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
1-88×104 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)-