post lab 9

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Arleny Paulino Post lab 9 Understanding the activity in a continuous assay and the results of inhibition of alkaline Biochemistry Professor Jessenia Lopez Due day 12/07/22

Materials and methods Determination of activity km and coax for alkaline phosphate The fraction in lab identified in lab 8, fraction number 8. the fraction was used for the assay. First, 1mM PNPP was placed in a micro tube. Then 750 minutes 1nM PNPP ul plus 659 ul of buffer, and 0.2 M tris -HCl, pH 8.0 was place in together a clean cuvette. This was our black which one was placed in the Kinect program to store the bio mate spectrometer to complete the absorbance measurement. The substances were measurement at 410nm every sec for three minutes. Then, 100ul of the enzyme was added to the cuvette with the final volume 1.5 ml and mixed probably. The cuvette wad place in the spectrometer for measurement the sample. Part B Determination of Km and Vmax for AP 0.5 mM PNPP was place in a micro tube as the purified AP. Then, 9 different sample were prepared with the concentration of PNPP solution in tris and 0.2M tris-HCL with a pH. Of 8. The nice sample were prepared with different values showed in the table number 2 in the appendix, with a final volume of 1500 uL. Each cuvette was the own blank, the addition of tris and PNPP first and have been measurement as a Blank. Then, the enzyme was added and measurement again as a sample measurement. Part II: Product Inhibition of Alkaline Phosphatase In this part, there were prepared five samples with different concentration of sodium phosphate (PI) by the used of the stock 1mM Pi, 0.5mM PNPP solution, water, and 0.3 tris- HCL, pH =8. The constants I this part was 0.5mM. The different values for the values are in the table 3 in the

appendix. The blank was the PNPP mixed with tris and-HCl and PI. Then the enzyme was added later for measurement the sample absorbances. The samples were measurement 410 every 30 sec for total 4 minutes. The final volume of the sample was 1500 after the addition of the enzyme. Then, another 5 cuvettes were prepared but with a higher concentration of Pi by used the stock 1mM Pi, 0.5mM PNPP solution, in water and 0.2 tris- HCl with a pH of 8. In this part the constant of PNPP is 0.150mM. The different values for the sample are showed in the appendix in table 4. Then, it will the same process then before, the blank was PNPP mixed with tris-HCl and Pi. Results This experiment focused on learning about the activity of the enzyme alkaline phosphatase (AP.) Use the fraction identification from Lab 8, fraction number 9. The activity of enzyme AP was calculated using Beer Lamber's law. AP. Convert A min to micromoles per minute [PNPP]. After determining the "amount of PNPP formed in nanomoles per minute in the reaction mixture (1.5-mL volume)" (Domashevskiy, 2022), we used this value to "calculate the enzyme activity as milliunits per milliliter of the enzyme," which resulted in enzyme activity of 246.06 mU/mL (as seen in Appendix 'Calculate the activity of the AP in your purified fraction.') This section of the experiment was designed to measure the Km and Vmax of alkaline phosphatase by using pure alkaline phosphatase from a standard 1 mM PNP in a pH 8.0 buffer supplied by Sigma. Inside polystyrene micro cuvettes, nine different concentrations of 0.5mM [PNPP] and para-nitrophenyl-phosphate were generated. The stock PNPP solution in TRIS is

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0.5mM, and the 0.2M Tris-HCl at pH 8.0 is used to make the other concentrations. After three minutes of utilizing the BioMates 3S to take readings at 410 nm every twenty seconds, 100 Each cuvette received L of Sigma's AP. Michalis-Menten Graph of alkaline Phosphate Curve Figure one. Michalis-Menten Graph of alkaline Phosphate Curve. Alkaline phosphatase's Michaelis Menten plot has a range of Vmax = 0.6032 Abs/min, Km = 0.1020 mM, and R squared = 0.9622. this data, this curve was generated in GraphPad Prism. Standardized final PNPP concentrations in millimoles per liter (mM) serve as the x- axis of the resulting concentration-time curve. The AP velocity (y-axis) was calculated from AP standard measurements taken every 20 seconds for 3 minutes at 410nm using a UV-spectrophotometer and an AP Kinetics software, yielding a slope in (A/min). The Michaelis-Menten Curve shown in figure one was generated using data in table 2 in the appendix, created with the program graph Prism, and a nonlinear regression fit. The program analyzed the relationship between alkaline phosphatase's velocity (A/min) at 410nm and final [PNPP] mM, yielding values for the enzyme's Km and Vmax. The Km value is 0.1020mM and the maximum absorption rate, Vmax, is 0,0632 A/min, with an R2 value of 0.9622. Km values were measured and compared to the 0.08mM value reported in the literature. The error was calculated to be 10.2 percent (Appendix ‘Km Percent error for Michaelis Menten Plot’). Vmax = 0.6032 Km= 0.1020 R 2 =0.9622

Figure two. Lineweaver-Burk of Alkaline Phosphatase with a pH of 8 This is a linear regression analysis, which compares various quantities of pure alkaline phosphatase produced by Sigma to one another. using the program prism to generate this graph at 410 nm. The value of the x-intercept was -19.18, while the value of the y-intercept was 2.725. The x-intercept was used in the calculation of Km, which yielded a value of 0.0521mM. It was determined that Vmax equaled 0.367abs/min after using the y-intercept in the calculation. The maximum possible coefficient of determination, R-Square value 0.6769. Figure two shows a graph of the Lineweaver-Burk of alkaline phosphatase in figure II. This graph was created using Graph Prism 9 and the linear regression fit. The graph was constructed by plotting the 1/final [PNPP] mM value with the 1/velocity at 410nm value. However, the y- intercept and the x-intercept were included in the data supplied and were obtained in the graph prism. To determine the values of Km and Vmax, the y-intercept and x-intercept formulas are found in the Appendix (Km and Vmax for Lineweaver-Burk Plot). Both the x-intercept was - 19.18 and the y-intercept 2.725. In Appendix 'Km and Vmax for Lineweaver-Burk Plot', the

calculations for Km and Vmax were carried out, and the results showed that the value for Km was 0.0521mM, while the value for Vmax was 0.367 abs/min. The R2 value was of 0.6769. Figure three . Dixon plot on alkaline sulfates conditions. For alkaline phosphates, the range of this D Dixon plot is from an equation for 0.5mM PNPP (y = 0.0005829X +0.1522) with an R squared of 0.8204 to an equation for 0.150mM PNPP (y = 0.0009290x + 0.03206) with an R squared of 0.4663 and a value of Ki =0.0020631474. To 0.5mM PNPP Y=-0.0005829 * X +0.-1522 R-square =0.8204 0.150Mm PNPP Y= 0.0009290 * X+ 0.3206 R-square =0.4663 Ki=0.0020631474

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create this graph, we used GraphPad Prism. We measured the AP standards generated at pH 8t over three minutes at 20-second intervals at 410 nm using a UV spectrophotometer and the AP Kinetics software. Discussion : Appendix:  Table 1. Determine Activity and Specific Activity  Volume (uL) Abs/min 1500 0.292 Table 2. Determine K m and V max Final [PNPP]mM Abs/ min 0.01 0.076 0.02 0.058 0.04 0.122 0.06 0.213 0.08 0.313 0.10 0.295 0.20 0.428 0.30 0.478 0.40 0.496  Table 4. for 0.05mM PNPP Final [Pi] uL Abs/min 0 0.159 3.33 0.142 20 0.143 50 0.123 100 0.133  Table 5. For 0.150mM PNPP

Final [Pi] uM Abs/min 0 0.363 3.33 0.251 20 0.322 50 0.286 100 0.220 1. Calculate the activity of the AP in your purified fraction.  [PNPP]/min = (A/min) / e * l = (0.292 a/min )/ 1.78* 10 -2 l. umol. 1cm = 16.404 umol .L -1 calculate the amount of PNP formed (in nanomoles per minute) in the reaction mixture (1.5-mL volume) = [PNPP]/min *1.5mL = 16 .404 uM. L -1 . Min -1* 1.5 uM. mL= 24.606 nanomoles / min = 1 enzymes = 24.606 mill unit x 1 mol / 0.1 = 246.06 mill units 2. Km and Vmax for Lineweaver-Burk Plot K m = x-intercept = -1/ k m K m = - 1) / x-intercept K m = - 1 / -19.18 = 0.0521376434 K m =0.0521mM Vmax = y-intercept = 1/ V max = Vmax = -1 / 2.725 Vmax = 0.367 abs//mim \ 3.. Ki 0.05 mM PNPP Slope = 0.0005819 Y-intercept= 0.1524Y=mx+b Y= 0.0005819x + 0.1524 0.150 mM PNPP Slope= 0.0009290

Y-intercept= 0.3206 Y=mx+b Y= 0.0009290x + 0.3206 0.0005819x + 0.1524 = 0.000929x + 0.3206 -0.00003471x = 0.1682 X = -0.00206361 Ki = -0.00206361 Um Error percentage I. K m Percent error for Michaelis Menten Plot For K m literature value = 0.08mM Percent error= experimental−theoretical × 100 theoretical Percent error= = 0.1020mM * 0.08mM / 0.08mM *100 = Percent Error = 10.2% K m Percent error for Lineweaver-Burk Plot For K m literature value = 0.08mM Percent error= experimental−theoretical × 10 0 theoretical Percent error=0.0521mM * 0.08mM / 0.08mM x 100 = Percent error = 5.21% Observation

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