Biology 450 - Animal Physiology Lab
Fall 2007
Laboratory Exercise 7 - Muscle Biochemistry
Samples of muscle that differ in their fiber composition (aerobic / anaerobic) will be
analyzed for activities of two enzymes using colorimetric assays. These enzymes
are metabolic, with one associated with glycolytic pathways and the other
oxidative. Some preliminary analyses of the muscle samples will also be performed.
The goals of these exercises are:

To learn how to carry out an assay of enzymatic activity.

To compare the activity of aerobic and anaerobic metabolic enzymes in two
muscle fiber types.

To relate differences in metabolic enzyme activity to contractile and
structural differences and to muscle function in vivo.
Background
Vertebrate muscle fibers vary considerably in their contractile, energetic,
morphological and biochemical properties. For instance, some fibers contain many
myofibrils but relatively few mitochondria; these fibers tend to produce a lot of
force but rely on glycolytic (anaerobic) pathways for energy production. Other
fibers have relatively high densities of mitochondria (at the expense of myofibril
content) and thus can remain active for long periods without fatiguing.
Skeletal muscle fibers can be separated into either two or three categories on the
basis of their contractile properties and the pathway used for energy production
(Table 1). The two category system classifies muscles as either Type I or Type II.
Type I are slow-twitch fibers that produce ATP primarily through the oxidation of
carbohydrates and lipids. Thus they are referred to in the three category system as
SO fibers - slow oxidative. Their high mitochondrial density gives them a dark color
and may be responsible for their fatigue resistance. Type II fibers can contract
more rapidly, and some kinds (Type IIb) obtain ATP mainly from the glycolysis of
glycogen, a glucose polymer. Type IIb are also known as FG fibers - fast glycolytic.
They tend to have fewer mitochondria and are much more susceptible to fatigue
than Type I fibers. (Type IIa fibers are FO - fast oxidative.)
In many animals, the different fiber types are located in discrete muscle masses in
different parts of the body, making them very easy to study. Type IIb fibers are
used for short bursts of intense activity (like chasing prey, escaping from predators
or fighting), but they cannot sustain such activity for very long. Type I fibers are
used during sustained low-speed activity. In addition, cardiac muscle, although
different from skeletal muscle in some respects, has many similarities to Type I
fibers.
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Table 1. Classifications of vertebrate muscle fiber types.
Type I: SO
Type IIa: FO
Type IIb: FG
Mitochondrial Density
High
High
Low
Glycogen Content
Low
High
High
Color
Red
Pink
White
Fatigue Resistance
High
High
Low
Contraction Velocity
Slow
Fast
Fast
Metabolic enzymes
A sizable number of enzymes are involved in the process of glycolysis and the
oxidative metabolism of the Krebs cycle. In this lab, you will examine just one
important enzyme from each of these biochemical pathways to determine whether
their activity differs between Type I and II muscles.
The first enzyme you will assay is malate dehydrogenase (MDH), a component of
the Krebs cycle that converts malate into oxaloacetate and generates NADH, which
mitochondria use to generate ATP:
MDH
Malate + NAD+
Oxaloacetate + NADH
This reaction normally proceeds from left to right in the Krebs cycle. However, for
ease of analysis, we will measure its activity in the reverse direction.
The other enzyme you will measure is lactate dehydrogenase (LDH), a glycolytic
enzyme that catalyzes the following reaction:
LDH
Pyruvate + NADH
Lactate + NAD+
During exercise this reaction proceeds from left to right, resulting in the buildup of
lactate and regenerating NAD+ for other steps in glycolysis.
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Lab Procedures
Note:

You will be working with a number of chemicals with the potential to
irritate skin and stain clothes. Please bring eye protection and
something to protect your clothes from possible splashes. Gloves will
be provided.
In this lab you will compare the specific activities of two enzymes, lactase
dehydrogenase and malate deydrogenate, in oxidative and glycolytic muscles. The
muscle samples you will use come from "an animal to be named later." In the past,
we have used a fish, because fish tend to have very discrete and distinct Type I and
II muscles.
As a first step in this lab, you will need to homogenize, or physically break down,
the muscle fibers in each sample in order to release these enzymes into a solution.
Ideally, every single fiber would be lysed, so that all the intracellular contents of a
particular muscle sample would end up released. However, it is typically very
difficult to lyse all the cells, so after homogenizing the sample you will need to run
an assay to determine how effective this process was. This involves using
spectrophotometry to determine the total quantity of protein released from lysed
cells, which we assume is proportional to the total mass of the cells that were
successfully broken open. This value will allow you to standardize the results of
your enzyme assays by the quantity of muscle from which the enzymes came.
In the main experiments, you will compare the activities of the two metabolic
enzymes by measuring the rate of conversion of NADH into NAD+, again using a
spectrophotometer.
Spectrophotometric examinations of reactions are often referred to as colorimetric
assays. The measure of total protein content in your homogenized solution will be
measured using one type of colorimetric assay, called an endpoint assay. In this
approach, a reaction is allowed to proceed either for a fixed period of time or to
completion, and the final color of the solution indicates the amount of reactant that
became product. Here, protein reacts with a "cocktail" of chemicals to produce a
purple product. Endpoint assays can also be used to determine enzyme activity by
allowing a reaction to proceed for a controlled period and the amount of color
change indicates the amount of reactant that was enzymatically converted to
product in a given time. Product(s) and the enzyme are placed in solution, along
with an indicator substance in some cases, and the color change (in the UV or
visible range) of the solution indicates the enzymatic activity.
The other type of colorimetric assay is a kinetic assay. In these assays, the rate of
the reaction is determined directly by following the conversion of reactant to
product through time. The concentration of one of the reactants or products is
tracked by recording output from a spectrophotometer using (in this case) a
PowerLab unit and Chart. In our experiments, we track the conversion of NADH to
NAD+
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The general steps are as follows:
1. Grind up samples of different muscle types into homogenate.
2. Determine protein concentration of the resulting solution using an endpoint
assay.
3. Prepare homogenate solutions of suitable concentration for enzyme assays.
4. Perform enzymatic assays using kinetic assays to determine reaction rates.
This is the nearest thing you will see to a "recipe" lab in Bio 450. In other words,
you will need to follow directions carefully or the assays will not work!
Important note:

Please leave the bottles of NADH, pyruvate and oxaloacetate on ice when not
in use. This will help prevent the breakdown of reagents before
measurements are made. Follow the specific steps for each assay carefully.
Be careful with the pipettes to avoid cross-contamination of samples - use
each tip on only one sample or reagent. Most important of all, clearly label
all tubes!
Setup
You will be using the Spec 20 to measure the color changes associated with these
assays. Some of these assays require looking the rates of change, others just the
final color of the test solution.
Procedure:
1. The spectrophotometer takes a while to warm up - turn this on first.
2. Answer the first set of questions on the worksheet.
3. Open Chart, and set up as follows (if not already done for you):

Set the display to show two channels.

Set the sampling rate to 100/s.

Under Channel 1, select “Input Amplifier…”, then “Units Conversion.”
For the calibration values, “Point 1” should have values of 0.0 and 0.0,
“Point 2” should have values of 1.0 and 100. Under units, choose
“%T” if it exists. If it doesn’t, select “Define unit…” and then type in
“%T”. For “Decimal places”, choose “1.” Click “OK” until you get back
to the main window.

Again under Channel 1, select “Smoothing…”, then set “Window width”
to 9 and click “OK.” This will smooth out small voltage variations from
the spec.

Under Channel 2, select “Arithmetic.” In the large box at the top, type
in “Log10(100/Ch1).” This equation converts the transmittance value
from Channel 1 into the absorbance value. For “Decimal places”,
choose “2,” and type in “Abs” for “Units name.” Deactivate autoscale,
then click “OK” until you get back to the main window. In the main
window, set the upper and lower scale for the Y axis of Channel 2 to
2.0 and 0.0.
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Tissue Preparation
Before running enzyme assays, the muscle tissue must be broken down to release
enzymes from the cells.
Procedure:
1. Obtain Type I and II muscle samples from the instructor or T.A. and weigh
the sample. If it is less than 0.4 g ask for some additional tissue; if more,
use a razor blade to remove some tissue.
2. Mince each muscle sample using a razor blade and petri dish. This step
makes the homogenization more effective.
3. Place each sample in a small test tube with roughly ten times its volume of
phosphate buffer (assume 1 g of muscle = 1 ml). The exact volume is not
critical since we will determine protein concentration later.
4. Homogenize each sample for 30-60 seconds at medium to high speed. Keep
the sample on ice (using a beaker) while homogenizing to keep it cool. Be
sure to rinse the homogenizer with distilled water between samples, and
clean it out with a brush if necessary, to avoid cross-contamination of your
muscle samples.
5. Centrifuge the samples at a speed of 30 for 3 minutes. Important note - be
sure the centrifuge is balanced by placing paired tubes opposite one
another! The substances you will assay are in the resulting supernatant.
6. Draw off about 1.5 ml of each supernatant and put them in separate tubes.
Use these supernatant samples for all subsequent work.
7. The full strength supernatant will be used for most of the assays, but you will
also need dilute samples for the LDH assay. In new test tubes, create a 5fold dilution of each supernatant sample by adding 200 µl of supernatant to
0.8 ml phosphate buffer. This is the sample you will use for the LDH assay.
Place all samples on ice.
Determining total protein concentrations
We will be using a "Total Protein Reagent" to determine the total protein
concentrations of our supernatants. This reagent is alkaline and contains copper
ions. The reagent reacts with peptide bonds to produce copper-protein complexes,
which have a purple color that can be quantified at 540 nm. The more peptide
bonds (and thus the more protein) in the sample, the more purple the color.
One problem with your muscle sample assays is that each assay will give us only a
number indicating the "purpleness" of the sample. In order to convert this number
to the protein concentration, you will need also to run the assay with a number of
protein standards (solutions of known protein concentrations). Then you will be able
to construct a standard curve - a graph of protein concentration vs. absorbance at
540 nm. This standard curve will then allow you to translate the absorbance
readings of your supernatants to actual protein concentrations.
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Reagents:
Total Protein Reagent - This solution changes color from blue to purple in
reaction with protein
Protein standards - Solutions containing 2, 4, 6, 8 or 10 mg protein/ml in
phosphate buffer
Phosphate buffer - a buffered reaction solution to control changes in pH
Procedure:
1. Label clean cuvette tubes for each of the two muscle types, a blank, and for
standards of 2, 4, 6, 8 and 10 mg protein/ml.
2. Pipette 2.5 ml of Total Protein Reagent into each cuvette.
3. Add 500 µl of the appropriate substance to each cuvette: undiluted red or
white muscle sample, protein standard of the appropriate concentration, or
phosphate buffer for the blank. Allow the reaction to proceed for 10 minutes
at room temperature.
4. While the reactions are occurring, set the spec to 540 nm. With the sample
chamber empty, adjust the transmittance to 0%.
5. When the solutions are ready, use the phosphate buffer + Protein Reagent
cuvette to set the blank value by inserting the cuvette in the spec and setting
the transmittance to 100% (= absorbance of 0.0).
6. Take a reading for each remaining sample, noting the absorbance in each
case. If the absorbance of a muscle sample is greater than the 10 mg/ml
standard, run a new muscle assay using a 5-fold diluted sample. Make sure
you correct for this dilution when calculating the original protein
concentration!
7. When finished, pour the contents of the cuvettes into the labeled waste
container and rinse out any residual solution.
Metabolic enzyme assays
The metabolic enzyme assays examine MDH and LDH activity using kinetic
measurements. In each case, one of the reactants in the reaction is NADH, which
becomes NAD+. NADH absorbs light at 340 nm, while NAD+ does not. The change
in absorbance at this wavelength can thus be used to measure the progress of the
reactions.
The MDH assay measures the rate of conversion of NADH to NAD+ when
oxaloacetate is converted to malate, while the LDH assay measures the rate of
NADH conversion when pyruavate is converted to lactate.
Reagents:
NADH solution - NADH in the phosphate buffer
Pyruvate solution - Pyruvate in buffer
Oxaloacetate solution - Oxaloacetate in buffer
Phosphate buffer
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Procedure:
1. Label clean cuvette tubes for each muscle and enzyme type. You should run
two replicates of each sample.
2. Add 3 ml of the NADH solution to each cuvette.
3. Place all the cuvettes in a beaker of room-temperature water, which will act
as a water bath.
4. Set the Spec 20 to 340 nm. With the sample chamber empty, adjust the
transmittance to 0%. Then use a cuvette containing 3 ml phosphate buffer
as a blank and set the transmittance to 100%.
5. Start recording in Chart. You may want to use the comment feature of the
program to note which samples are being tested in each case.
6. When ready to run each sample, do the following, noting that speed is
essential once the final reagent in added:
o
Wipe the water from the bath off the cuvette.
o
For MDH, add 20 µl of the undiluted muscle sample, and then 200 µl of
oxaloacetate solution to each cuvette.
o
For LDH, add 20 µl of the 5-fold diluted muscle sample, and then 200
µl of pyruvate solution to each cuvette.
o
Rapidly mix the sample and place it in the spectrophotometer. With
gloved hands, you can just place a thumb over the mouth of the tube
and shake it. (Be sure to wipe your thumb off before doing the next
sample.)
o
Repeat for each sample.
7. Find the reaction rate for each sample as change in absorbance per second.
Do this by determining the time it takes for the reaction to proceed between
two fixed absorbance values for each reaction type (MDH or LDH). For
example, find the time needed for the absorbance to change from 0.6 to 0.4
in each case.
8. Each reaction rate should then be divided by the muscle sample’s total
protein concentration to standardize the rate for differences in cell content
yield.
9. Wastes from these assays can be poured down the drain with running water.
Note:

The reactions should take a between one-half and five minutes to run. If
faster or slower, you may need to adjust your sample dilutions. But be sure
to use the same dilution for both muscle types so that reaction rates will be
directly comparable.
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