CHEM322 Foundations of Chemical Biology Laboratory
β-Lactamase: Protein Kinetics and Stability, Week 3
You have now characterized the purity and relative activity of the protein that you purified. This week you will utilize your pooled protein samples to examine two things:
Kinetic parameters for the purified enzyme using a continuous enzyme activity assay. Review the continuous activity assay.
Protein stability/activity following treatment with a number of reagents commonly utilized in chemical biology such as urea and hydrogen peroxide.
Why Study Enzyme Kinetics?
We will measure the amount of product formed at different substrate concentrations plotted as a function of time. The concentration of hydrolyzed nitrocefin product will be measured using absorbance at 486 nm. The molar extinction coefficient of hydrolyzed nitrocefin at 486 nm is 20,500 M-1 cm-1. Molar absorptivity, the molar extinction coefficient, is a measure of how well a chemical species absorbs a given wavelength of light. It allows you to make comparisons between compounds without taking into account differences in concentration during measurements.
Prelab Work:
What is Vmax and why does the activity plot level off at high substrate concentrations?
Learn about the reagents that we will utilize in our stability assays. Provide the chemical structure and 1-2 sentences about each of these reagents as they relate to protein structure, function and/or stability. Why are they commonly used in chemical biology?
Hydrogen peroxide
Urea
Guanidine thiocyanate
1,4-dithiothreitol
How does pH affect protein activity (hint: look up pH rate profiles)?
Materials Needed:
β-lactamase
75 µL – 8 M urea
75 µL – 8 M guandidine thiocyanate
75 µL – 30% hydrogen peroxide
75 µL – 5 mM dithiothreitol
75 µL – 20% sodium dodecyl sulfate (SDS)
15 µL – 1 M HCl
15 µL – 1 M NaOH
1 mL – 0.05 M phosphate buffer, pH 7.0
600 µL – 0.15 mg/mL nitrocephin solution (continuous assays)
50 µL – 0.5 mg/mL nitrocephin solution (spot test assays)
15 µL – 15 mM Bis-ANS (4,4′-bis-1-anilinonaphthalene-8-sulfonate) in MeOH
17 eppi tubes
parafilm
96 well plate (ROW E)
Half of the class should start with running the protein stability assays and the other half with the kinetics assays to prevent long lines at the plate reader.
Continuous Activity Assay on Purified Protein
Important Notes: A fresh stock solution of nitrocefin at 0.15 mg/mL in 0.05 M phosphate buffer, pH 7.0 is provided. For Michaelis-Menten kinetics, substrate concentrations ranging from 15 to 150 µg/mL will be ideal. Use the same amount of enzyme in all reactions; only substrate concentration will vary.
Perform continuous enzyme assays on your purified enzyme sample.
These will be performed in the 96 well plate with the plate reader.
Be sure to use the 0.15 mg/mL nitrocefin stock, not the 0.5 mg/mL stock. Be sure the nitrocefin is warmed to room temperature.
Be sure to mix each well with your pipettor before adding enzyme to ensure that the concentration of nitrocefin is homogeneous.
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).
Set up the plate (ROW E) as follows:
| Well 1 | 2 | 3 | 4 | 5 | 6 | 7 | |
| Nitrocefin Solution | 149 µL | 145 µL | 115 µL | 57.5 µL | 29 µL | 14 µL | 0 |
| Phosphate Buffer | 1.0 µL | 0 | 30 µL | 87.5 µL | 116 µL | 131 µL | 145 µL |
| DO NOT ADD ENZYME TO ANY WELL UNTIL YOU ARE AT THE PLATE READER | |||||||
| Enzyme | 0 | 5.0 µL | 5.0 µL | 5.0 µL | 5.0 µL | 5.0 µL | 5.0µL |
Use the nitrocefin-only well to autozero the spec.
You will need to do a full 2 minute experiment on each well to ensure that you get data immediately after enzyme addition.
Add your pooled fractions to a reservoir well and use the multi-channel pipettor to add 5 µL of enzyme to wells 2-7. For each well, add 5.0 µL of enzyme ALL AT THE SAME TIME, quickly mix, and immediately place in the plate reader and begin recording A486.
Look up information either in the text or in a credible source on graphing your data and calculating Km and Vmax. Remember to properly cite any references.
Plot A486 time for each reaction to determine the initialvelocity 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.
Use the slopes to calculate μM of product formed per minute for each reaction. This is the velocity.
Velocity = μM/min
Use the velocities to generate a Michaelis-Menten plot and a Lineweaver-Burk plot.
Using the Lineweaver-Burk plot, calculate the Kmand Vmax.
Protein Stability and Activity
We will examine the effects of several reagents commonly utilized in protein manipulation. Most of these reagents interfere with the tertiary structure of the protein in some way, often denaturation, and may also affect catalysis. We will utilize the qualitative spot test assay to discern the extent to which these reagents alter the ability of our enzyme to hydrolyze nitrocefin. Additionally, we will use Bis-ANS ((4,4′-bis-1-anilinonaphthalene-8-sulfonate) to determine if any inhibition of the enzymatic activity is due to protein denaturation. Bis-ANS is a fluorescent compound -but only under certain circumstances. Bis-ANS fluorescence is related to the polarity, viscosity and temperature of the environment it is in. It has low fluorescence in polar environments, and binding to non-polar portions of a protein amplifies this fluorescence allowing for visualization of this binding. Denaturation of the protein causes loss of the required non-covalent interactions and dissociation of the probe, decreasing fluorescence. Thus, this probe is ideal for evaluating folded state of proteins.(Extrinsic Fluorescent Dyes as Tools for Protein Characterization, Pharm Res. 2008, 25, 1487–1499)
Obtain seven eppi tubes and label them with the name of each reagent to be assessed. Add enzyme to each tube as below.
Reagent Table for protein stability assay
| Tube 1
Urea |
2
Guanidine |
3
H2O2 |
4
SDS |
5
DTT |
6
Heat |
7
Control |
|
| Reagent Amt | 50 µL | 50 µL | 15 µL | 50 µL | 50 µL | 0 | 0 |
| Phosphate Buffer | 0 | 0 | 35 µL | 0 | 0 | 50 µL | 50 µL |
| Enzyme | 50 µL | 50 µL | 50 µL | 50 µL | 50 µL | 50 µL | 50 µL |
Mix the tubes by gentle inversion, do not vortex. Let samples #1-5 and 7 stand for 15 minutes at room temperature with occasional mixing. Heat treat sample #6 for 5 minutes at 95 °C in the heat block and then let it cool to room temperature until the other samples are done incubating.
For the spot test assay be sure that you are using the 0.5 mg/mL nitrocephin solution.
Use 1 µL of each sample + 3 µL of nitrocefin (0.5 mg/mL solution) and combine into a spot on a parafilm wax sheet. You will need to make sure that each drop is mixed, so add the sample material first, then the nitrocefin and pipette up and down briefly for each sample. Make sure to keep track of each spot.
Begin timing the reaction once the substrate (nitrocefin) is added. Monitor the reaction and look for the color to change from yellow to red. Note the time it takes to turn red and record a qualitative score for each fraction, using a scoring system such as:
more than 2 minutes
1-2 minutes
30-60 seconds
5-30 seconds
Instant
Next examine the influence of pH on the rate of catalysis. To do this you will need to place two additional spots of 5 µL of nitrocefin on the parafilm. To one spot, you will add 1 µL of 1M NaOH and to the other spot, add 1 µL of 1M HCl. Mix each spot well using the pipettor. Add 1 µL of enzyme to each spot, mix well and record the reaction color change as before.
Next, you will examine denaturation of the proteins using the Bis-ANS. To each Eppendorf tube add 0.67 µL of the Bis-ANS solution and mix well. You can visualize this using the UV table. Normal protein will fluoresce a bright blue color while denatured protein will be dull blue or have no fluorescence. Use qualitative metrics to determine state of protein (i.e., folded or denatured)
It is always important to run controls. What happens when you add these reagents to nitrocephin? How would these results influence your ability to interpret the experiments in the presence of enzyme?
You have ~ 15 µL of extra nitrocephin solution (0.5 mg/mL). How can you use this to answer the question above?
Perform these experiments.
| Protein Stability Studies | ||||
| spot tests | Bis-ANS | |||
| Reagent | Final Concentration | qualitative score | qualitative score | Nitrocephin Controls |
| Urea | ||||
| H2O2 | ||||
| Guanidine | ||||
| DTT | ||||
| SDS | ||||
| HCl | ||||
| NaOH | ||||
| Enzyme Control | X | |||
