BioC 4025W Fall 2017 Lab 8

Lab Manual LAB 8

ENZYME KINETICS OF LDH, PLASMID PREPS OF RECOMBINANT

PLASMID Pet15b/LDH AND ORIGINAL VECTOR Pet15b/rKGA,
DIGESTION OF BOTH PLASMIDS, SEQUENCING OF Pet15b/LDH

INQUIRY-PreLab-Lecture all rolled into one, so watch it, take notes, and prepare.

https://www.khanacademy.org/test-prep/mcat/biomolecules/enzyme-kinetics/v/an-
introduction-to-enzyme-kinetics

There is no formal pre-lab for this week.

Enzyme Kinetics
Enzyme kinetics is the study of enzyme rates and how these rates relate to substrate,
inhibitor and activator concentration. The enzyme rate is expressed as the change in
concentration of the substrate or product (this is usually what we look at) for a given
time interval.

If we consider the reaction A + B  P, the reaction rate at a given time is proportional

to the product of the concentration of the reactants raised to an approximate power,

Rate α [A]a[B]b or Rate = k [A]a[B]b

Where k is the proportionality called the rate constant and a and b are constants that
must be determined experimentally. If we consider the reaction A  B, the rate
equation is

Rate = k [A]1

This is said to be a 1st order reaction (exponent is 1) with respect to A. An example of

this reaction is radioactive decay where amount of radioactive substance is always
based on the amount of the substance and the decay constant.

If we look at the reaction (which is the same as the LDH reaction), A + B  C + D, it

is described as

Rate = k [A]1[B]1

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Where the reaction is first order with respect to A and first order with respect to B
(second order overall) and therefore the rate of the reaction should increase with
increase in either A or B.

The Michaelis-Menten
(M-M) Equation is the
foundation model for
enzyme kinetics. It posits
that an enzyme
reaction goes through an
enzyme-substrate ES. If
we consider the enzyme
catalyzed reaction (at
right), we can see the
formation and breakdown
of the ES. Conventionally, experiments are run for short periods of time and there is
little product build-up, so the equation simplifies to,

where kcat is the catalytic rate constant for formation of product, (breakdown of ES).

The reaction rate is dependent on the concentration of enzyme and of substrate, but the
dependence is different at different substrate concentrations, as shown below:

Velocity vs [Enzyme] Velocity vs [S]

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The hyperbolic Velocity vs. [S] plot above to the right is the observation that led to the
Michaelis-Menten model. The M-M equation relates the velocity to the substrate
concentration, KM and Vmax. These parameters can be seen on the Velocity versus [S]
plot below.

The KM tells us the substrate concentration

required to give the ½ Vmax. This is important
when you are trying assay the quantity of enzyme
present, in this case, you need to be well over the
KM in order to have the enzyme as the only
variable. The KM gives an idea of the affinity of
the enzyme and substrate. Knowing the KM of an
enzyme allows you to know whether its activity is
subject to changes in substrate concentration, in
vivo. If the in vivo concentration of the substrate
is close to the KM, then the enzyme activity will
change with changes in the substrate. A change
in KM can also indicate a post translational
modification of an enzyme that changes the enzyme’s affinity for substrate.

Vmax is also important parameter (but not a constant). The rate law is,

v0 = kcat[ES].
At Vmax, all the enzyme is in the ES form, so

kcat = Vmax/[Et]

by calculating the Vmax and the actual molar concentration of the enzyme, we can
calculate the kcat (also called the turnover number), which is the number of substrate
molecules converted to product per unit time by one enzyme molecule.

Values of these parameters (KM and Vmax) can be determined graphically from the linear
transform (y = mx + b) of the M–M equation by construction a Lineweaver–Burk plot.

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We can put the M–M equation in a linear form (y = mx + b) and then plot the data with
1/V on the y-axis and 1/[ES] on the x-axis.

There are other linear plots that can be

useful for analysis of kinetic data. In an
Eadie–Hoftee plot the M–M equation is
linearized as follows, and the v versus v/[S]
plot is shown.

Enzyme inhibition has been used to study enzymes, and from the way the inhibitors
interact with the enzyme we can determine enzyme mechanism. The study of enzyme
inhibition is also essential in screening for new drugs. Inhibitors can interact with
enzymes in different ways, competitively, noncompetitively and uncompetitive. Below
these 3 types of inhibition are illustrated in Lineweaver-Burk plots

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In lab you will run LDH reaction at varying NAD+ concentrations to determine KM for
NAD+ and the Vmax of the enzyme. You will also do this in the presence of an inhibitor
of LDH. By plotting these runs in a Lineweaver-Burk plot you should be able to
determine what type of an inhibitor it is. In lecture we went over how each type of
inhibition effects the enzyme reaction mechanism and how the inhibitor is
accommodated in the M-M equation.

Prelab questions – There are no prelab questions, but still write out an abbreviated
protocol and a summary of last week’s lab.

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Please note, start with Procedure 2 (Plasmid Preps), NOT Procedure 1.

After you have prepared your plasmids, set up your digestions (Procedure 3) then
do Procedure 1.

Procedure 1
Kinetics

PART ONE
• Use the concentrated AFC sample for the kinetic study. Choose the one that
had the best activity.

• Using the normal reaction cocktail mix, titrate your LDH fraction to determine the
volume needed to give a Δ Abs/min ~0.15-0.25 (in part 2)
a. Use 2, 3, 5, 7, 8, 10 µl of LDH
b. Adjust H2O volume so total volume stays the same – 1 ml
i. (e.g. 900 µl reaction cocktail, X µl LDH, X µl H2O)
ii. Once you find the correct amount of LDH to use, hold this value
constant for the duration of the experiment.

3. Use the following parameters for the NanoPhotometer (every 10 sec for a 1 min
assay)

4. You will use this volume for part two.

PART TWO
1. Dispose of your premixed reaction cocktail – you can’t use it for this part.

2. Mix each reaction cuvette separately: 633 µl CAPS buffer,

167 μl lactate, ___µl NAD+ (volume according to the table on the next page),
___μl LDH (volume determined in part one), add H2O so the total volume is 1 ml
– be sure to mix reaction well before adding LDH.

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3. Decrease NAD+ until no activity is seen (≥6 measurements). Collect LDH

activity data for each concentration of NAD+.

NAD+ H2O LDH LACTATE CAPS TOTAL

µl µl µl µl µl µl
167 167 633 1000
83 167 633 1000
40 167 633 1000
20 167 633 1000
10 167 633 1000
5 167 633 1000

4a. Repeat the above kinetic run substituting the lactate+inhibitor solution for the
lactate. (Lactate + 6 mM Oxamate) or (Lactate + 6 mM Oxalate)

NAD+ H2O LDH LACTATE + CAPS TOTAL

Inhibitor
µl µl µl µl µl µl
167 167 633 1000
83 167 633 1000
40 167 633 1000
20 167 633 1000
10 167 633 1000
5 167 633 1000

4b. Repeat the above kinetic run substituting the lactate+inhibitor solution for the
lactate. (Lactate + 12 mM Oxamate) or (Lactate + 12 mM Oxalate)

NAD+ H2O LDH LACTATE + CAPS TOTAL

Inhibitor
µl µl µl µl µl µl
167 167 633 1000
83 167 633 1000
40 167 633 1000
20 167 633 1000
10 167 633 1000
5 167 633 1000

Collect LDH activity data for each concentration of NAD+.

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Procedure 2

pET15b/rKGA – 7.3 kb
F 6xHis
NdeI

rKGA
(1.6 kb)

BamHI
pET15b
(5.7 kb)
R

AmpR

Plasmid preps of pET15b/LDH and pET15b/rKGA

(We decided that it would be best if each group could do their own sequencing, thus,
each group circled two colonies.)

The colonies you circled were used to inoculate tubes with 5 ml of LB/Amp. The tubes
for the entire lab were grown overnight in a shaking 37°C water. You will also get a tube
with pET15b/rKGA (in a 1.5 microfuge tube) that was also grown overnight and spun
down.

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YOU WILL DO THREE PLASMID PREPS:

You will do TWO plasmid preps of your pET15/LDH (given to you in 50 ml screw cap
tubes and ONE of pET15b/rKGA given to you in 1.5 microfuge tube already spun down.

Be sure to keep track which plasmid you are working with. And it is a good idea to label
the plasmid prep column to indicate LDH or rKGA.

1. For the pET15b/LDH culture, put 1.5 ml into a clean 1.5 microfuge tube and spin
at high speed for one minute. Remove the supernatant and add another 1.5 ml
pET15b/LDH culture to that tube and spin at high speed for one minute. Remove
ALL the supernatant.

For the pET15b/rKGA already in a microfuge tube, spin at high speed for 1
minute and remove ALL the supernatant.

You are ready to start with the step below for your three cultures:

2. Resuspend the pellet in 250 µl of Buffer P1. (P1 is in your ice bucket) To
resuspend, pipette up and down to break up the pellet and do a quick vortex to
make sure it is totally resuspended. It is important to get the pellet totally
resuspended.

3. Add 250 µl of Buffer P2. (P2 is on your bench in tube rack) Mix thoroughly by
inverting the tube 4-6 times.

4. Add 350 µl of Buffer N3. (N3 is on your bench in tube rack) Immediately invert
the tube 4-6 times.

5. Centrifuge for 10 minutes at high speed.

6. Put the supernatant into a spin column. (columns are on your bench in microfuge
tube rack). Be careful to only take the supernatant.

7. Centrifuge for 30-60 seconds. Discard the flow-through.

8. Add 0.75 ml of Buffer PE and centrifuge for 30-60 seconds. (PE is on your
bench in tube rack) Discard the flow-through.

9. Centrifuge on high for an additional minute. This is to remove residual wash

buffer.
10. Put the column into a new 1.5 microfuge tube.

11. Add 50 µl of Buffer EB to the center of the spin column. (EB is on your bench in
tube rack)

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12. Let it sit for 1 minute then centrifuge for 1 minute at high speed. Discard column.

Label the recombinant plasmids (vector with insert): pET/LDH#1, pET/LDH#2

Label the plasmid that was your parent vector: pET/rKGA

Procedure 3

One hour Digests of pET/LDH #1 and #2 and pET/rKGA

Set up the digests as listed below:

pET15b/LDH pET15b/LDH
dig #1 dig #2 pET15b/rKGA
Component Amount Component Amount Component Amount
Sterile Water 6 μl Sterile Water 6 μl Sterile Water 6 μl
10X buffer 2 μl 10X buffer 2 μl 10X buffer 2 μl
pET15b/LDH 10 μl pET15b/LDH 10 μl pET15b/rKGA 10 μl
NdeI 1 μl NdeI 1 μl NdeI 1 μl
BamHI 1 μl BamHI 1 μl BamHI 1 μl
Total 20 μl Total 20 μl Total 20 μl

After all components have been added, do a quick flick of the tubes, then a quick spin to
bring all reagents down, and put into a 37° water bath.

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Procedure 4

1% Agarose in 30 mL 0.5X TBE gels to check results of digestions

After one hour you can run your digests on a 0.8% 0.5X TBE gel. 2 groups per gel.

To set up plasmids to run on gel, do the following:

To your digested and undigested plasmids:

20 µl plasmid (this is the entire volume of your digests)

2 µl 10X loading dye

(Put 20 µl of your uncut plasmid into a 0.5 microfuge tube and add the loading
dye.)

Load 10 µl into the wells on the gel.

Run the gels at ~ 125V about ½ the distance to the bottom.

YOU WILL RUN YOUR DIGESTED SAMPLE AND AN UNDIGESTED SAMPLE OF

THE SAME PLASMID TO COMPARE.

Please note, you will just run one sample of your undigested plasmid pET15b/LDH
rather than both plasmids. This way we can have 2 groups per gel.

Lane 1 1st digested plasmid pET/LDH #1 (GROUP 1)

Lane 2 1st digested plasmid pET/LDH #2 (GROUP 1)
Lane 3 1st undigested plasmid pET15b/LDH #1 or #2 (GROUP 1)
Lane 4 2nd digested plasmid pET15b/rKGA (GROUP 1)
Lane 5 2nd undigested plasmid pET15b/rKGA (GROUP 1)
Lane 6 1 Kb Ladder
Lane 7 1st digested plasmid pET/LDH #1 (GROUP 2)
Lane 8 1st digested plasmid pET/LDH #2 (GROUP 2)
Lane 9 1st undigested plasmid pET15b/LDH #1 or #2 (GROUP 2)
Lane 10 2nd digested plasmid pET/rKGA (GROUP 1)
Lane 11 2nd undigested plasmid pET15b/rKGA (GROUP 2)
Lane 12 Empty

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Procedure 5

Sequencing of pET15b/LDH

DO NOT USE THE pET15b/rKGA PLASMID YOU JUST MADE.

Use 0.5 microfuge tubes and WRITE ON THE LIDS-not the tubes…the LIDS

You will use forward and reverse primers.

1. You have a chart at your station. Write on the lid of the tube the initials and numbers
that correspond to the sample you will submit.

WRITE EXACTLY WHAT THE CHART AT YOUR STATION TELLS YOU TO PUT
ON THE LID, e.g. BMP101. THIS IS A UNIQUE LABELING SYSTEM AND THE
SEQUENCING CENTER REQUIRES THREE INTIALS FOLLOWED BY THREE
NUMBERS. DO NOT WRITE ON THE SIDE OF THE TUBES -ONLY THE LIDS.

You need to send in ~600 ng, therefore, you need to spec your samples using the
Nanodrops. If you have 400 ng and above, use 1µl and adjust the water, see chart.
If you have less than 400 ng, use 2 µl as shown in the chart below. Your final volume is
12 µl.

Set up your sequencing as shown below: If you use 1 µl of plasmid, adjust water to 9 µl.

Sequencing #1 (Forward Primer: 5’ – GTG AGC GGA TAA CAA TTC CCC TC – 3’)
Reagent Amount
Undigested pET15b/LDH plasmid 2.0 µl
ET15P1 (50 ng forward primer) 2.0 µl
Sterile Water 8.0 µl

Sequencing #2 (Reverse Primer: 5’ – GAC CCG TTT AGA GGC CCC – 3’)
Reagent Amount
Undigested pET15b/LDH plasmid 2.0 µl
ET15P2 (40 ng reverse primer) 2.0 µl
Sterile Water 8.0 µl

3. Put your tubes into the ice bucket where the TA sits. There is also a sheet with the
ice bucket and please check off the list so we know you have given us your tubes.

Your samples will be sent to St. Paul for sequencing. You will analyze your results
next lab period.

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What’s Due Next Lab

Data and Summary/Conclusions Lab 8, Procedures 1 - 5

Lab Reports 8a and 8b including data and questions (Note it is one lab report but two sections
worth high points so be sure not to miss doing both!)
Bring both as “completed” so we can compare graphs and data for discussion and you
will still be able to complete your lab report to turn in the next lab period.

Prelab write-up in notebook Week Lab 9, Procedures 1-4

Manuscript LLR Keep writing and bring Methods and Results 3-8 for review
Post by ~10pm the night before your lab section and bring completed printed copy for
peer review in lab.

CLEAN UP: Blah blah blah same routine…clean your stations 

NO INQUIRY Check out just think about and write in lab notebook:

THIS IS A DETAILED LAB SO WORK ON LAB REPORTS TOGETHER AND WITH TA

AS MUCH AS POSSIBLE BEFORE LEAVING

INQUIRY: Design an experiment with the His-tag using chromatography. How does
the protein bind and how would you get it off the column once it bound?

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