Enzymes lab report

Paper details v The following questions are about a lab that was done- i Will be attaching the results and the lab manual ( they must be both used) it is lab number three in the lab manual under results and discussion- (pg 63) R1- Convert the corrected absorbances from step 7 into amounts of PNP. In your calculations, you should assume a molar absorptivity for PNP of 1-88x104 M-1cm-1 (£405), a path length for the cuvette of 1 cm and a final assay volume of 2-5 mL (This final volume takes into consideration the 2 mL of KOH that was added for stopping the reaction)- From the volume of the incubation mixture (05 mL aliquot: before the addition of KOH) and the substrate concentration, calculate the percentage of substrate converted- Show your calculations for a given time- From the concentration of the provided stock solution of enzyme, calculate the concentration of acid phosphatase in your assay. Summarize your results in the following Table 4- R2- Plot the amount of product (pmol PNP) versus time (minutes) for your results in Table 4- Estimate the initial velocity of the reaction- For how long does the reaction rate remain constant? Indicate the highest level of substrate conversion (in %) that you can observe in the linear range- Can you explain why plots sometime deviates from the straight line (reach a plateau)? Determination of KM and Vmax R3- Use the [PNPP] values calculated for Table 3- Convert the corrected absorbances from step 19 for both experiments 2 (control) and 3 (inhibitor) into amounts of PNP as above- Calculate the corresponding reaction rates (v)- Show all your calculations for one substrate concentration for the control (exp- 2)- Summarize your results in two tables (Table 5/6) With the following format: R4- From Tables 5/6 complete Table 7 for the analysis of results by the Lineweaver-Burk method, a linear form of the Kinetic equation- Show all of your calculations for one substrate concentration for the control- R5- From Table 7, construct the Lineweaver-Burk plot for the enzyme in the presence and absence of inhibitor. (Prepare one graphic With the two Lineweaver-Burk plots- Draw for each plot the best-fit straight line, also called the linear-regression trend line)- R6- From the linear equations of the two best-fit lines found in your Lineweaver-Burk plot, determine the KM and Vmax parameters corresponding to each condition- Show your full calculations for the control plot- Summarize your results in Table 8- Indicate the type of inhibitor that you have used- Justify your answer. Remember to use a 25°16 cut-off for your inhibitor identification (please see Appendix C4 for information on how to calculate the percent difference)-