Alkaline Phosphatase

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Alkaline phosphatase, or AP, is an enzyme that is responsible for dephosphorylation, or removing phosphate groups, of various types of molecules such as proteins and nucleotides. It has a molecular weight of 140-160 kilo-Daltons and as said in the name, alkaline phosphatase works in an environment with pH values ranging from 7.5-9.5, which is an alkaline, or basic, environment. And according to Sigma-Aldrich, alkaline phosphatase can be used to “to dephosphorylate the 5'-termini of DNA or RNA to prevent self-ligation” (sigmaaldrich.com). Alkaline phosphatase in gram-negative bacteria can be found in the periplasmic space, which is found outside of the cell membrane. Because of this, it is subject to various types of environmental activity, making alkaline phosphatase more resistant to being denatured, inactivated, and degraded, while also giving AP a higher rate of activity.
Every cell has a plasma membrane, which is a protein-lipid bilayer. It forms a barrier that separates the cell contents from the outside environment. Amphipathic lipids make up the plasma membrane, meaning they have hydrophilic and hydrophobic parts. The hydrophilic heads and hydrophobic tails form a “sheet” containing lipids whose hydrophobic tails face each other. In order to obtain something from inside the cell, or in this case alkaline phosphatase, one could use the process of cell lysis. Cell lysis is a process that breaks down or destroys a cell wall due to an outside force. Cell lysis can occur both naturally and artificially. Natural lysis can occur through viral infections whereas artificial lysis can occur by adding a substance that can break down a cell wall.
One such substance that can cause artificial cell lysis is lysozyme. Lysozyme, or N-acetyl...

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...tions collected, a spot test can be used. The spot test is a qualitative determination to see if a molecule exists in solution. For this spot test, para-nitrophenylphosphate, PNPP, in solution was used to determine fractions from IEC had AP or not. PNPP was used specifically because when PNPP comes into contact with AP in basic conditions, a phosphate from PNPP is cleaved off, becoming para-nitrophenol, PNP. PNP turns yellow in basic conditions. If AP was in a fragment, the yellow tint shown would indicate such.
The experiment was performed using mutated E. coli, cell lysis, two centrifugations, two dialysis processes, heat denaturation, salting out via ammonium sulfate, anion exchange chromatography, and spot testing. By following these procedures, one should be able to obtain a lot of purified alkaline phosphatase and should see a yellow tint during the spot test.

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