol was diluted in a buffer, NAOH, and PNP buffer to establish the standard curve for p-nitrophenol and the extinction coefficient. A time course was then established to determine over what length of time the reaction should occur. Substrate PNP and the pH buffer 10 were added to test tubes and placed in the incubator at 5 minute increments. With the information obtained through the experimentation, the hypothesis was ready to be tested. The amount of time it took the AP enzyme to convert PNP to p-nitrophenol was recorded to determine if there was a difference in the effect of pH on normal and mutant AP in heterozygous individuals. To ensure validity of the functional differences between normal AP and mutant AP, polyacrylamide gel electrophoresis was used. The center lane of gel with a pre stained protein standard was loaded first. Homozygous normal AP extract (15ul), heterozygous AP extract (15ul), and purified mutant AP (15ul) were the other three samples loaded into the wells. After the samples ran far enough, they were transferred to staining trays and washed with 50 ml of gel wash buffer, AP buffer, and pH 6. Next 50 ml of carbonate buffer, pH10 was added to one tray and 50 ml of citrate, pH6 was added to the other tray. Lastly 1 ml of AP dye and substrate was added to each tray and placed in 37 degrees Celsius water to incubate. Once the incubation was complete 50 ml of PBS was added to wash off the excess staining solution and the gels were ready to be

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