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Alkaline Phosphatase Lab Report

Alkaline Phosphatase Lab Report - Agustin Garcia Lab Report...

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Agustin Garcia Lab Report Biology 2107 Introduction Alkaline phosphatase is an enzyme that is seen in the human body. Its role in our metabolism is somewhat unclear as of now though. It is known to have a structure of a zinc metalloenzyme with two identical subunits and serine at the center of the molecule (2). It is responsible for the removal of phosphate groups from molecules in the body. It is found in the placenta and in the intestines for humans (3). It also plays a large role in bone resportion, as well as human calcification. In bone resportion, it alkaline phosphatase retards the rate of deposition of calcium and phosphate. Although not clear yet, alkaline phosphatase may also play a role in DNA synthesis and growth. (3) A possible role in the development of our genetic material makes alkaline phosphatase a very relevant enzyme. It has also been found in bacteria and many other mammals. Scientists hope to get a better understanding of the enzyme in the near future and develop ways to possibly control and discover its actual function soon. Recently, alkaline phosphatase has become a part of the medical field. Its serum content is helpful to people suffering from skeletal disorders (2) Materials and Methods Reagents Alkaline phosphatase enzyme and para-nitro-phenyl-phosphate (PNPP) substrate will be used for performing assay. BPA or BSA will be used to slow self-degradation of alkaline phosphatase. This stabilization of the alkaline phosphatase has already been performed by adding 0.1 mL stock alkaline phosphatase to 9.9 mL of BSA in 100 fold dilution factor. PNPP Assay
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Agustin Garcia Lab Report Biology 2107 Colorimetric assay will be performed with spectrophotometer to identify ideal temperature for enzyme activity in alkaline phosphatase. pH conditions must be kept constant throughout using only pH of 11 for all temperature experiments. Spectrophotometer should be set to absorbance at a wavelength of 410 nm. Spectrophotometer must be blanked before each temperature experiment. Prepare first blank by adding 4.5 mL PNPP substrate at conditions of pH 11 and 0 degrees C to 5 mL BSA at 0 degrees C. Mix and return to ice bath. Fill cuvette 2/3 full with mixture and zero spectrophotometer. Prepare enzyme tube by combining 4.5 mL PNPP substrate
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