1

BCH 372
Modern Concepts in Biochemistry Laboratory

Laboratory 4
Assay for L-Lactate Dehydrogenase
The purpose of this laboratory session is to learn how to measure the activity of L-lactate
dehydrogenase (LDH) and to define some of the basic factors that affect the rate of the reaction.
As noted earlier, L-lactate dehydrogenase catalyzes the reduction of pyruvate to form L-lactate in
the presence of NADH. It also catalyzes the oxidation of L-lactate to form pyruvate in the
presence of NAD+ as an electron acceptor.

While the reaction can be measured in either direction, it is more convenient to assay LDH
activity in the direction of L-lactate oxidation because NADH absorbs light at 340 nm and NAD+
does not. The reaction thus can be measured as an increase in A340. For this experiment, you
will be provided with a stock solution of a purified sample of LDH from bovine heart. You will
determine the activity of this solution first. You will then determine the substrate specificity of
the reaction and measure the effects of pH on its activity using buffers.
I.

PreLab Preparation

Before the lab, please read Chapter 4 in the lab manual Experiments in Biochemistry: a
hands-on approach by S. O. Farrell and L. E. Taylor. You do not need to understand all of the
details related to enzyme purification for this lab session, so concentrate on sections 4.1, 4.4, 4.6,
and 4.8. Pay particular attention to the calculations shown in Practice Session 4.2 on page 97
and to the additional calculations on page 99 of the lab manual. Students sometimes find the use
of the terms rate, activity, and specific activity confusing and have difficulty doing the
appropriate calculations. The section on pages 8-12 at the end of this handout summarizes the
calculations that will be done throughout the semester. At this point in the course, you only need
to pay attention to those related to initial velocity and enzyme activity.

II.

Laboratory Procedures

A.

Measurement of L-Lactate Dehydrogenase (LDH) Activity

The purpose of this part of the experiment is to learn how to measure the activity of LDH
using a standardized protocol. The standard protocol or assay involves combining a buffer (0.15
M CAPS, pH 10.0), a solution of L-lactate (the primary substrate), a solution of NAD+ (the
electron acceptor), and the enzyme L-lactate dehydrogenase (LDH). An alkaline pH buffer
favors the reverse (L-lactate -----> pyruvate) reaction. While the amounts of the buffer, Llactate, and NAD+ will usually be the same, the source and the volume of the enzyme solution
can be varied. The reaction will be followed by measuring an increase in absorbance at 340 nm
as NAD+ is reduced and as NADH is formed. You will start with step 4 of the experiment
described on page 102 of the lab manual.
1.

Turn on the Genesys 20 spectrophotometer and allow it to warm up for 15 minutes. Set
the wavelength to 340 nm.

2.

You will be provided with the following stock solutions for the LDH assay:
Assay Buffer Solution = 0.15 M CAPS, pH 10.0
NAD+ = 6 mM
L-lactate = 150 mM, pH 7.0
stock bovine heart LDH solution = an ammonium sulfate preparation of bovine heart
LDH which was diluted about 1/200 in 0.1 M KPO4 buffer, pH 7.5

3.

To set up a standard assay, add 1.9 ml of the Assay Buffer to a 4.5 ml methacrylate
cuvette. Then add 0.5 ml (500 l) of the NAD+ solution and 0.5 ml (500 l) of the Llactate solution. Note that the total volume at this point is 2.9 ml. It will be easiest if
you use a 5 or 10 ml pipet to add the buffer and a P-1000 micropipetter to add the NAD+
and L-lactate solutions. Be sure to use a clean tip for each solution so you do not
contaminate the stocks.

4.

Since you do not know how much activity is present in the stock solution of LDH, you
will probably need to try several different volumes in order to find a volume that gives a
reasonable rate of reaction. The final total volume in the cuvette should always be 3.0
ml (3000 l), so you can add up to 100 l (0.1 ml) of the enzyme solution. You will
make up the difference (up to 100 l) with water.

5.

Start by trying to detect LDH activity with a small volume of the stock enzyme solution.
After adding the CAPS buffer, NAD+, and L-lactate to the cuvette, add 90 l of water.
Place a piece of Parafilm over the top of the cuvette and invert the solution several times
to mix everything together. Insert the cuvette into the spectrophotometer and set the
instrument to zero absorbance.

3
6.

Then remove the cuvette from the spectrophotometer and add 10 l of the stock enzyme
solution. Rapidly place a piece of Parafilm over the top of the cuvette and invert the
solution several times to mix everything together. Place the cuvette back in the
instrument and read the absorbance at 340 nm. Take readings at 15 second intervals for a
total of 3 minutes (180 seconds). Record the absorbance values in your lab notebook.

7.

If the reaction occurs at a reasonable rate - an increase in A340 of about 0.1 or 0.2 per
minute - repeat the assay two more times, so that you have a total of three (3) replicate
assays.

8.

If the rate of the reaction with 10 l of the stock enzyme solution is too slow, try adding a
larger volume of stock enzyme such as 25 l or 50 l to the reaction. Be sure to reduce
the amount of water, so that the final total volume in the cuvette is 3.0 ml in each
case.

9.

If the rate of the reaction with 10 l of the stock enzyme solution is too fast, make an
additional 1/10 dilution of the enzyme solution by adding 100 l of the enzyme solution
to 900 l of 0.1 M KPO4 buffer, pH 7.5, in a microcentrifuge tube. Then assay 10, 25, or
50 l volumes of the diluted enzyme using the standard protocol. Again, the total volume
should be 3.0 ml. Once a suitable volume of the enzyme solution has been identified, do
three (3) replicate assays.

10.

Once you have obtained three good replicate assays of the LDH activity in the stock
enzyme solution, plot the results on a piece of graph paper as shown in Figure 4.3 on
page 97 of the lab manual. Note that Absorbance (at 340 nm) is plotted on the Y axis as
a function of Time in Seconds on the X axis. Make a separate graph for each of your
three reactions. You can also make graphs using Excel. Be sure to do this as XY
scatterplots, again with A340 on the Y axis and Time in Seconds on the X axis. While you
may find that a reaction continues linearly for the full three minutes, it is more likely that
it will start off at a linear rate but then slow down. This is commonly observed with
NAD+-dependent dehydrogenases.

11.

You should only use the initial linear rates of reaction in all of your calculations. If you
make the rate plots on graph paper, use a ruler to identify the linear portion of the rate
curve for each reaction. Draw the best-fit straight line to as many points in the linear
region as you can. The line does not have to go through the origin because some
additional absorbance at 340 nm may be introduced when the enzyme solution is added.
If you make the graph in Excel, you can fit a linear trendline to the data points. You may
need to select which data points to use in order to ensure that you only have a line
through only the linear region of the rate plot.

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B.
1.

Calculation of the Initial Velocity and Activity of the LDH Reaction

Use the linear part of each rate curve to determine the initial velocity (Vo) or rate of the
reaction. This value corresponds to the slope of the straight line. You can calculate this
Vo value by taking the difference between any two absorbance values within the linear
portion of the curve and then dividing by the difference in time between these values.
Vo

A340 (time 2) - A340 (time 1)

time 2 (seconds) - time 1 (seconds)

Vo

A340
second

If you are making graphs with Excel, you can use the equation for the trendline that you
fit to those points within the linear region. Be careful, however, to fit the line to only
those data points in the linear region.
2.

Then convert the initial velocity for each reaction to a change () in the absorbance at
340 nm per minute (A340/minute) as follows:
Vo

A340
second

A340
minute

60 seconds
minute

3.

Then determine the average initial velocity as A340/min. That is, calculate the mean of
the three replicate assays with a particular volume of the stock enzyme solution or a
dilution of it.

4.

Next, express the average initial velocity in terms of the actual number of moles of
NADH formed per minute. The molar extinction coefficient for NADH is 6220 A340 M-1
cm-1 (refer back to Laboratory 3). This means that a 1.0 M solution of NADH in a 1 cm
light path cuvette will have an A340 of 6220. Absorbance is usually expressed on a 1.0 ml
basis, so an absorbance of 6220 also represents what would be observed with a 1
mmole/ml solution. Since the total volume of the reaction mixture in the cuvette was
actually 3.0 ml, the total amount of NADH formed per minute is

Vo

XXX A340
minute

XXX A340
minute

XXX moles
minute

3.0 ml total

3000 moles
6220

1 mmole
6220 A340

1000 moles
1 mmole

As shown on page 99 of the lab manual, 3000/6220 = 0.48, so

Vo

XXX A340
min

0.48

XXX moles
minute

0.48 is a simple conversion factor that allows you to easily convert a A340/min value to a
mole/min value. While using this number is simple, you always need to remember what
it means. It would have a different value if you had a total reaction volume of 1.5 ml or if
the molar extinction coefficient of the light absorbing chemical had a value of 4569.
5.

Finally, calculate the activity of the stock LDH solution in CAPS buffer at pH 10.
Activity is defined as the rate of the reaction (moles/min) per ml of the enzyme
solution. For example, if you find that 10 l of the stock LDH enzyme solution gives a
rate of 0.247 moles/min, the activity is
0.247 moles
__
min x 10 l enzyme

1000 l
ml

24.7 moles
min ml enzyme

Note that you must be careful to distinguish between the total volume of the reaction
mixture (3.0 ml or 3000 l in this case) and the volume of enzyme added to the
reaction mixture (in this example, 10 l). Be sure to use the volume of enzyme you
actually used in your three replicate assays.
6.

If you found it necessary to make a dilution of the stock enzyme, be sure to correct for
the dilution factor (1/10) by multiplying the activity rate by 10. For example, if you used
50 l of a 1/10 dilution and found the rate to be 0.192 moles/min, the activity would be
0.192 moles_____
min x 50 l enzyme

7.

1000 l
ml

10

38.4 moles
min ml enzyme

Finally, express the activity in units/ml of enzyme. In the case of L-lactate

dehydrogenase, 1 unit of LDH activity is defined as the amount of enzyme needed to
form 1 mole of NADH/min. So in the case of the calculations shown in step 5 and step
6, the activities would be 24.7 units/ml and 38.4 units/ml, respectively.
This is a way of saying how much activity is present in the stock enzyme solution. If you
had a total of 350 l of the stock enzyme and found the activity was 38.4 units/ml, you
would have a total of
350 l

1 ml
1000 l

13.44 units of LDH altogether.

38.4 units
ml

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C.

Specificity of the L-Lactate Dehydrogenase Reaction

The purpose of this part of the experiment is to determine the specificity of the reaction
with respect to its substrates. As part of this section, you will measure the rate of the reaction
with D-lactate (the enantiomer of L-lactate) as the substrate and with NADP+ (the modified form
of NAD+) as the electron acceptor. NADPH has an extra phosphate group but still absorbs light
at 340 nm.
1.

Set up a new reaction by adding 1.9 ml of the Assay Buffer to a 4.5 ml methacrylate
cuvette. Then add 0.5 ml (500 l) of the 6 mM NAD+ solution and 0.5 ml (500 l) of the
150 mM D-lactate solution. Note that the total volume at this point is 2.9 ml. Again, it
will be easiest if you use a 5 or 10 ml pipet to add the buffer and a P-1000 micropipetter
to add the NAD+ and D-lactate solutions. Be sure to use a clean tip for each solution.

2.

Then add the amounts of water and stock enzyme which you found in Part A gave you a
reasonable rate of reaction and which you used for your replicate assays. Place the
cuvette back in the instrument and read the absorbance at 340 nm at 15 second intervals
for a total of 3 minutes. Record the absorbance values in your lab notebook. If there is
no increase in A340, record the rate of the reaction as zero. If there is an increase in A340,
repeat the assay two more times so that you have three replicate assays.

3.

Now set up a new reaction by adding 1.9 ml of the Assay Buffer to a 4.5 ml methacrylate
cuvette. Then add 0.5 ml (500 l) of the 6 mM NADP+ solution and 0.5 ml (500 l) of
the 150 mM L-lactate solution. Note that the total volume at this point is 2.9 ml. Again,
it will be easiest if you use a 5 or 10 ml pipet to add the buffer and a P-1000
micropipetter to add the NADP+ and L-lactate solutions. Be sure to use a clean tip for
each solution.

4.

Then add the amounts of water and stock enzyme which you found in Part A gave you a
reasonable rate of reaction and which you used for your replicate assays. Place the
cuvette back in the instrument and read the absorbance at 340 nm at 15 second intervals
for a total of 3 minutes. Record the absorbance values in your lab notebook. If there is
no increase in A340, record the rate of the reaction as zero. If there is an increase in A340,
repeat the assay two more times so that you have three replicate assays.

5.

Calculate the activities of the LDH with these substrates as described in Part B and
compare to those found with the standard combination.

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D.

Effect of pH on the Rate of the L-Lactate Dehydrogenase Reaction

The purpose of this part of the experiment is to determine the effect of pH on the rate of
the L-lactate dehydrogenase reaction. Each group will determine the activity of the enzyme with
the two buffers at different pHs. The class will then pool the data so that the results with all of
the buffers can be determined.
1.

Set up a new series of assays for LDH using the volume or dilution of the stock
enzyme found in Part A to give a reasonable rate of reaction. Use 150 mM L-lactate
and 6 mM NAD+ as the substrates. In this case, instead of adding 1.9 ml of CAPS, pH 10
as the buffer, you will add 1.9 ml of one of your buffers instead. Again, the total volume
of the reaction mixture should be 3.0 ml.

2.

Do the LDH assay three times with each of your buffer solutions. Again, plot the data,
determine the initial velocities (Vo), and calculate the average initial velocity of the
reaction for that buffer as A340/min.

3.

For each buffer, convert the initial activity from a A340/min to mol/min as described
above. Again, the conversion factor of 0.48 takes into account the facts that 1) the molar
extinction coefficient of NADH is 6220 M-1 cm-1, 2) the path length of light through a
standard cuvette is 1.0 cm, and 3) the total volume of each reaction is 3.0 ml (0.003 L).
E.

Calculation of LDH Activities at Different pHs

1.

Express the initial velocity with each buffer as units of activity as described in Part B.

2.

Exchange contact information with the other groups in the class, so you can make
arrangements for exchanging data for the purposes of pooling.

3.

Make a graph in which you plot the LDH activity (in units/ml) with each buffer as a
function of pH. What does the graph look like? Draw a best fit line through the data
points to determine the pH optimum of LDH. Why do you think that the use of different
buffers to achieve the various pH values might affect the results?

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BCH 372
Modern Concepts in Biochemistry Lab

Basic Calculations Used in the Measurement and Purification of

L-Lactate Dehydrogenase
There are a number of simple calculations that are used throughout the course to study
the activity of L-lactate dehydrogenase from an animal tissue and to follow its purification. The
following is a quick summary of the formulas used in these calculations. WWW, XXX, YYY,
and ZZZ just represent numbers found in an experiment. More detailed descriptions of each
calculation are then given in the rest of this handout. All of the calculations are also described in
the lab manual.
A.
1.

Basic Formulas for Calculations

initial velocity (Vo) =

Vo

=
=

__XXX___ moles/min

Vo (run 1) + Vo (run 2) + Vo (run 3)

3
average A340/min

average A340/min x 0.48 =

2.

3.

__XXX___ moles/min ml

enzyme activity

activity

__YYY__ moles
min x __ZZZ__ l enzyme

__XXX__ moles
min ml enzyme

total enzyme units

total units

4.

__ XXX___ moles/min

__YYY__ moles
min ml enzyme

__XXX__ moles
min
=

1000 l
ml

__XXX___ moles/min

percent recovery

_ ZZZ__ total ml of enzyme fraction

total units in fraction K

x
total units in first fraction

100%

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5.

protein concentration

6.

specific activity

__XXX ___ moles/min mg

sp. act.

__YYY__ moles
min ml

__XXX _ moles
min mg

7.

ml
WWW mg protein

purification factor

specific activity in fraction D

specific activity in first fraction

purification factor

__XXX________ moles/min mg
__YYY________ moles/min mg

N fold

B.
1.

____WWW______ mg/ml

Descriptions of Calculations

Initial Velocity

The rate of the L-lactate dehydrogenase reaction is expressed in terms of the initial
velocity (Vo) in the presence of L-lactate and NAD+. In the reaction, L-lactate is oxidized to
form pyruvate and NAD+ is reduced to form NADH. This results in an increase in absorbance at
340 nm since NADH absorbs light at this wavelength and NAD+ does not. The initial velocity is
determined by plotting the absorbance of a standard 3.0 ml reaction mixture at 340 nm as a
function of time in seconds. The best straight line is fitted to the initial set of data points and the
slope of the line is expressed as A340/min. This slope is the initial velocity (Vo) since it
represents the amount of product formed per unit of time. See Figure 4.3 in the lab manual on
page 97. Because there will be some variation in the initial velocity, LDH reactions are normally
run in triplicate and the average (mean) of the three values is used.
Vo

Vo (run 1) + Vo (run 2) + Vo (run3)

3

Vo

__________ A340/min

The initial velocity can be converted to an actual amount of product in moles/min by using the
absorption characteristics of the NADH. The molar extinction coefficient of NADH at 340 nm is
6220. This means that a 1.0 M solution of NADH will have an absorbance of 6220 in a 1.0 cm
light path. Since the total volume of the reaction mixture is 3.0 ml (0.003 liter), you can convert
A340/min to moles/min as follows:

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Vo

__________ A340/min
(6220 M-1 cm-1) (1 cm)

106 moles
moles

__________ A340/min

0.48

__________ moles/min

0.003 liter

This is shown on page 99 of the lab manual. You can also think about the calculation in the
following way. Since a 1.0 M solution of NADH has an absorbance of 6220, a 1.0 mM solution
of NADH will have an absorbance of 6.220 and a 1.0 M solution will have an absorbance of
0.00622.
Vo

__________ A340/min

1 mole
x
liter x 0.00622 A340

__________ A340/min

0.48

__________ moles/min

0.003 liter

Suppose you measure the initial velocity of a crude homogenate and find the A340/min
to be 0.135/min, 0.159/min, and 0.146/min. The average A340/min is thus 0.147 A340/min.
This can be converted to 0.147 A340/min x 0.48 = 0.0704 moles/min.
2.

Enzyme Activity

The rate of the LDH reaction will, of course, depend on the amount of enzyme added to
the reaction mixture. Enzyme activity is normally expressed in terms of the initial velocity of the
reaction per ml of solution. Suppose that you measured the initial velocity of the reaction
described above on using 10 l of a crude homogenate. The enzyme activity of the solution is
thus:
0.0704 moles
min x 10 l

1000 l
ml

7.04 moles
min ml

This is important because different solutions will have different amounts of LDH in them. If it
took 50 l of enzyme solution to get this same velocity, the activity of the solution would be only
0.0704 moles
min x 50 l

1000 l
ml

1.41 moles
min ml

In the lab manual, 1 unit of activity is defined as the amount of the amount of enzyme that
converts 1 mole of L-lactate to pyruvate/min (or that reduces 1 mole of NAD+ to NADH/min).
We would therefore say that the first solution has 7.04 units of enzyme/ml and the second
solution only 1.41 units of enzyme/ml.

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3.

Total Units and Percent Recovery

During the purification of LDH from beef heart, some of the activity is lost along the way
as fractions are discarded or as the enzyme is denatured. To monitor the recovery of the enzyme,
the amount of enzyme in each fraction is expressed in terms of total units. The total number of
enzyme units is simply the enzyme activity in (moles/min ml or units/ml) multiplied by the
volume of each fraction. If the crude homogenate has 7.04 units/ml and a total volume of 110
ml, the total amount of activity is:
7.04 units
ml

110 ml

774 units

Suppose that much of the LDH in this crude homogenate is precipitated with ammonium sulfate
and recovered in a fraction that has 66.5 units/ml and a total volume of 4.8 ml. The total number
of units in this fraction is:
66.5 units
ml

4.8 ml

319 units

The percent recovery of the activity in this fraction compared to the crude homogenate is:
319 units in ammonium sulfate fraction
774 units in crude homogenate
4.

100% =

41.2%

Protein Concentrations and Specific Activity

Most of the fractions obtained during the purification of LDH contain many different
proteins. The total amount of protein is determined using a protein standard curve and is
expressed in mg/ml. See pages 70-72 in the lab manual. The specific activity of LDH is the
amount of activity per unit of protein. It is expressed as moles/min mg or units/mg. See pages
94-95 in the lab manual. Suppose that you determine that the crude homogenate has 7.04
units/ml. If the protein concentration of this fraction is 1.2 mg/ml, then the specific activity is
7.04 units
ml

ml
1.2 mg

5.87 units
mg

If the ammonium sulfate fraction has 66.5 units/ml and 6.5 mg/ml of protein, then the specific
activity of this fraction is
66.5 units
ml

ml
6.5 mg

10.2 units
mg

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5.

Fold Purification

The extent of purification is expressed in terms of the increase in specific activity that
occurs as extraneous proteins are removed from fractions containing LDH. It is expressed as the
ratio of the specific activity in the fraction of interest to the initial specific activity. In the
example shown above, the fold purification is
10.2 units/mg
5.87 units/mg

See page 95 in the lab manual.

1.74 fold