BioChemistry

Inhibitor Analysis Protocol Materials Needed:
β-lactamase
2 mL – 0.05 M phosphate buffer, pH 7.5
1.5 mL – 0.15 mg/mL nitrocephin solution (continuous assays) eppi tubes
96 well plate (ROW F, G, AND H)

You have been given a stock of one of the following inhibitors, located in your eppi rack:
300 µM penicillin (labelled 300 µM Pen) 300 µM ampicillin (labelled 300 µM Amp)

Continuous Activity Assay on Beta-Lactamase

A fresh stock solution of nitrocefin at 0.15 mg/mL in 0.05 M phosphate buffer, pH 7.5 is provided. For Michaelis- Menten kinetics, substrate concentrations ranging from 15 to 150 µg/mL are ideal. Use the same amount of enzyme in all reactions; only substrate and inhibitor concentrations will vary.

Required concentrations for penicillin and ampicillin: [High] = 50 µM
[Medium High] = 25 µM [Medium] = 12.5 µM [Medium Low] = 7 µM [Low] = 3.5 µM
[Very Low] = 1.7 µM

REMEMBER TO CONSIDER USING SERIAL DILUTIONS!

1. Perform continuous enzyme assays on your purified enzyme sample. You have been given a new solution of enzyme labelled B-lact that is located in your eppi rack.

a. Be sure to use the 0.15 mg/mL nitrocefin stock and that it is at room temperature.
b. Be sure to mix each well with your pipettor before adding enzyme to ensure that the concentrations of nitrocefin and inhibitor are homogeneous.

c. Once you add enzyme to any well it will be critical that you move extremely quickly so that you can take measurements within seconds. DO NOT add enzyme to any well until you are ready to read it immediately (i.e., standing at the plate reader).
2. Set up the plate (Rows F, G, and H) as follows:
a. In row F, add 20 uL of your enzyme to wells 1 – 10.
b. For rows G and H, use the following table:
Row G Well 1 2 3 4 5 6 7 8 9 10
Nitrocefin Solution 0 100 µL 100 µL 100 µL 50 µL 50 µL 50 µL 25 µL 25 µL 25 µL
Inhibitor none [H] [MH] [M] [H] [MH] [M] [H] [MH] [M] Volume needed 0
Phosphate Buffer 150.0 µL
DO NOT ADD ENZYME TO ANY WELL UNTIL YOU ARE AT THE PLATE READER
Enzyme 0 5.0µL 5.0 µL 5.0 µL 5.0 µL 5.0 µL 5.0 µL 5.0 µL 5.0 µL 5.0 µL

Row H Well 1 2 3 4 5 6 7 8 9 10
Nitrocefin Solution 100 µL 100 µL 100 µL 100 µL 50 µL 50 µL 50 µL 25 µL 25 µL 25 µL
Inhibitor none [ML] [L] [VL] [ML] [L] [VL] [ML] [L] [VL] Volume needed 0
Phosphate Buffer 45 µL
DO NOT ADD ENZYME TO ANY WELL UNTIL YOU ARE AT THE PLATE READER
Enzyme 5.0 µL 5.0 µL 5.0 µL 5.0 µL 5.0 µL 5.0 µL 5.0 µL 5.0 µL 5.0 µ L 6.0 µ L

c. For each well, use the multichannel pipettor to add 5.0 µL of enzyme to rows G and F, quickly mix by pipetting up and down 3 times, and immediately place in the plate reader and begin recording A486.
i. NOTE: Do NOT add enzyme to well G1. You will need to manually remove the first tip from the multichannel BEFORE adding enzyme to row G. After, quickly reload the multichannel to add enzyme to row F.
d. You will need to do a full 2 minute experiment on each well to ensure that you get data immediately after enzyme addition.
e. The absorbance should increase at a linear rate for most of this time period. If the absorbance of a sample maxes out the detector during your run, you will need to dilute your enzyme and try again.
f. The data from these trials will be used to perform kinetic analysis using Michaelis-Menten kinetics. Transfer your data to a flash drive before leaving the lab!

1. Calculate the percent inhibition of the compound against the nitrocefin control well as follows: 100 x [1.00 – (A480 of the well containing inhibitor/A480 of the control well)]. Only make these plots for the inhibitor experiments performed with the same amount of nitrocefin as the control well. Papanicolaou, G. A. et al. (1990) Antimicrob Agents Chemother 34, 2184-2192.

2. Plot A486 vs. time for each reaction to determine the initial velocity
for each substrate concentration. Remember that the velocity is estimated using the linear range of the reaction; therefore you can only use those values of the reaction to generate a best-fit line. Remember that the slope represents the initial velocity of the reaction, which is what we need to determine Km and Vmax.

3. Use the slopes to calculate μM of product formed per minute for each reaction. This is the velocity. Velocity = μM/min

4. Use the velocities to generate Michaelis-Menten plots and Lineweaver-Burk plots with each inhibitor.

5. Using the Lineweaver-Burk (L-B) plot, calculate the Km and Vmax for each inhibitor. Based on these values and their comparison to data you obtained in the absence of inhibitor, what type of inhibitor is each of the compounds you assessed? Competitive, uncompetitive or noncompetitive inhibitor?

6. Calculate the Ki values for the antibiotic.

L-B plot for a competitive inhibitor.

It is important to note that in calculations to solve for the Ki value of each compound, you need to use the Km and Vmax values obtained WITHOUT inhibitor. For example, if you determine that the inhibitors were competitive, the equation is written as follows:

Slope(with I) = [Km(no I)/Vmax(no I)] x (1 + [I]/Ki)

1. Fill out table 1 in the word doc I provided
Note there is 13 data tables ( In the excel file). Each table represents 10 second time interval. So, we start
from 0s for table 1, 10s for table two, ………, 120s for table13.
2. Calculate the percent inhibition of the compound against the nitrocefin control well as follows: 100 x [1.00
– (A480 of the well containing inhibitor/A480 of the control well)]. Only make these plots for the inhibitor
experiments performed with the same amount of nitrocefin as the control well. Papanicolaou, G. A. et al.
(1990) Antimicrob Agents Chemother 34, 2184-2192
3. Use the slopes to calculate μM of product formed per minute for each reaction. This is the velocity.
Velocity = μM/min
4. Use the velocities to generate Michaelis-Menten plots and Lineweaver-Burk plots with each inhibitor
5.Using the Lineweaver-Burk (L-B) plot, calculate the Km and Vmax for each inhibitor. Based on these
values and their comparison to data you obtained in the absence of inhibitor, what type of inhibitor is each of
the compounds you assessed? Competitive, uncompetitive or noncompetitive inhibitor?
6. Calculate the Ki values for the antibiotic.
please provide the excel file for the work with the calculation