Km And Vmax Of Alkalne Lab Report

Jay A. Torres
With David Shbeeb and Matt Shinsky
Liberty University
1003 Misty Mountain road apt.507, V.A. 24502 [e-mail] words count: 1,313

Determination of Km and Vmax of Alkaline Phosphatase
Graphs will be used with the enzyme alkaline phosphatase of the unknown in the enzyme solution to Determine Km and Vmax

To Dr. Kimberly A. P. Mitchell, Editor, Liberty Journal of Cell Biology, 1971 University Blvd, Lynchburg, VA 24502.

Materials and Methods

Dilution: Alkaline Phosphatase
The objective of this laboratory is to determine the velocity of alkaline phosphatase and make a graph of rate vs substrate concentration (Michaelis Menten plot). After the wavelength was determined the para-Nitrophenyl phosphate (PNPP, Sigma Aldrich)
was obtained to get the absorption spectrum. To obtain a reaction of .5 A/min, a 0.5 of substrate solution was used and 0.015-0.07 ml of enzyme solution as well to bring a total of volume of 1 ml with the buffer. With the Genesys spectrophotometer the blank was set and diluted solution with a wavelength of 410 nm and set on the option of kinetics. The interval was set on 10 seconds from 0 seconds to a minute (60 seconds), and for the end the alkaline phosphatase was added. Then enzyme solution was saved for the other parts of the laboratory. As a result from the dilution, .492 absorbance/ minute was given.
Different Concentrations of PNPP
After calculating the amount of solution for the rate, with the formula M 1V 1=M 2V 2, 5 different dilutions for PNPP that varied by at least 100-fold was made. For the process of this enzyme, the procedure used the same as the first assay, which was the same spectrophotometer with the kinetic option.

Graphs to determine Km and the Vmax
With the different 5 trials and the time (intervals) trials of the assays results a graph to determine the Vmax and the Km was created .using the rate from the other graph and concentration of the substrate a new graph was created. The extinction coefficient was obtained from the standard curve. To determine the standard curve we used 25µM of PNP, but in order to get the results a graph of PNP vs time was created to determine the rate at each substrate concentration, then a graph for the rate vs the substrate concentration to also determine the Km and Vmax. The Km of alkaline phosphatase and V max. Making Lineweaver-Burke plot graph was calculated.

Results
Creation of the Standard Curve of PNP to Find Extinction Coefficient The extinction coefficient is basically used to obtain the slope of the line. This is essential to get the concentrations of PNP (5 trials) and PNPP. To find the extinction coefficient, we did a graph, which is the absorbance at 410nm versus the concentrations of the sigma Aldrich solution (PNP) and the absorbances were obtained from the Genesys spectrophotometer. The Lambert-Beer Law (A=εcl) was used for the concentration of the trials. As the final results the extinction coefficient 18.2 mM-1cm-1

Graphing PNP Concentration vs Time The only way to get the rate of the reaction is graphing the PNP concentration using the formula showed before (A=εcl) to get the concentration that we are looking for, the equation was rearranged.
After getting those results and calculating the extinction coefficient a graph was made to calculate the rate of reaction with the axes PNP (Sigma Aldrich) versus the time. The formula used to calculate the rate of reaction was v=Vmax[S]/Km+[S] (Vmax) is the maximum velocity of the enzyme, Km is the Michaelis-Menton, and [S] is the concentration of the substrate). The concentrations used in the graph are 0.5, 0.3, 0.05, 0.03, 0.005 mM. And the results for the rates of reaction were 0.4519, 0.4694, 0.2186, 0.1126, 0.0502 Mm/s.

Determining the Vmax and Michaelis-Menton constant for Alkaline Phosphatase After getting the result of the rate of reaction, the figure #3 was made with the axes of rate of reaction versus the PNPP. The reason why this graph was made is to determine the V max and the Michaelis-Menton constant using the formula K m=[S] at 1/2Vmax. The highest rate of Vmax was 0.4700 mM/s. using the formula ½ Vmax the value for the Km was determinate. After that the value of Y, which was 3.0739, was used to get the X value; and then using the equation of the hyperbola the Km was calculated. The value for the Km was 0.0500
mM

Determing the Vmax and Michaelis-Menton Constant for Alkaline Phosphatase Using Lineweaver-Burke plot
According to the laboratory book the Y intercept is 1/Kmax, for the X intercept is -1/ Km and for the slope is Km/Vmax.
The formula used to get the results was 1/v=1/Vmax+Km/Vmax*1/[S]. In order to get the Lineweaver-Burke plot, we need to get from the graph before (graph #3) the data. After solving for the Km and Vmax with the formula provided, the X and Y were solved to obtain the reciprocal values with the formula provided. The result for the y intercept was 3.0739 and for the x value was -35.5775463, and then the Vmax and Km were calculated with those results. The Vmax was 0.3252 mM/s and the Km was 0.02811 mM. As we could see the results were completely different from the Vmax and Km obtained from the Michaelis-Menton so we assume that could be on the graph that is not accurate when it tries to interpret the
enzymes.

References
DeWitt, D. A. and Mitchell K.A, 2014. Exploring Cell biology Laboratory Manual. 3rd ed. Academx Publishing Services, Inc., Sagamore Beach, MA. pgs. 21-26. Legends

Figure 1: Concentrations of PNP (mM) Vs Absorbance at 410 nm that calculated Extinction Coefficient of 18.2 M-1cm-1.
According to the book 410 nm wavelength was selected to calculate the absorbance for the concentration. With the slope we get from the graph, we calculated the different PNP concentrations versus the absorbance at 410 nm to then have the results for the extinction coefficient. It gave a final answer of 18.2 mM-1cm-1
Figure 2: PNP Concentration (mM) Vs. Time (Seconds) which showed us the final rate of reaction of the five different concentrations of the PNP
After getting the results of the 5 different concentrations using the formula c=A/εl (Lambert-Beer Law). So a new graph was created using the concentration plotted versus the time (s). The final rates of the reactions for the five different trial were first trial was 0.5 mM/s , second trial 0.3 mM/s , third trial 0.05 mM/s , fourth trial 0.03 mM/s , and for the fifth was 0.005 mM/s. the final result of the curve line was not that accurate because of the trial 4th that was a little off.
Figure 3: A Michaelis-Menton Graph with theresult of Vmax being 0.4700 mM/s and the Km being .0500mM.
The rate of reaction was used to graph them versus the substrate of the PNPP. Km=[S] at 1/2Vmax was the formula used for to the results for Km, but in order to that, the Y value was calculated to also calculate the X value using the formula ½ the Vmax. The highest rate of reaction was the Vmax and the value was 0.3 mM to get the value of Km, the x value was calculated first, which was -35.58 and then using the formula for the hyperbola showed the result for the Km that was 0.0500 mM

Figure 4: A Lineweaver-Burke Plot with a final result for Vmax of 0.32532 mM/s and the Km of .02811 mM.
Using the previous results from the graph #3, the inverse was made using the formula (1/v=1/Vmax+Km/Vmax*1/[S] to make Lineweaver-Burke plot. The final result for the Km and Vmax were 0.3253 mM and 0.028mM/s to get this results we solve for the X and Y. The Y intercept is 1/Kmax, for the X intercept is -1/ Km and for the slope is Km/Vmax