Enzyme Kinetics: Investigating Alkaline Phosphatase Activity

The extinction coefficient that was found of the phenolate ion was 19.825M^-1cm^-1 and the pKa of the p-nitrophenol was 7.05. This was found from the previous lab Photometry in order to demonstrate the Kinetics lab.

The kinetic assay used the enzyme; alkaline phosphatase, the substrate p-nitrophenyl phosphate, the product p-nitrophenolate ion, and the inhibitor used is orthophosphate. A concentration equation was used to identify the concentration of substrates that were used in the cuvettes. In order to determine the equation.

The volume of the substrate that was used was 2.7mL, the total volume was 3mL, and the concentration was the changing variable in the stock solution.

The concentrations of substrates used from the concentration equation was determined as 0.9mL in 1 mM stock solution, 0.68mM in 0.75mM stock solution, 0.45mM in 0.5mM stock solution, 0.23mM in 0.25mM stock solution, 0.09mM in 0.1mM stock solution, 0.045mM in 0.05mM stock solution, and 0.023mM in 0.025mM stock solution.

These concentrations were helpful when fining out the amount of product that formed an uninhibited full concentration enzyme, uninhibited half concentration enzyme, and the inhibited enzyme.

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When alkaline phosphatase is added to p-nitrophenyl phosphate the reaction will begin, meaning the substrate is being used and a product will begin to form. For that reason, the product that is being formed is recorded every 10 seconds for 70 seconds as an absorbance.

The uninhibited full concentration of an enzyme, the uninhibited half concentration of an enzyme, and the inhibited enzyme all were graphed using excel with a y-axis and x-axis labeled as absorbance vs.

time. A line was plotted, and slope was found to equal the velocity of the enzyme.

Next, the slope was multiplied by 60 minutes to give the absorbance/minute. After, the absorbance/minute value was multiplied by 1,000,000 ?mols/min. This value was multiplied by 0.003L and that would give you the final value of V0 in ?mols/min.

A Lineweaver-Burk plot was created to find the slope which is known as Km/Vmax and y-int which equals 1/Vmax. The equation for Vmax is known as 1/y-intercept. When u find the value of Vmax then you can divide that by 2 to find Km. The Km is known as the Michaelis constant which is helpful when determining the rate of both binding and release of the substrate and release of the product from the enzyme.

A Michaelis Menten plot is a non-linear regression plot which is most accurate when compared to the Lineweaver-Burk plot which is a linear regression plot. This is due to the fact that at a low substrate concentration the linear plot doesn't work as well, meaning there is no reliable data. The Michaelis Menten plot is most accurate because it demonstrates the rate of change in different substrate concentrations. In this particular lab, low substrate concentrations were used in this case.

From what was graphed and plotted, no sources of errors were found. This may not always be the case. Some sources of error that could have happened was not putting the cuvette back in the spectrophotometer fast enough after enzyme had been added.

This is due to the 10 second time slot that was given. Another error that could have occurred is the cuvette not being mixed well enough when the enzyme was added. One other error to consider is the uninhibited full enzyme was recorded from a separate group. We don't know if they're absorbances are accurate or if there were mistakes made when doing the experiment.

There are many ways in which this lab can be considered in a clinical significance. Alkaline phosphatase is applied to a group of enzymes which all share the capability to hydrolyze the phosphate esters in an alkaline (AACC 2019). A test of alkaline phosphatase is used to help detect liver disease and bon disorders. This is helpful when detecting early. It can help detect malnutrition, which can be caused by diseases. Overall, this lab is useful when treating someone.