Introduction to Proteins

The first section of the lab focuses on proteins and the measurement of some of
their properties. The first few experiments deal exclusively with the determination of
the activities of an enzymes using galactosidase or Alkaline Phosphatase as
representative enzymes. The techniques we shall learn in these experiments are of
general utility and can easily be adapted them to the measurement, or assaying, of the
activities of other enzymes. These enzymes are easily obtainable, stable and detectable
by an inexpensive, sensitive assay. After learning the measurement basics we will then
go on to learn how to isolate Alkaline Phosphatase from a crude bacterial mix. Along the
way to this we are going to learn how to manipulate bacteria so as to get them to
preferentially produce relatively amounts of a foreign an enzyme, -galactosidase. We
will also learn to use SDS and Native gels to analyze protein mixtures for purity and to
determine subunit and native molecular weights of proteins. We will learn lots of new
lab skills and make practical use of information we have encountered in our lecture
courses.
Spectrophotometry
Before we do all the above interesting things we must learn to use the
spectrophotometer because the spectrophotometer is the instrument of most use in
almost every Biochemistry lab. Spectrophotometry, measuring the absorption of light
by a molecule, is one of the most common, as well as the most useful and powerful of all
analytical techniques. It is mainly used as a means of detecting concentrations or
changes in concentration.
Basis of Spectrophotometry
The basis of spectrophotometric measurement of concentration is the absorption
of a photon by a chromophore when the energy of the photon exactly matches the
energy difference between two energy levels of the compound. In the gas state the
distance between these energy levels will be almost the same in all molecules and so we
get a line spectrum. In a solution however, there are interactions with the solvent, and
energy transfers result, so that there is a range of distances between the energy levels
of the different molecules of the same compound, with a wide range of photons being
absorbed. We therefore get an absorption band in solution instead of a line spectrum.
Usually the first thing one does when working with a particular chromophore is to
determine the range of wavelengths over which absorption takes place. The wavelength
with the highest probability of absorbing a photon is the most sensitive one to use when
detecting the chromophore and is called the max.
1

Figure 1 Absorption Band of Tryptophan

The Spectrophotometer and Beers Law
We are going to use a spectrophotometer to measure the concentration of
chromophores. Spectrophotometers do this by measuring the number of photons that
reach the photon detector in a give time; this is called the intensity of the photon
stream. The instrument will measure the intensity in the presence and absence of the
absorbing molecule. The log of this intensity ratio, log Io/I, where Io is the intensity in
the absence of absorber and I is that in the presence of the absorber. The number,
Log Io/I, is the absorbance of the system, and the way it is defined makes it always a
positive number. Thus absorbance is directly proportional to the concentration of the
absorbing molecules because it is a measure of the photons that do not reach the
detector in a given time. These, being absorbed by the molecules in the solution, are
prevented from reaching the detector. Any parameter, which increases the chance of a
photon being absorbed by the absorber, will increase the absorbance of the solution. So
increasing the path length of the solution, through which a photon must pass, will
increase the absorbance of the solution. It also seems obvious that the particular
nature of an absorber must make some contribution to the probability of a photons
being absorbed so the nature of the absorber has an effect on the absorbance. All of
these absorbance contributions are summarized in the well-known Beers law, which
relates absorbance to all of these parameters. Beers law states that:
2

A = BC

(1)

where A, the absorbance, is the measured experimental parameter, (epsilon) is the

extinction coefficient, a physical property of a compound (equal to the absorbance of a
1M solution of that compound measured in a 1 cm path length), B is the path length,
the distance traveled by the light through the solution (for most regular cuvettes, the
ones used in this course, this path length is 1 cm) and C is the concentration in
moles/L, or some other appropriate units. Beers law is true at any wavelength at which
simple absorption takes place.
The Absorbances of Acid-Base Functional Groups and pKas
With both max and it seems we are now ready to use Beer's Law to calculate
concentration. However, a further complication in arises when the chromophore is a
weak acid that can compartmentalize into its acid and base forms in the pH range we are
using to determine its concentration. This problem arises when some pH-dependent
fraction of the initial product of the reaction, a nitrophenol, is ionized almost
immediately to the nitrophenolate as shown in Figure 2.

Figure 2 Partitioning of Nitrophenol Product

As we detect only the nitrophenolate we need to correct for the fraction still
present as the nitrophenol. We correct by using the pH and the pKa of the system. The
3

proper form of the absorbance relation when acid/base partitioning is present is shown
below.
= 1 + 2

or

CT = 1C1 + 2C2

(2)

Where , 1 and 2 are the extinction coefficients of the system, the acid and the
base form of the system respectively, and CT, C1 and C2 are the total
concentration of the compound and the concentration of its acid and base forms
respectively.
Reflection on Assays in General
Usually enzyme assays are good example of what an assay should be. They are
specific, sensitive, precise, accurate, and convenient. Let us reflect on the meaning of
these terms:
Specificity means that we are measuring the change in concentration of only one of a
single type of reactant, in this case, B-galactosidase, an enzyme that hydrolyzes only galactosidic linkages. There is virtually no interference from enzymes that catalyze
other reactions, even hydrolysis reactions involving closely related sugars!
Sensitivity refers to the smallest amount of a substance that can be reliably
discriminated from zero. Since the enzyme assay involves absorbance of light, the
limiting factor is the smallest change that our instruments can reliably detect. Greater
sensitivity can be obtained by changing the assay conditions (for example, assaying for a
longer time, or using a greater concentration of either reactant).
Precision refers to the reproducibility with which measurements can be made. Precision
also reflects the skill of the experimenter, and can be measured statistically, by
repeating an assay measurement several times and observing the scatter of the data.
Accuracy refers to the degree to which a measured value in an assay conforms to the
absolute or true value. Lack of accuracy reflects non-random error, or bias in the assay
technique, and cannot be measured statistically. There is no simple way to guard against
this possibility, but if one understands the chemistry of the assay, it is often possible
to make intelligent guesses about what kinds of problems may arise and what kinds of
control experiments might be useful. It is our hope that the end of the course your
assays will be characterized by sensitivity, precision, and accuracy not to mention
grace and style!

We will first do two experiments to introduce you to some critical and quantitative
aspects two of the most basic processes in all lab work the proper use of the pipetman
to measure volumes and the making of buffers.

Experiment I Learning to use the pH meter, making up and understanding the

behavior of, buffers.

Experiment II the pipetman, the statistics of their delivery and the reliability
of Spectrophotometric measurements.

Experiment I Understanding Buffers

Material

Six small beakers.

Some plastic test tubes.
Solid Tris base, Mr 0f 121.1 Da and pKa of 7.5.
2M HCl for adjusting the pH of the buffer.
0.1MHCl and 0.1M NaOH solutions to check buffering capacity of buffer.
pH meter and standard calibration buffer sets.
Ten ml graduated cylinder.
50ml conical test tube.

Method.

Calibrate the pH meter as per protocol. You will find the protocol for this
in the appendix of the lab manual.
Using the Tris base and the concentrated HCl make up 60ml of 0.1M
TrisHCl, pH 7.5. Making Buffers. on p.69 of the lab manual will guide you
through this process.
Place 5ml of your buffer in a test tube and incubate on ice for about 15
minutes.
Measure the pHs of the concentrated buffer at both room temperature
and at ice temperature. Record in lab book.
Take 5ml of the concentrated buffer and add 45ml of distilled water, mix
and measure the pH of the diluted buffer.
Set up the following data tables:

Beaker

Diluted
Buffer
20
20

A
B
C
D

Water

0.1M
NaOH

0.5

Final
pH

0.5
20
20

0.1M HCl

0.5
0.5

Mix and record final pH.

Flow Sheet for Experiment I
Calibrate pH Meter
Distilled Water
20ml

5ml

20ml

add 0.5ml 0.1M NaOH add 0.5ml 0.1M HCl

Measure pH
Measure pH

Make 60ml of 0.05M TrisHCl

5ml
cool on ice
measure pH

5ml

5ml

Add 45ml water

measure pH

Dilute Buffer
20ml
add 0.5ml 0.1M NaOH
Measure pH

20ml
add 0.5ml 0.1M HCl
Measure pH

For next time:

Be able to use the Henderson-Hasselbalch equations to calculate the

concentrations of the acid and base forms of Tris at pH 7.5 in the
concentrated buffer and in the diluted buffer. (6)

Explain how weak acids plus their salts buffer reactions? An equilibrated
chemical equation is necessary.(5)

What two properties of the system might affect the pH at different

temperatures? Explain. (4)

Look at the magnitude of the pH shifts on adding acid and base to your
buffer and explain what this tells us about the buffering ability of your
Tris buffer. Explain why we see this type of behavior? (10)

Experiment II The Accuracy and Precision of Pipetman and

Spectrophotometers.
Pipettors are designed to accurately dispense volumes ranging from their
maximum volume to 10% of their maximum volume. For a P1000 pipetman the
range is 1000L to 100L, for the P20 the range is 20 to 2 L. These various
sizes all have their maximal volumes displayed on the barrel. Become familiar with
the sizes and shapes of the various pipetman. Trying to dispense volume outside
these ranges is not only inaccurate but will damage the unit.
Care and Feeding of the Pipetman

Choose the correct unit for the measurement being taken.

Fit the proper tip onto the barrel of the pipetman.
Press the plunger to the first sop, place the tip under the liquid being
dispensed, and release the plunger slowly to pull up the liquid into the
barrel.
Wipe the tip off with paper towels, the correct volume of the liquid is that
inside the tip.
Discharge the contents completely by placing the tip against some surface
of the container, or just under the surface of the liquid in the container.
Always look at your delivery to make sure the contents have been
completely transferred.
Part A - The statistics of Pipettors (and Balances)

Materials:
P200 and P1000 pipetman.
Small and large tips.
Deionized water.
Balance.
I.
II.
III.
IV.
V.

Make five measurements of what a P200 pipetman says is 0.200ml.

Make five measurements of what a P1000 pipetman says is 0.200ml.
Make five measurements of what a P200 pipetman says is 1.000ml.
Make five measurements of what a P1000 pipetman says is 1.000ml.
Find the mean, the standard deviation, the standard error of the mean and
the 95% confidence level of the data.

Method

Place the appropriate tip on the P200 pipetman.

Place a weighing boat in the balance and tare to 0.000g.
Pull up the 200L volume into the pipetman, wipe the tip and discharge the
volume into the weighing boat.
Record the final mass change in the table.
Repeat four more times.
Find the individual masses by difference.
Repeat this process for the P1000 pipetman dispensing 200L and the P200
and P1000 pipetman dispensing 1000L.
Data Display with Legend to be redrawn in lab book
200uL Delivery by P200 and P1000 Pipetman

P200

Measured

P1000

Difference

.
.

X-bar
s
SE(X-bar)
95% confidence

P200
Measured

Measured

Difference

X-bar
s
SE(X-bar)
95% confidence

.
.
.
.

1000mL Delivery by P1000 and P200 Pipetman

P1000
Difference

Measured

Difference

X-bar
s
SE(X-bar)
95% confidence

.
.
.

X-bar
s
SE(X-bar)
95% confidence

.
.
.
.

Questions for next time

What is the number called the 95% level of confidence really telling us? (10)
Which is the best way of dispensing 200uL? Explain your choice. (5)
Which is the best method for dispensing 1000uL? Explain your choice. (3)
What does best mean in this context? (3)

Part B - Statistics for Spectrophotometers at 400nm

Materials
p-Nitrophenolate solution.
Six one ml cuvettes.
Shimadzu Spectrophotometer.
Method

Turn spec on and set to 400nm in photometric mode.

Clean cuvettes by washing in water and alcohol.
Add about one ml of water to two clean cuvettes.
Place cuvettes in compartment in proper orientation for measurement.
Close compartment and autozero cuvettes.
Remove the sample cuvette the cuvette closest to you, completely remove
the water with a Pasteur pipet and rubber bulb, add about one ml of pnitrophenolate (NP), put back into the spectrophotometer, close the
compartment and measure the A400.
Pour the NP back into a clean beaker as it can be re-used.
Repeat the above experiment four more times with the same cuvette and fresh
NP and record the data.
Determine the absorbencies of the same volume of NP successively decanted
into five different cuvettes, each autozeroed independently and record the
data.

10

A Note on Data Collection using Spectrophotometers

The spectrophotometer in the photometric mode will keep a running
list of the most recent twelve data points on its display and will
print these out on request. On request should be interpreted to
mean, when the display is full or when the experiment is over,
whichever comes first. The point of this is that the data should be
continually generated; dont stop between each measurement to
record the data. The function of the person making the
measurement should be to generate data. This means adding NP,
inserting into the compartment, closing the lid and pressing the
measure button. Write the data into the data grid already in your lab
book.
Spectrophotometer Statistics
A400

X-bar

s
SE(X-bar)
95% confidence

.
.

Cuvette Statistics
Cuvette
1
2
3
4
5

A400

X-bar
s
SE(X-bar)
95% confidence
11

.
.

.
.

All calculations to be done by hand.

From the data infer the lower limit of an absorbance measurement on your
Spectrophotometer. Explain your decision. (5)

12

Experiment 3: Spectrophotometric Measurements

Today we are going to learn a little bit about the theory and practice of some
spectrophotometric measurements. We are going to use the Shimadzu
spectrophotometer to do the following set of experiments and thereby gain some
experience in the potentialities of spectrophotometry.
(A). Use the spectrum mode to examine the effect of composition on the
absorption of light.
(B). Use the Spectrum Mode to measure the wavelength of maximal
absorbance (max) of the p-nitrophenolate anion.
(C) Use the Photometric mode to calculate the extinction coefficient, , of the pnitrophenolate anion.
(D). Use the photometric mode to determine the pKa of the acid, p-nitrophenol.
(E). Use the kinetic mode to measure the rate of the -galactosidase reaction and
use this data to do some statistical analysis of the assay in your hands.

We are doing four experiments and each aisle has its own spectrophotometer and
includes four members. We are therefore going to do these experiments by aisle.
One student in each aisle should be responsible, i.e. be in charge of making solutions
and changing the parameters on the spectrophotometer for a single experiment. So
first, organize the work and then get on with the preparations. Each student is to
hand in their own report on all experiments and should be familiar with all aspects of
all experiments.
With the exception of a few dilutions to be done by the group, all reagents will be
provided as ready to go. Be sure to read the instructions provided and understand
the diagram of the spectrophotometer provided in the handout.
A. Examine the Effect of Composition on the Absorption of Light
Use the spectrum mode to measure the absorbance of a glass, a plastic and a quartz
cuvette from 500-240nm. These absorbances are to be measured versus air i.e., no
blank cuvette in the reference compartment.
Sketch all curves for your lab book on an Absorbance versus wavelength plot. Fill in
suitable units.

13

B. Use the Spectrum Mode to measure the wavelength of Maximal

Absorbance (max) of NP, the p-nitrophenolate anion,.
1.

Selecting the Wavelength Range over which the Solution will be

Scanned

Turn instrument on and select Spectrum.

Set s to 500nm and e to 360nm.
Set scan speed to medium

2. Allow spectrophotometer to Match Cuvettes

The cuvettes we are using are not matched and so their absorbencies at different
wavelengths will be different. If we want to accurately measure concentration we need
to correct for this difference between cuvettes. The program Baseline will run the
cuvettes through the spectrum range and record the differences at each wavelength.
It will then correct absorbencies for the differences in cuvette properties.

Go back to basic modes on the screen and select condition set.

Fill the two cuvettes with water and place in sample and reference
compartments of the spectrophotometer.
Select baseline and press start. After the baseline calibration is finished,
go back to he spectrum mode and press start. Check that the instrument
has done the calibration properly by running a spectrum of the cuvettes. The
base line should be zero along the wavelength range.

14

3. Running the Spectrum

Add the nitrophenol solution to the sample cuvette and run its spectrum.
After running the spectrum the instrument will give you an option of finding
the max of the spectrum and will print it out on the chart.
Make a copy of the spectrum for each group member.

The following experiments, C and D, are analytical experiments so care must be taken in
making solutions, wipe pipette tips off after dipping them into the concentrated
reagents and make sure the contents of the pipette tip are delivered into the contents
of the test-tube. This is done by looking to see what you are doing; making sure the tips
are under the solution before delivery.
The stock concentration of the p-nitrophenol (Mr is 139.1) we are using is 1.39mg/ml. Mr
is the IUPAC designated symbol of relative molecular weight in Daltons (Da). Daltons
are the units we used to know as grams/mole

C. Use the Photometric mode to calculate the Extinction Coefficient () of the pnitrophenolate anion.
The unprotonated nitrophenolate anion has a much larger extinction coefficient than
the protonated or neutral form so the absorbance and extinction coefficient of the
anionic form will be used to determine concentration in our assays. Our stock of
nitrophenolate solution will be 1.39 mg/ml. The Mr of nitrophenol is 139.1Da.

I.

Setting up the Reactions.

Set up the following in labeled 13 X 100mm test tubes. Use this tabular form in
your lab book to organize the setting up of the experiment and recording of the
data.

Nitrophenol Stock (L)

5.0
10.0
15.0
20.0

Base (ml)

NP Conc. (mM)

15

A400

II. Setting the Spectrophotometer to the Photometric Mode

The Photometric mode is used to measure absorbances at a fixed wavelength. The
photometric mode is what is used to determine the concentration of a chromophore
directly when we know its extinction coefficient, or indirectly when we need to run a
standard curve to relate absorbance to concentration.

Use the mode key and follow the screen instruction to get into the
Photometric Mode.
We need to change the 1 wavelength setting to the max of the
nitrophenolate anion. We do this by going to parameter 5, the processing
parameter. In the processing mode press 1 and the cursor will allow you to
impute max in the 1 space.
Fill both cuvettes with base and press the Auto Zero pad on the screen.
This will zero the cuvettes so any absorbance differences we measure are
indicative of changes in only the absorbencies of the solutions in the cuvettes
and not from the cuvettes themselves.
Remove the sample cuvette and use a Pasteur pipette to decant and discard
the contents.
Add one of the nitrophenolate solutions you have made and press Start. The
screen will display the absorbance of that sample. Repeat the last two
instructions for all your nitrophenolate solution. Record the absorbencies in
you lab book.
Note: By holding the base volume constant and changing nitrophenol volumes,
we are changing the properties of the resulting solutions by about 1%. But, we
are banking that the change in blank absorbance values at 400 nm between the
most concentrated nitrophenol solution and the least concentrated nitrophenol
solution will be very small.
Plot absorbance versus concentration in mM on graph paper. Do a least
squares fit to your data and append the equation and r2 values of the fit.

D. Using the Photometric mode to determine the pKa of p-nitrophenol

We are going to use the photometric mode to measure the absorbance at 400nm of a
fixed concentration of nitrophenol at different pH values. We will use these
absorbance values to calculate a pKa for nitrophenolate.

16

Mathematical Theory - we can use the following equations to derive a relationship

between measured absorbance and the pH. A plot of that data will give us the pKa of
the system.
The Henderson-Hasselbalch equation,

Total Absorbance (A) = CT = 1C1 + 2C2. Divide by CT to give

= 1f1 +2f, where f is fraction of the requisite form of the functional group.
Conservation of Mass, CT = C1 + C2, Where 1 and 2 refer to the acid and base
form of the functional group respectively. Divide by CT to give 1 = f1 + f2.
Manipulate the Henderson-Hasselbalch equation by plugging in the new forms of

pH = pKa + log

A
. (1-1)
HA

the conservation of mass and the absorbance equation to give pH = pKa + log

- 1
2

and subsequently, manipulation:

log

1
= pH - pKa.
2

(1-2)

Where is the absorbance divided by CT. 1 and 2 are the absorbances divided
by CT at our lowest and highest pHs. These two numbers are estimates of the
extinction coefficients of the acidic and basic forms of the functional group. This
equation is essentially an absorbance versus pH equation and we use it to find pKa of
p-nitrophenol. Below you will find an Excel sheet with the absorbance and pH data
for a sample spectral titration. Use this form for reporting your own data.

17

Sample Data from Original Experiment

1.39

0.0100

0.0001

e-eHA

eA-e

0.007
0.012
0.027
0.139
0.631
0.969
0.991
0.999
1.011

70.7
121.2
272.7
1403.9
6373.1
9786.9
10009.1
10089.9
10211.1

5.7000
56.2000
207.7000
1338.9000
6308.1001
9721.9001
9944.1001
10024.9001
10146.1001

10229.3
10178.8
10027.3
8896.099986
3926.899936
513.0999021
290.8998999
210.0998991
88.89989789

2.0000
1.5000
1.0000
0.5000
0.0000
-0.5000 4
-1.0000
-1.5000
-2.0000

pH

5.04
6.01
7.01
8.04
8.99

-3.2540
-2.2580
-1.6837
-0.8225
0.2058
1.2775
1.5338
1.6787
2.0574

10

y = 0.8607x - 5.9382
R2 = 0.9759

6.90

Fig. 3 Spreadsheet for Organization of or pKa Determination Data

For the above graph we used 65 to estimate the 1. This value is small enough that
the -1 value is always positive in our calculations. The 2 value used was
approximately 10,300. This estimate of 2 made the 2- value always positive. We
used the log

1
values bracketing the S axis to plot and from which to determine
2

the pKa.
Protocol
We have provided a set of 0.1M buffers ranging from a pH of 4.0 to 11.0 and a small
volume of 1.39mg/ml nitrophenol in water.

18

Each group will be given a cuvette rack and 9 clean 2ml cuvettes. Set out copy of the
data grid below in your lab book. It should serve to organize the setting up of the
experiment and to record the data.

Place 10X75 test tubes in a rack, number them and add one ml of each buffer to a
cuvette starting from the most acidic buffer. Add exactly 10L of nitrophenol into
each test tube and mix using parafilm.
Place a reference cuvette filled with water into the reference compartment of the
spectrophotometer and a second water-filled cuvette into the sample compartment.
Hit autozero to zero both sample and reference solutions and cuvette.
Remove the sample cuvette, decant all the water, add the contents of the most
acidic solution to the cuvette and measure its absorbance, to two decimal places.
Record the absorbance in the appropriate slot in the data grid in your lab book.
Repeat the above procedure; remove the cuvette, decant ALL of its solution, add the
next most acidic nitrophenol solution and measure and record its absorbance. Use
the data to determine the pKa for the system as described in lab. All the data are to
be manipulated and plotted by hand on graph paper. After this you can use Excel to
manipulate and chart the data to compare your analysis to Excels.

Set up the grid below in your lab book to contain your data.
Sample Data
Volume Nitrophenol
(ml)
0.010
0.010
0.010
0.010
0.010
0.010
0.010
0.010
0.010

Vol. Buffer
(ml)
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0

19

No.
1
2
3
4
5
6
7
8
9

pH

A400

E. Use the kinetic mode to measure the rate of the -galactosidase reaction and use
this data to calculate some population statistics for spectrophotometry.
We are going to use the kinetic mode of the spectrophotometer to determine rate
the reaction of the enzyme -galactosidase with its substrate p-nitrophenol--Dgalactose, PNPG. The kinetic mode fixes the wavelength and measures absorbance as
a function of time i.e., it measures how the reaction progresses in time. The
instrument will show the progress curve of the reaction on the screen and print out
the rate as dA/dT on the chart paper. There is no need to copy the progress curve,
just record the rate given in the data grid in your lab book.
We are going to measure the rates of four independent assays under the same
conditions. We will then calculate the average value and the standard deviation
expected from such an assay. We will then use the class data and do some population
comparisons with the averaged data from the other section.

Protocol
A ONE-STUDENT PROCESS FROM
START TO FINISH.

Set up five 10X75mm test tubes in a plastic rack.

Put 1.0ml of substrate concentration into a test- tube.
Set the Shimadzu to the kinetics mode and the to 400nm.
Set the lag time to 0, the rate T to 60 seconds, and the interval to
60seconds. This saves chart paper, as the instrument will only print out the rate
determined by the initial and final (60 second) absorbances, and not all the rates
at 10 second intervals.
Fill a cuvette with water and zero it against a second water-filled cuvette, remove
the cuvette from the sample compartment and decant the water.
Add 10uL of the enzyme solution to the substrate in the test-tube, and pour the
contents into the cuvette. The act of pouring will provide enough perturbation to
mix the contents of the test tube. Place the cuvette into the sample compartment
and press Start.
If the progress curve is linear over the entire time course this setting will give us
the rate of the reaction. Inspect the curve; if it is linear the value dA/dT will be
printed out by the instrument. If the curve is non-linear, tell the TA and you will
be shown how to linear rates.
Record the linear dA/dT from the chart paper in the data table slot in your lab
book.

20

Sample Data Grid

Stock PNPG
Testtube No.
(ml)
1

Z Buffer
(ml)

[S]
mM

dA/dT

2
3
4
5

B. In the Lab Book

A plot of absorbance versus wavelength with max appended.

A plot of Absorbance versus concentration in nM. This plot is to be done by hand
on graph paper. All statistical numbers for the least squares plot of the data
should be hand-calculated and displayed on the plot. Your calculated should be
prominently . Look up published values of and comment briefly on differences.
A plot of the log ratio versus with the pKa appended. The plot should be done by
hand. You will be allowed later to use a commercial program to manipulate your
data, but first, and primary, is the hand-developed data. Look up published pKa
and comment on differences.

Questions to be answered:

What are you seeing when you examine the effect of composition on light
transmission?
What is the molecular basis of the differences in light behavior of the different
cuvettes?
What practical (lab-related) lesson can we learn from this effect?

21

Experiment 4 - Finding the Specific Activity of Lactases

Purpose:

To set up a rate assay

To do the Bradford assay for total protein
To calculate the specific activity of an enzyme solution.

Specific Activity - We have already been introduced to a rate assay for -galactosidase
in experiment I. In most cases we need some more detailed information about the
enzyme, other than the fact that it is active. In enzyme isolation, in particular, getting
some idea of the amount and quality of the enzyme we are isolating is very important.
When we isolate enzymes we need to monitor each step of the isolation to see how it
affects the enzyme we are trying to isolate. Ideally we want to get rid of all the junk
proteins without losing any enzyme. Unfortunately, despite our best intentions, this
ideal behavior is seldom realized but we do need to do our bookkeeping whenever we
attempt a protein isolation to see how well we are doing. Perhaps one of our columns has
stopped doing what it should do or perhaps some other unforeseen variable has affected
our yield so badly that we need to start over again. One of the experimental numbers
we use to monitor the success of our isolation is called the specific activity. Specific
Activity (S.A.) is defined as the rate of an enzyme reaction divided by the amount of
protein in the sample and has units of

umoles product
. It is important because it is a
min - mg enz.

measure of the quality of an enzyme. A relatively high specific activity implies an

enzyme that is either very pure or very active, or both. In an enzyme purification
procedure the starting material has a relatively low specific activity, the S.A., being the
ratio of reaction rate (proportional to the concentration one of the proteins, the
enzyme, we are looking at) divided by the concentration of all the proteins in the
solution. This latter concentration is determined by a different method than the
former and so the ratio of reaction rate to protein concentration is not unity. At the
beginning we have lots of unwanted proteins relative to our enzyme protein so the S.A.
is low. At the end of the isolation we have only the pure protein of choice, having
removed all the other proteins, and the S.A. should be a maximum. The ratio of final
specific activity to starting specific activity is defined as the purification factor. Your
ability to understand and calculate specific activities in today's experiment is a critical
skill that you will use again when you do your bookkeeping on your -galactosidase
purification.
22

The Rate Assay - The measurement that allows us to obtain information about the
concentration of an enzyme is the rate of its reaction with a substrate. The measured
rate of -galactosidase reaction is governed by a rate equation that simplifies to the
Michaelis-Menten form:
v=

kcatEoS
Km + S

(2-1)

So if we hold [S] fixed we see that v is proportional to [E0]. At concentrations lower

than the Km the rate varies substantially with substrate concentration. These would not
be good conditions to use for a quantitative assay, because the rate would change as the
substrate was depleted in the reaction and small errors in determining the substrate
concentration would be give rise to day-to-day differences in rate. On the other hand,
at high concentrations of substrate (well above the Km) the rate hardly varies with
substrate concentration changes. These are the most sensitive conditions for a
quantitative assay. Provided you don't let the reaction run too long or use too much
enzyme, substrate depletion, or inaccuracies in determining the initial substrate
concentration, will hardly affect your measurements at all. Whatever substrate
concentration we use, if fixed, will allow us to get some relative measure of enzyme
concentration. As the equation above reduces to:
v = k[E0],

(2-2)

where k is a constant, kcat/Km. Raising or lowering substrate concentration merely

affects the sensitivity of the assay. However, if all we want is relative enzyme
concentrations, any fixed [S] will do. We could always increase the assay time to
increase the sensitivity of the assay.
-galactosidase (Lactase) Assay - The enzyme activity we will measure in this part of
the course is that of Lactase. It hydrolyzes the 1-4 glycosidic bonds in its natural
substrate, lactose as shown in fig. 5.

23

Fig.5 Hydrolysis of PNPG

The possession of this activity allows E.coli to utilize lactose, when it encounters it, as a
fuel. The enzyme does not need to be made regularly, as E.coli only infrequently
encounters lactose under conditions where glucose is also not also available. As it is
metabolically expensive to synthesize a protein, E.coli has evolved a means to synthesize
-galactosidase only when it encounters lactose. -galactosidase synthesis is therefore
under control of a lactose metabolite. Such production, in response to the presence of a
low molecular weight intermediate is called Induction. We are going to look at the
synthesis of lactase as a function of time, in the presence and absence of an inducer,
isopropyl--D-galactose (IPTG). We could measure -galactosidase activity on its
natural substrate, lactose, if we had an assay that could distinguish a disaccharide
reactant from its monosaccharide breakdown products. One such assay, using thin layer
chromatography, for instance, can separate and detect the different sugars but is not
very sensitive or convenient. What we will use as an assay is the breakdown of an
alternate substrate, p-nitrophenyl--D-galactose, or PNPG. Hydrolysis of PNPG, by galactosidase, produces galactose and nitrophenol. Neither of these immediate
products is easily quantitated. However, as discussed in experiment I, the nitrophenol
is partly ionized under physiological pH's to form the nitrophenolate anion and it is this
secondary product that is responsible for the ease and sensitivity of the assay. The
diagram for this reaction is shown in figure 1-2 in the introduction to
spectrophotometry section.
Initial Velocity Conditions - another assay consideration is product accumulation or,
"How long shall we run the reaction?" Obviously we need to run the reaction long enough
so that the desired sensitivity of signal is obtained. To make measurements easier to
24

interpret we also run our reactions under conditions of constant substrate

concentration so that our rate is linear. So combining these two considerations we run
the reaction only so long in the linear mode as is needed to get a good signal. Enzymes
can do several things make their progress curves non-linear. If there is a significant
accumulation of product, the reverse reaction might occur and influence your forward
rate measurement. In addition there could be enzyme Denaturation or substrate
exhaustion. All of these could result in a rate curve that would fall away from linearity
and we might have difficulty in determining the rate of the reaction. One of the
assumptions in Michaelis-Menten kinetics is that the product concentration is
insignificant over the time course of the reaction. Of course, the product concentration
cannot be always be insignificant in a real reaction and a practical guideline is that
measurements should be made under conditions where less than 5% of the substrate
has been converted to product. This condition will still show a linear rate - the reactant
concentration in is approximately constant over the time course of the assay. A
representative plot of an enzyme reaction is shown in the figure below. Note that the
enzyme reactions are not linear with time over the entire time in which they are
assayed. The rate of greatest interest is that one indicated by the dotted line. This is
called the initial rate or velocity (zero time rate) and is the rate of the reaction during
which both conditions above will apply, and consequently, the rate we use in all our
calculations. The Shimadzu will display the graph of the reaction on the screen as the
reaction progresses and print out a hard copy of the average rate on the chart paper.
This average rate is the line marked B. If your time course is non-linear the hard copy
rate will be less than the initial velocity and you need to input instructions to select the
time period giving the linear velocity to use in your calculations. In the picture you will
ask the instrument to read the tangent to the progress curve at times close to zero
time- the curve A. This is the initial velocity for the reaction and what should be
reported in the lab book. You do NOT need hard copies of the progress curve in
your lab book so do not ask the instrument to print any.

FIG. 6 -Galactosidase-PNPG Progress Curve

25

Other Reaction Variables - Enzyme reaction rates are influenced by temperature, pH, and
frequently, by other reaction variables, such as ionic strength. Although there is no
inherently "right" or "wrong" set of conditions under which to do an assay, it is important
that conditions be constant from one set of assays to the next. In this way, results with
different batches of enzyme or from different experimenters can be compared.
(a) Temperature effect - enzymatic reaction rates (like all chemical
reactions) increase with temperature up to a certain point. Beyond this
optimum temperature, however, the rate will decline because the enzyme
denatures. In terms of Michaelis-Menten rate expression, E0, the
concentration of active enzyme, can be said to decrease. Usually,
reactions are run well below the temperature that denatures the enzyme.
This helps assure that E0, and hence reaction rates, remain constant
during the period of the assay. Many E.coli enzymes work best at or near
37C, reflecting the fact that this organism is adapted to grow in the
human intestine. For convenience, we will run our assays at room
temperature, which is about 23C.
(b) pH Effects - the pH of the reaction also influences the rate. Changes in pH
may change the effective enzyme concentration by protonation or
deprotonation of critical residues. In addition, pH's well outside the
physiological range will denature the enzyme, sometimes irreversibly.
Because pH often has a major influence on reaction rate, it is important
that pH be controlled at a constant value throughout the reaction. This is
achieved by use of a buffer, which minimizes pH changes (or buffer) in
solution. We therefore look for buffers whose pKa's are close to the pH
optimum for our enzyme assay where their buffering capacities are at a
maximum.
Outline of the Specific Activity Experiment
A. Extraction of Lactase from Lactaid Tablet and solution of E.coli Lactase.
B. Rate Assays on Small Volumes of Extracted Lactase.
C. Bradford Assay on Different Volumes.
This experiment will be done in groups of two. Each group will receive one Lactaid tablet,
a small volume of E.coli Lactase, a volume of 1.0mM PNPG in Z buffer and a volume of Z
buffer for the dilutions.

26

A. Extraction of Enzyme from Tablets

The protocol for extraction is as follows:

Grind up the tablet well in a mortar and pestle.
Add 5 ml Z buffer and continue grinding for a further 5 minutes.
Decant about 1.5ml of the slurry into a labeled microfuge tube and microfuge for one
minute at 3000rpm.
Decant the supernatant into a clean eppendorff and use this extract for the
experiments 2 and 3.

B. Assaying Small Volumes of Enzyme

The volumes of extract we are using are small enough so that the concentration of PNPG
in all assays is essentially constant. When an instrument becomes available, check that
it is in the kinetics mode and that the settings are correct before doing any assays.

Set up a test-tube rack with two sets of four 10X75mm labeled test tubes. One
set is for the tablet lactase, the other for the E.coli Lactase.
Rate Assay

Zero the cuvette against water

Add the volume of extract to one of the substrate solutions in the test tubes
using a pipetman. Hold the test-tube and pipetman up at eye-level and discharge
the contents of the pipetman directly into the PNPG solution by having the
pipetmans tip under the liquid surface. Quickly remove the pipetman and pour the
reacting solution into the cuvette, place the cuvette in to the sample
compartment and record its rate. The act of pouring will serve to mix the enzyme
and substrate thoroughly. Assay from the smallest to the largest volume of
extract.

Pour out the spent reaction into the waste container and use the same cuvette to
determine the rates of all your dilutions.

Look at the dA/dT on the chart and record it in your lab book in the form of the
table shown above.

27

PNPG
1.0
1.0
1.0
1.0

Z Buffer (uL)
45
40
35
30

lactase (ul)
5
10
15
20

A400/min/

The recorded is rate i.e., the slope dA/dT the rate of the enzyme reaction in units
of change in absorbance/change in time/volume of enzyme in the assay. The total
assay volume is held constant over the course of all assays. The instrument will
calculate and record the slope of the curve and print it out on the thermal paper. So
let's review what we want to do with this instrumental slope.
A400
. For
min - cm - ml enz.
units
publication, the rate needs to be reported in dimensions of
, the IUPACml
approved dimensions. We need to convert our observed initial rate measurement into
its IUPAC equivalent.

The rate assay, the observed slope (or rate), has dimensions of

Example of a Specific Activity Calculation

This calculation is just an exercise and has nothing directly to do with the assay
conditions for experiment 4. Use this calculation only to understand the process of
getting to units/ml from the raw data.
Conditions of the assay - Put 1.5 ml of substrate in a test tube. Add 1.5 ml of buffer
and 0.050 ml of an enzyme solution that we have diluted by a factor of fifty. Dilution
by a factor of fifty means that we have taken one part by volume and added forty-nine
parts of buffer. The observed rate (dA/dT) is 0.43/min. The assay is being run in a
buffer with a pH of 6.5 and the chromophore which we are measuring has a pKa of 7.12.
Step One - manipulate the raw rate by dividing it by the extinction coefficient and
multiplying by the assay volume, in liters. This converts the raw rate into a rate in
terms of a change in the mass, in moles, of the product in the cuvette, per min, per ml
enzyme.

0.43
mmoles - cm 0.00305L
mmoles
X
X
= 1.43X10-3
cm - min - 0.050ml
18.4L
min - ml

28

Step two - manipulate the rate equation to get the rate in terms of units/ml. The unit,
the official IUPAC dimension for rates, is defined as moles product/min under optimal
assay conditions. The equation thus reduces to:
units
1.43X10 - 3mmoles 1000moles unit - min
X
= 1.43
X
ml
min - ml
mmole
mole

Step three - the rate in step two is that for the fifty-fold diluted enzyme solution and
we must correct it if we want the rate of our original enzyme solution. This is done by
multiplying the rate from step two by the dilution factor of fifty. The dilution factor is
a ratio of concentration units and has no dimensions.
1.43 units 50
units
X
= 71.5
ml
ml

If we had used one ml of the concentrated solution in the assay we would have
determined an activity of 71.5 units.
Step four - We have done the assay at a pH where the product is partitioned between
an ionized species that absorbs at the wavelength of observation and whose
concentration we can determine, and an unionized species that does not absorb. We now
correct the rate for the absence of the non-absorbing species by dividing by the
fraction ionized at the pH of the assay. For our example the correction factor is:

fa =

10 ( 6.5 7.12)

1 + 10 (6.5- 7.12)

= 0.19

Thus the units/ml of the original sample is:

units
71.5 units
X
= 376
ml
ml
0.19

C. Bradford Assay on Dilutions

Accurate determination of protein concentration is inherently difficult. The reason
should be obvious, with a little thought. Unlike the nucleotide bases in RNA and DNA,
which are chemically quite uniform, the amino acids in proteins vary greatly in charge,
size, hydrophobicity and ultraviolet absorbance.
Sometimes protein concentration is estimated by ultraviolet absorbance at 280
nm. Three of the 20 naturally occurring amino acids, tyrosine, phenylalanine, and
tryptophan, absorb at this wavelength. This method is all right for pure protein with a
29

known extinction coefficient. However, the proportion of UV-absorbing amino acids

varies considerably from one protein to the next. More importantly, biological samples
often contain other substances - particularly nucleotides and polynucleotides - that
absorb significantly in the same range. Thus, UV absorbance is of limited value for
estimating protein concentration in complex mixtures.
The most common method in use today for estimating total protein concentration
is the method of Bradford [Anal. Biochem. 72:248(1976)]. In this method, an organic
dye is allowed to bind to protein in the sample. The color changes from brownish purple
to brilliant blue, a change which can be measured, and quantitated, in the
spectrophotometer.

Fig.8 Spectrum of Protein-Bound And Unbound Coomassie Blue

The reaction is shown below:
30

The exact basis of the binding specificity is not certain, but it is clear that the dye
binds to proteins more or less uniformly, so that the amount of absorbance we read is
proportional to total protein mass. In addition, the Bradford assay is quite specific for
protein, and is not subject to interference from nucleic acids, most detergents, buffers,
or other common substances found in biological samples. Because the amount of
absorbance may vary somewhat from day to day and depending on the batch of reagent,
the Bradford method is always used in conjunction with a standard curve constructed
at the same time that the unknown samples are tested. A standard curve is made by
measuring the absorbance values obtained using samples of Bovine Serum Albumin (BSA)
of known concentration. Our unkown protein concentration is determined by comparing
the absorbance of the unkown with those of the BSA standard set. The results are only
relative measures of the concentration of our unkown as different proteins might have
different specific absorbances when bound to Coomassie Blue.
Setting Up the standard curve
The standard curve will consist of 5 points ranging from 5g to 50 g BSA per tube.
The concentration of the BSA standard protein solution provided will be 50 g/ml. The
unkowns solutions are to be mixed and read at the same time as are the standard BSA
solutions. Record the data and construct a standard curve consisting of A595 versus g
protein. Use this curve to determine the concentration of the various dilutions of your
unkown protein. Use only absorbance values within the range of the standard curve for
your calculations. You need at least two acceptable values for your report. If no values
are in the accepted range then use a smaller volume of the original and repeat the assay.
Remember you have two unknowns for specific activity determination.

Tube No.
Std.0
Std.1
Std.2
Std.3
Std.4
Std.5
Unknown

BSA
mL
0
0.20
0.40
0.60
0.80
1.0
X* L

Water
mL
1.0
0.80
0.60
0.40
0.20
1.0

Bradford
mL
1.0
1.0
1.0
1.0
1.0
1.0
1.0

31

g BSA

A595

* the volumes of the unknowns are small enough so they do not siginificantly
alter concentrations or volumes of any important components in the assay.
Initial, Speculative volumes to use for Unknowns
You do not know how concentrated your protein extracts are, so it is best to do an initial
say with a small volume of extract, say 2-5L and compare this A595 value with those
from your standard curve. If the low volume value is within the range of the standard
curve values, well and good; if it is too concentrated, i.e., its values are above the range
of the standard curve values, you need to make dilutions of the extract to bring it
within the acceptable range, i.e., those that lie within the range of the standard curve
values. Dilute the extract by 5-10X using extract buffer, and check the diluted samples
A595 against acceptable values, Again, you need at least two unknown values within the
range of the standard curve to determine a minimally acceptable value for the protein
concentration of your extract. Use the Unknown row in the data table above to set up
your unknowns for assay. Record exactly and explicitly in your lab book what you had to
do in order to get acceptable values for your concetration calculations. This includes
recording those dilutions or volumes which were out of range. All these should be
organized under the title of Getting Unknow Values in your data section of the lab
book. Construct a standard curve by hand and insert it into your lab book in the results
section.

Fig. Standard Curve for Bradford protein Asay

32

Calculations to Determine Protein Concentration

We have already showed you how to calculate one of the values, units/ml, needed for
your specific activity. The remaining number is the concentration of protein in mg/ml. An
example of a protein concentration calculation is shown below:
We put 5Lml of a 50-fold diluted protein extract into one ml water and added
one ml of Bradford dye. After 10 minutes we found the A595 to be 0.37. According
to the standard curve this absorbance corresponds to a protein (BSA) mass of 8
g. Therefore the concentration of the diluted protein is 67g/0.005ml or
(1600g/ml). This is the mass of the protein in the volume we added to the assay
divide by that volume. To find the original extract concentration multiply by the
dilution factor and convert g to mg.
1600ug 50
mg
mg
X
X
= 80
ml
1000ug
ml

Step six - we now have both numbers needed for calculating the specific activity. So
dividing the activity in units/ml of the original solution by the
The figure below is what we would expect of a Bradford standard curve. Note the nonlinearity and the limits imposed on measurements with meaning. If your unknowns are
not with in the range of the standard curve you should make up new ones that are within
the limits.
protein concentration of the original solution in mg/ml will give us the specific activity
of the original solution in units/mg.
376units
ml
units
X
= 4.7
ml
80mg
mg

Lab Book Information

In the Results section of your lab book under the title Specific Activities of
Lactases Construct the following table of Specific Activities and Protein
Concentrations

33

Extract
Volume
L
5
10
15
20
Tablet
E.coli

Rate
(dA/dT)

Units/ml

mg/ml

Spec. Act.
U/mg

Show an explicit specific activity calculation for the sample with the highest rate.

Report the value of your specific activity you deem most accurate.

Use the uncorrected rates for your various dilutions and their protein
concentrations to construct a graph of rate (dA/dT) versus protein concentration in
g/ml. Comment on the form of the plot.

34

Experiment 5 Subunit and Native Molecular Masses of Lactases

Uses SDS Polyacrylamide Gel Electrophoresis (PAGE) in an 8% gel to determine

subunit Mrs of E.coli and Tablet Lactases.
Use non-denaturing PAGE in a gradient gel to determine functional Mrs for E.coli
and Tablet Lactases.

The Basis of Polyacrylamide Gel Electrophoresis

Charges to move in an in an electric field according to their:

Charge/mass.
The sieving effect of the gel which will affect mobility.

Polyacrylamide Matrix
The retarding matrix is a polyacrylamide gel. This is made by adding a together
acrylamide, the polymer-forming compound, with bis-acrylamide a cross-linker,
persulfate, an initiator, and TEMED a catalyst. The sieving effect of the resultant gel
depends on the pore size of the gel matrix which, in turn, depends on the concentrations
of both the acrylamide and the cross-linker.
35

Migration of Ions
Migration distance = Charge/(size, shape) sieving effect of the matrix. In SDS PAGE
all proteins have a constant charge/shape contribution and therefore the separation
depends on inverse size as larger molecules are more retarded by collisions with the
matrix material and have smaller migration distances. Native gel electrophoresis is a far
more complicated function of molecular and matrix properties but still follow the
log(Mr) vs. migration distance plot..

Reprinted from D.J. Holme and H.Peck, Analytical Biochemistry.

36

We will use two kinds of gels:

An 8% gel for the SDS PAGE separation. An 8% gel will resolve between 200,000
and 43,000 Da
A 4%-20% gradient gel will be used for the native gel. The resolution should be
between 200,000 and 18,000 Da. Note that the resolution in a gradient gel may
not be as fine as in a single-concentration gel because the resolution ranges
overlap.
SDS Gel Electrophoresis

SDS Gel SeeBlue Plus 2 Prestained standards. This standard needs no

preparation and 10ul/well is the recommended volume for our size gels.

SDS standards

37

Prepare Unknowns for SDS Gel Electrophoresis

Dilute both lactases to about 1ug/uL in water in a small eppendorff tube.

Loading Buffer (uL)
10

Diluted Lactase (uL)

50

Boil for 2 minutes.

Cool to room temperature.
Load 15-20 uL on 8% gel.

Running Gels
SDS gel four groups per SDS gel and there are 10 wells in the gel. The two outer lanes
will contain SDS standards at the volumes suggested. The eight inner lanes will contain
20uL aliquots of the two lactases from each group in the order E.coli lactase then
Tablet lactase from left to right. Thus we can accommodate four groups on a single gel.
Three electrophoresis units will be needed for the SDS gels.

Running Voltage
We will run the gel at constant voltage of 100-150V. The electric Field is defined as
volts/gel length. Use this formula to calculate the electric field under which we are
separating our lactases.

38

Native Gel Electrophoresis

Native Gel Standards.

Standards

Mol. Masses (Da)

Loading Volume (uL)

Carbonic Anhydrase
Chicken Egg Albumin
Bovine Serum Albumin

29,000
45,000
66,000 (monomer)
132,000 (dimmer)
272,00 (trimer)
545,000 (hexamer)

20
20
15

Jack Bean Urease

100

The table below shows how the Native standards were prepared for you, all you have to
do is load the appropriate volume in the wells.
Standard

Volume Std (uL)

Loading Dye volume uL)

Carbonic Anhydrase
Chicken Egg Albumin
Bovine Serum Albumin
Jack Bean Urease

20
20
20
30

10
10
10
5

Native gels There are two kinds of proteins on the gel lactases and standards. The
lactases will be identified using specific substrates which will allow visualization of the
all active bands of the lactases. The standards need to be identified using the harsh
general protein stain - Coomassie blue dye. The Coomassie conditions preclude using the
same visualization technique on both sets of protein on the same gel. We thus need to
run a separate Coomassie gel along with the we only have five electrophoresis units so
that the remaining two have to be used for the native gel. In order to accommodate nine
groups o two gel it is necessary for one group to share another groups data. Group 9, in
aisle 5 will share the data of groups 8 and 7 in aisle 4.

39

Loading Native gels

Lane
1
2
3
4
5
6
7
8
9
10

Sample
Carbonic Anhydrase
Egg albumin
Bovine Albumin
Urease
E.coli Lactase group X
Table lactase group Y
E.coli Lactase group X
Table lactase group Y

After running the gels we will cut off the top of the gel at the wells and the bottom of
the gel at the migration distance of bromphenol blue the fastest running component on
the gel before we cut the gel in half to visualize the two sets of proteins appropriately.
We can thus determine rf vaues for standards and unknowns to use these rF values to
calculate relative molecular weights.

40

Experiment 4 - Isolation of E.coli Alkaline Phosphatase (AP)

Reading: Ostland and Janson et al., Protein Expression and Purification, Parts I
and II, 1(1997)

In the olden days, those times prior to 1970, when one attempted to isolate
almost any protein they were constrained by the fact that, in all probability, their
protein was coded for by the chromosomal DNA and present in only one copy per cell.
Thus the protein was expressed in a one-to-one ratio with every other protein
expressed at that time by the cell. Any concentration differences obtained between
proteins depended entirely on the strength of the particular promoter used for
transcribing the gene for that protein. This made for tedious, complicated isolations, as
the protein of choice was generally a minor component in a complex mixture of proteins.
In addition the means for isolating one protein from a mixture, depended on subtle
differences in size, shape or some constellation of charges on the protein surface. Thus
isolating a pure protein was tedious and success was not guaranteed.
However there was still the problem of the proteins natural abundance. The
proteins concentration would be decided by its relative efficiency of transcription and
translation, and it could well be present in relatively small amounts in the original mix.
In the years 1972-1973 Boyer, Cohen and Berg, were able to devise a method that
ensured that the protein of choice could synthesized in relatively large amounts by some
microorganism. By taking advantage of earlier discoveries of restriction endonucleases,
which would hydrolyze DNA at specific sites, and, DNA ligases would could re-join them,
Cohen and Berg were able to excise relatively specific DNA sequences from one source
and insert them into another source, thereby transferring genetic information from one
source to another, in a routine fashion. This insertion of foreign DNA was called cloning
and the resulting DNA was called recombinant DNA. If the cloned DNA coded for a
protein and were ligated into a plasmid in a particular promoter's reading frame,
bacteria carrying that plasmid could express the protein. Plasmids are natural
information-carrying pieces of DNA that are replicable by normal cellular machinery. In
addition they can replicate independently of their hosts' chromosomal DNA, and
therefore are able to be maintained in multiple copies inside the cell. These multiple
copies of the plasmid will each express their coded proteins resulting in an increase
concentration of plasmid proteins in the cytoplasm of the organism. Thus they can
produce much more protein than could be produced by the expression of chromosomal
DNA. This increased relative concentration, called over-expression, makes for an easier
purification because there is a larger fraction of the over-expressed protein present in
the protein pool.
We are not going to use recombinant DNA to produce our protein in bulk, but
rather a technique somewhat older. Our source of the enzyme will be an E.coli strain
called C90, a strain which has been genetically modified to produce AP constitutively.
41

This means that as long as the cell is viable it will churn out the enzyme regardless of
cellular needs resulting in AP being a larger fraction of the cellular proteins than is
normal. Even though the AP is a larger fraction of the protein it is still very similar in
composition to some of the other proteins and we need to remove them. The bases of
protein separation are; solubility, size/shape, biological affinity, and charge
constellation and/or net charge and. We are going to use the latter properties in an
isolation technique called ion-exchange chromatography
Overview and Organization of the Experiment.
This experiment will be done in groups of four. A volume of a crude AP extract will be
given to each group. An aliquot of this crude extract will be run over a DEAE-cellulose
column which will separate proteins according to differences in their charges. We will
construct a purification table along the way to measure the success of the isolation.
This is a multi-session project and its logistics are given below.
Step I Revive and grow C90.
Extract and retain a 5ml volume for the experiment.
Prepare and pour DEAE column.
Step II Run aliquot through the column.
Elute the activity with NaCl.
Analyze all test tubes to find to find active fractions and inactive fractions.
The compilation of this data is called the column profile.
Combine the active test tubes content and designate it the Purified Fraction.
Label and retain for purification table measurements.
Concentrate a 2ml of the purified fraction for SDS gel analysis.
Step III
Run SDS gel of all fractions.
Visualize protein bands on the SDS gel using the Sypro Red protein stain.

Specific Protocols for Isolation

A. Growing up the Culture.
Add 0.2ml of the stock strain C90 to 200ml of LB medium, shake overnight at 37o.
The genotype of E.coli C90 is: (garB10,fhuA22, ompF627(TR2),fadL701(T2R),relA1,

pst-9, rrnB-2, mcrB-1, creC(510), pit-10, spoT1,)

42

B. Extracting Proteins by Sonication

Line voltages are converted to very high frequency AC which is fed into a titanium tip of
the sonotrode. The tip vibrates rapidly in unison leading to the rapid back and forth
movement of the surrounding liquid. This movement creates small bubbles within the
liquid which rapidly implode. This implosion causes transient regions of high temperature
and pressure and a rapidly moving (400km/hr) liquid stream which impacts, and destroys
the integrity of, the cell surface.
Extracting From the Pellet

Resuspend the frozen pellet thoroughly in 10ml of cold water.

Sonicate the suspension in an ice bath for 30 seconds at a power setting of 8.
Allow the sonicate to cool for about one minute.
Repeat the last two steps three more times.
Centrifuge the sonicate for about 5 minutes at about 10,000rpm on the Sorvall
centrifuge.
Decant and retain the supernatant and discard the pellet in the trash.
Record the volume of the supernatant (the crude extract) in the appropriate of
your purification table in the lab book.
Determine the activity of the crude extract as follows:

1mM
NPP
50uL

1M TrisHCl. pH 8.0
and 1mM MgsO4
0.9ml

enzyme

A400/min

50uL

NPP is nitrophenylphosphate a substrate for the phosphatase.

Label your crude extract and store it in the refrigerator until Tuesday.

Making Buffers

Each aisle will make 400ml of 0.003mM TrisHCl, pH 7.4, containing 1mM MgSO4.
Take 50 ml aliquots for the stock buffer above and add solid NaCl to make two
additional buffers containing 0.08M and 0.2M NaCl.

Equilibrating the Column.

43

Each aisle will be provided with 10ml 0f the preswollen DEAE in 24% ethanol.
Follow the manufacturers instructions on Preparing the gel.
Pour the gel into the column provided and pack as instructed.
We will not be using an adaptor.
Equilibrate as per instructions checking the pH of the eluate using the good pH
meter in lab M225 which has already been standardized.
When the DEAE has been equilibrated, store the column at room temperature
until next Tuesday.

Locating Active Fractions and Making Column Profiles

After the isolation you have a collection of test tubes, some containing enzyme
activity, some containing no activity but containing other proteins and some containing
both. We are initially interested in constructing a column profile for the isolation and so
need to measure both enzyme activity and protein concentration in every test tube we
have collected. A column profile is a diagram of the enzyme activity and protein
concentration as a function of elution volume or fraction number, and is a graphical way
to assess the isolation. A column profile is shown in the figure below. After the rate
and protein assays for the individual test tubes are completed we will use this data to
compile a column profile. We will then combine the contents of the most active test
tubes in the flow-through set to make one large fraction for our purification table. This
fraction is called the flow-through fraction. We then combine the most contents of the
most active test tubes from the eluted set to give us one large fraction for the
purification table. This fraction is called the purified fraction. Aliquots of the two above
fractions, along with an aliquot from the crude fraction, will be assayed for enzyme
activity and protein concentration to make the entries in the purification table. The
tasks outlined below allow us to organize the work necessary to go from a set of test
tubes containing solutions of unknown composition to the information in the purification
table. Look over these tasks and organize the group to do the work collectively and
efficiently. Make sure you retain two ml of the active fraction for the SDS analysis.
Take this aliquot out first and store it in a safe place.
Location protocol

We will be assaying fractions as they come off the column. This will do this by
allowing about five fractions to collect in the test tubes, removing these from the
rotor, numbering them, and placing them in a rack.
Set up about twenty 10X75mm test tubes containing exactly one ml of
nitrophenylphosphate (NPP) made as follows:
44

Stock NPP 6.67ml of 3mM NPP in water.

Stock Buffer 1.0M TrisHCl, pH 8.0 containing 1mM MgSO4
Stock NPP
6.67ml

Water
193

1.0M TrisHCl
200

Calculate the final or assay concentration of NPP and record in your lab book.

Add 100uL of each fraction to a test-tube, mix, and determine the reaction rate of
that fraction.
Record the rate in the appropriate column in a 40 entry master table in your lab
book. The table should be arranged as follows:

Fraction No.

A400/min/0.01ml

A595

1
2
3
4

Determining Protein concentration for Column Profile.

Locate Protein-containing fractions

There are three possible large-scale fractions in this isolation; the initial 30ml
volume, the 3mM elution or wash; the 20ml 80mM elution and the 20ml 200mM final
elution.
We will wait until each large-scale elution has been assayed for activity and then we
will use the Eliza rack method from the lab book to assay each for proteins.
The Eliza rack method is as follows:
o Add 1ml Bradford to as many slots in the Eliza rack as you have test-tubes
in a large fraction.
o Add 0.1ml of each fraction to a slot in the Eliza rack and mix gently.
o Wait for about ten minutes.
o Read at 595nm, versus a Bradford blank.

45

Record the data in your lab book in a table as shown below:

Fraction No.
Blank
1
2
3

Bradford (ml)
1.0
1.0*
1.0
1.0
1.0

Fraction (ml)
0.1

A595-Blank

* Blank contains 0.1ml water instead of fraction.

Record your (A595 blank) value in the A595 column in the mater table.
Use this to construct the column profile and to select active volumes from the
large-scale fractions.

There is no need to be waiting for the rate information before measuring the protein
concentration. No standard curve is needed for this measurement. What we want is
relative protein concentration.

Do Formal Rate and Bradford Assays for Purification Table

We now have all the information we need for the column profile and we need to combine
the individual active test tubes for our bookkeeping calculations. This bookkeeping will
involve doing rates and protein concentration of both the flow-through and the salteluted fractions in a formal manner as we did for the specific activity experiment. The
four or five test-tubes containing the most enzyme from the flow-through set form one
large fraction also called the flow-through fraction . The four or five test tubes from
the affinity set combine to give the purified fraction. These will form two entries for
the purification table. Record the volume of each of these two large fractions in the
purification table in your lab book.
Assays for the Purification Table

46

Do a rate assay by adding 2.0 ml of 0.1mM NPH to a 10X75mm test-tube. We dont

know how active each fraction and we want to assay small volumes of each major
fraction as well as have a constant assay volume. To do this we will add a volume of
buffer to the NPH in the test tube and, when we are sitting at the
spectrophotometer ready to assay, the small aliquot of fraction. The total volume of
fraction + buffer should not exceed 0.5ml. All of these combine to give a final assay
volume of 2.0ml. Do this assay on all fractions the crude, the flow-through and the
salt-eluted fractions. Note there is nothing magical about an assay time of one
minute. If your enzyme is active enough to assay in one minute, good. If not, (usually
the affinity fraction won't cooperate in this manner), do a fixed time assay on it
using a larger volume of enzyme solution or wait longer for the absorbance to
increase. You may need to wait for up to thirty minutes to get a decent absorbance
change. Also, the fact that I am asking for a total of 2.50ml for the assay volume
does not mean you initially have to use 0.5ml of any fraction. The volume assayed
depends on the activity of the enzyme. In some cases you might need to add only
40uLto get a decent rate, in other cases 0.4ml. In all cases make up the missing
volume with buffer.

Do a Bradford assay, including a new standard curve, on all fractions as we did in

the specific activity experiment.

From the data, construct a purification table. Some of the entries should be
familiar. The activity, protein concentration and specific activity measurements and
calculations have already been discussed. Keep in mind that these entries are for a
particular fraction. Entries which might be unfamiliar are:

Volume - this refers to the entire combined volume of the active fractions
selected.

Total Activity - this is a measure of the enzyme activity remaining in each

fractionation step. This parameter is calculated by determining the activity, in
units/ml, in a particular fraction, and multiplying by the volume of that fraction.
This leaves us with dimensions of units. Units are, as we recall from the
specific activity experiment, are proportional to enzyme concentration. A low
value for total activity indicates possibly that our isolation methods may need
to be modified to recover more enzyme.

Percentage Yield - the % yield is not included in the purification table in the lab
manual but should be incorporated into the purification table in your lab book.
This number is calculated for each column fraction by dividing the total activity
of that fraction by the total activity of our original crude solution and then
47

multiplying by 100. Note that the total activity of the crude fraction serves as
a benchmark - 100% activity - to determine percentage yield for all later
actions. The assumption of 100% recovery of enzyme assumes that we have
broken open our cells perfectly and that, not having had time enough, we have
done nothing yet to inactivate the enzyme and so have lost none of its activity.
As operations on the enzyme solutions take place and time increases, the
percentage yield usually drops.

Fraction

Vo
l.
ml

Activity
Units/ml

[Protein]
mg/ml

Sp.Act.
Units/mg

Crude

Activity
units

Yiel
d
100

Flow-thro
0.08Meluate
0.2M eluate
Figure 27: Data Matrix for Purification Table.

Task IV - Concentration of Proteins by Filtration

Add about 2 ml of your purified fraction to the filtration device and centrifuge for
about 120 minutes at less than about 5000 rpm. The filtration unit has a selectively
permeable membrane that will only allow proteins with molecular weights smaller than a
certain value - in our case roughly about 30,000 Da, to pass through. Thus the solution
48

retained in the filter is enriched in higher molecular weight components such as our
fusion protein which weighs in at about 150,000 Da. As the solvent volume goes down,
the concentration of the retained components goes up.
DAY IV - SDS Gel Electrophoresis of Fractions
Set up and run SDS as gels before. Each group will need three slots for their fractions
so two groups may cooperate in pouring and running a gel. As an exercise put some very
dilute fraction in one slot of the gel to check the sensitivity of the Sypro stain. Don't
forget to include a standard with each gel.

Lab Information for the Isolation Experiment

a. Complete all the entries in the purification table. Find a
reference in the literature to an isolation of .coli Alkaline Phosphatase and
compare purification factors for your affinity purified -galactosidase and
the one from the literature and comment on the differences.
Purification factor = specific activity pure .
specific activity crude
b. Complete the column profile.
c. Comment on the purification looking at the information contained in both the
purification table and the gel. Do they roughly give the same information
about the purity of the final enzyme solution? Any other bands on the final
gel? Did we remove much junk protein using our column?
d.

What is the molecular weight of the protein?

49

Kinetic Analysis of E.coli Alkaline Phosphatase

Do a kinetic analysis of the Alkaline Phosphatase reaction with NPP as substrate

following the protocol in the lab manual. Each group will do its own substrate
preparation and do duplicate assays at every substrate concentration. I will
provide a common enzyme prep for the occasion.
Each group will pour a fixed percentage gel for the electrophoresis experiments
on Thursday.

Kinetics
Each group will make, in clean 10X75mm test tubes, 2X1ml volumes at the substrate
concentrations shown in the table below in a final volume of 1 ml. Each group will be given
an aliquot of 16.3mg of NPP dissolved in 43.9ml water, and the relevant buffer, to make
the substrate concentrations as specified. Fill in the missing entries in the table The Mr
of NPP is 371.1Da,
Solution
A
A
B
B
C
C
D
D
E
E
F
F

Buffer
(ml)
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500
0.500

H2O
(ml)

NPP
(l)

[NPP]
mM
0.003
0.003
0.010
0.010
0.020
0.020
0.050
0.050
0.100
0.100
0.200
0.200

A400
/min/0/050ml

Data Analysis - do both the direct linear plot and the non-linear Least Squares program
from Michaelis-Menten mechanism from Enzfitter.
Non-Linear Least Squares Analysis of Kinetic Data
Use Enzfitter from Biosoft to find least squares values for alkaline phosphatase using NPP as
substrate.
50

Start Enzfitter from the Start menu.

Select NEW from the Enzfitter menu, click on the folder to to open it and click on RAW
DATA which will open a spreadsheet.
Enter your data into the spreadsheet, your [S] values to the left and their corresponding
rates to the right.
From the menu INSERT, choose DATA SET.
Select your data points in the newly opened Data Set Wizard, just check that your ranges
are valid.
Enter a name for your data.
New Folder opened for your data. Click on folder and open DATA to open a spreadsheet
with the data set.
Select DATA SET/CURVE FIT and enter your choices into the CURVE
FIT WIZARD as flows:
o Equation Michaelis-Menten.
o Weighing SIMPLE.
o Independent Variables s(=X0).
o Select NEXT for step 4 of the wizard.
o Select AUTO ESTIMATE for step 5.
o Select MARQUARDT-LEVEVBERG for step 6.
o FINISH will get initial estimates for the curve fit, hit OK to accept the
estimates.
Will get a FIT folder.
Open folder and look at and print:
o Fitted Parameters Details of your data and its analysis.
o Best Estimates get best fit Km and Vmax values and statistical parameters relating
to the goodness of their fit to the data.

Enter FITTED DATA/INSERT/GRAPH FIT.

o On ADD PLOT menu select: X-axis X0.
o Select Y-axis observed y.
These entries will give you a graph of your data points on an appropriate set of axes called GRAPH 1.
Select GRAPH/PLOT FIT and enter X-axis X0, Y-axis fitted y.
Print all four pages.

Data Analysis
Explain the meaning of all the statistics listed on your FIT 1 Results.
Compare your values with literature values.

51

Molecular Weights of E.coli Alkaline Phosphatase

Pouring Acrylamide Gels at Home

Each group will be assigned a gel to pour for next time. The gel would either be a
8.0% SDS gel or a single percentage gel for the Ferguson method of determining
native molecular weight by electrophoresis. Each group should have two spacers, one
silica notched plate and one glass plate and some clay.
Thoroughly clean and dry both plates, the notched and the glass plates by washing in
soap, thoroughly rinsing in water and then cleaning with 95% ethanol. Dry thoroughly
and leave for about 15 minutes. .
Lay the notched plate down on a rack with the notch to the top.
Slightly grease the outer edges of the spacers and lay them down on either side of
the notched plate.
Place the glass plate over the notched plate and seal the bottom of the plates with
clay. Label the glass plate with the gel %. This arrangement should form a
compartment into which we will pour a polymerizing solution called the separating gel.

Spacers

Glass Plate
Notched Plate

Glass Plate

Stacking Gel
Separating gel poured
to below the lower part
of the notched plate.

Separating ge

Notched Plate

Clay Plug

Solutions for Making Gels

Separating Gel Solution -1.5M TrisHCl, pH 8.8. This contains some bromophenol
blue to aid loading.
Stacking gel Solution 0.5M TrisHCl, pH 6.8.
52

10X Electrode Buffer- 30.3g Tris base+ 144g glycine per liter. The final pH
should be about 8.3. Each aisle making native gels will make 500ml of a 1X

solution of Electrode buffer for the next time.

Native Gel Loading buffer (5X) 15.5ml Stacking gel solution, 8.25ml water, 25ml
glycerol and bromophenol-blue to taste.

Use a clean 15ml centrifuge tube for your solutions. Acrylamide is a neurotoxin WEAR GLOVES. Wash out the tube afterwards and dispose of the waste in the
plastic bottle on each aisle. The same formulation will be used for the separating
gels in both SDS PAGE and the native PAGE.

% Gel

Water (ml)

38.5% BisAcrylamide (ml)

Separating Gel
Soln (ml)

4
5
6
7
8
9
10

7.75
7.4
7.1
6.8
6.5
6.2
5.9

1.25
1.6
1.9
2.2
2.5
2.8
3.1

3.0
3.0
3.0
3.0
3.0
3.0
3.0

Add 50L APS, ammonium per sulfate, and 5L TEMED, mix and pour the gel
smartly.
Tilt the plates at an angle and use a Pasteur pipette to add the bis-acrylamide
separating gel solution to the compartment formed by the plates, spacers and
clay. Fill the compartment up about one-half an inch below the lower edge of the
notch. This should take only about 7 ml. Watch out for bubbles in the
compartment. Place unit upright in a rack, layer t-butanol carefully over the
surface and wait for polymerization. This should take about 20 minutes.
When it is polymerized, wash off the surface with dilute separating gel solution,
cover with saran wrap and store in the refrigerator until next time.
Next Time

Run SDS Gel. Aisles 3 and 4 will handle these.

Finish making and Run Native Gel
I have concentrated your Purified fractions in Amicon Concentration Units.
Prepare and load 25uL aliquots of this concentrated fraction in both gels.
53

Amicon Ultrafiltration Unit

Concentration Factor =

Original volume
Retentate volum

3000Da Cut-off Filter

One person in an aisle attends to the SSD gel while another attends to the
Native gel.

Native Gel Preparation

Each aisle will make 500ml of a 1X Native gel electrode buffer for todays
run.
Wash out the surface if the gel and blot dry with 3M Paper.
Add 100uL of APS to 12 ml of stacking gel solution. This already contains all
ingredients necessary for polymerization except APS.
There is enough stacking gel solution for more than one gel so 5%, 6%
and 7% native gels will be poured I aisle 1.
When all these gels, with surface washed and blotted dry, with comb and
pasteur pipette and rubber bulb ready, only then will 100uL of APS be
added to the stacking gel mix to initiate polymerization.
Each % gel will then remove a pasteur-pipette-ful of the reaction mix and
add it to the surface of the separating gel.
54

Insert the comb to a point about 2-3mm above the surface of the
separating gel.
Allow about 40 minutes for proper polymerization.
The 8%, 9% and 10% gels will work in aisle 3. Aisle 3 will be responsible for
adding the APS to the stacking gel solution.

Loading Samples

SDS unknown samples- as for SDS gels 30UL+ 10uL SDS loading buffer, boil 2
minutes, load when cool. Use Prestained standards at either end of the gel and
unknowns from left to right in order of crude extract, purified.
Native gels standards and unknowns 30uL of standard or unknown, add 10uL 5X
native gel loading buffer, mix and load as much as you can. Load standards and
unknowns as follows:
o
o
o
o
o
o
o
o
o
o

CA
Ov
BSA
Urease
Aisle 1, purified
Aisle 2, purified
Aisle3, purified
Aisle 4, purified
Aisle 5, purified
Prestain.

There should be a set like this for every percentage gel.

55

Run all gels at 150-200V until the bromophenol blue line reaches the bottom of
the gel.

Staining

SDS gel stain over week-end with Coomassie Blue.

Native gel will l be cut in half: the standards will be stained with Coomassie blue
as usual and the purified fractions with an activity stain for phosphatase activity,
BCIP and NBT. The activity stain will be left overnight.
Reporting Data

Each Aisle should record rf values for all bands on the gel they have run.

Bottom of well or
top of separating
gel.

a
A
b

rf, a = a/l

l
B

rf, b = b/l
Bromophenol-blue
migration distance.
SDS Gel Report

Each aisle should complete one of these. We will copy and distribute.
Rf Values
Lane
Band
1
2
3
4
5
6

56

10

Native Gel Report

Each aisle should complete one of these. We will copy and distribute.
.
Percentage Native Gel Band

1
CA

2
Ov

3
BSA

4
Ur

5
UK1

%
6
UK2

7
UK3

8
UK4

9
UK5

Band 1
Band II
Band III
Band V
Band VI

Plot data and determine Subunit and Native molecular weight for the E.coli Alkaline
Phosphatase.
Comment on data analysis.

57

Exp.5 A quasiindependent experiment to be done in groups of four.

Each aisle will repeat the experiments of Silverstein and Blomberg in

Denaturation of Chymotrypsin by Guanidinium Chloride

Will check the denaturation of a-chymotrypsin as a function of Guanidinium
hydrochloride concentration [GuHCl], by looking at both activity changes, using NPA
(nitrophenylacetate) as substrate, and the structural changes, by following the emission
spectrum of the tryptophan residues in the protein as a function of [GuHCl].
Can only get fluorimeter on Thursday and so we will spend the afternoon checking some
properties of the system.

Not so sensitive an assay. Will check a pH 7.0 assay system.

Will look at the approach to equilibrium.

I will provide:

30mM NPA in methanol.

mg/ml a-chymotrypsin in 10mM HEPES, pH 7.0

Each aisle will make:

200ml of 10mM HEPES, pH 7.0

100ml of 9.0M GuHCl in 10mM HEPES, pH 7.0

I Approach to Equilibrium
Use the following table to set up a series of solutions of chymotrypsin in GuHCl
concentrations ranging from 0M to 3.0M in 0.5M Increments. The final volume of each
solution will be 3.0ml.
58

Enz (ml)
1.5ml

9M GuHCl

HEPES

Assay
We want to measure the approach to equilibrium as a function of [GuHCl] so as
soon as a solution is made remove one ml, add enough NPA solution to give final
concentration 1mM. Measure the rate for one minute and pour the assay solution
back into the test-tube. Repeat the measurement about 10 minutes later.
Repeat the above assays for all GuHCl concentrations.
Keep repeating until one hour after the addition of the denaturant to the enzyme
solution.

Spontaneous Hydrolysis of NPA

Check spontaneous hydrolysis of NPA in HEPES-GuHCl at the same concentrations as
these will be found in the assay. Check spontaneous hydrolysis over the same time as
the assay.

Hitachi Fluorimeter
I. Powering Up Instrument

Turn left-hand switch on fluorimeter panel on.

Wait 10s and ignite Xenon lamp. Do not press switch for longer than 5s. If the
lamp does not ignite wait 1omin then repeat the ignition.
After lamp is lit, turn right-hand switch on fluorimeter on. Allow 20min for
lamp to warm up.
59

II.

Setting up Instrument Parameters for Measurements

Photometric Measurements
Select Photometry from main menu.

Set Data Mode = Intensity.

Set Num WL= 1.

In Test

Setup
Set ex and em maximum.
Set Init Delay = 0.
Set Integ Time = 0
Set replicates = 5

In Instrument Setup
Set Response = 0.5
Set Bandpass = 10
Set PM Voltage = 400 or 700
Set Text Print = off

Measurement of Spectra
Obtain Spectra
Select Goto/ex on keypad.
Select WL Scan from the Main
Menu. In the Test Set-up Mode:
Set ex and ex and stop and stop
s
Set up low scale
Set up scan speed not
1200nm/min.
In Instrument Set-up

Set Response = auto select

Set Bandpass = 10nm
Set PM voltage = 400 or 700
Set Graph /Text Print = off
Save parameters = catalog 1.

Press Forward to get to

Measurement Screen.
Press Start ex to get
excitation spectrum.
Rescale data to fit screen.

Emission spectra the same as above

except
Select Goto/em on keypad.
Select WL Scan from the Main
Menu. In the Test Set-up Mode:
Set em and em start and and
stop s
Set up low scale
Set up scan speed not
1200nm/min.
In Instrument Set-up
60

Set Response = auto select

Set Bandpass = 10nm
Set PM voltage = 400 or 700
Set Graph /Text Print = off
Save parameters = catalog 1.

Press Forward to get to

Measurement Screen.
Press Start em to get emission
spectrum.
Rescale data to fit screen

III.

Measurement

Photometric Mode
After setting up the instrument as above:
Press forward key to get to the measurement screen.
Insert sample in fluorimeter cuvette, into holder. The sample volume should
be about 1 ml.
Press Start, either the ex or em key, and the intensity value should
appear on the screen. Repeat the measurement four more times on the
sample.
Measure all your other samples.
Record fluorometric data in following form:
[GuHCl]

61

Average

IV.

Power Instrument Down

Turn right-hand side power switch off.

Turn left-hand side power switch off.
Turn right-hand side power switch on for 10 minutes to allow the lamp to
cool.
Turn left-hand side power switch off.

62

Making Buffers in a Biochemistry Lab

The first lab periods will be devoted to making buffers which to be used during
the coming semester. In order to save space, and make enough of a given buffer for the
entire semester we usually make the buffers at a higher concentration than we use
them at. In the lab terminology we describe the situation above as, we make buffers at,
say a, 10X concentration (ten-fold higher than we need) and dilute them to 1X (the
concentration of use). To make buffers we need to brush up on some basic calculations
from genchem, to relearn the use of chemical balances, and to understand and use pH
meters.
Choosing a Buffer
As you recall from genchem, a buffer is a solution of a weak acid and its conjugate base,
the combination of which is able to minimize changes in pH caused by the addition of
external acid or base. You also will, no doubt, also recall that the pH of the solution is
determined by the pKa of the acid and the ratio of the concentration of the conjugate
base to the concentration of the acid. The Henderson-Hasselbach expresses this
relation:
pH = pKa + log[(conjugate base) / (acid)]
Biological systems, including enzyme reactions, often have an optimal pH so that the
most sensitive assays are usually be carried out near that optimal pH. So, knowing the
optimum pH, we need to choose an appropriate buffer i.e., one which would buffer (or
hold the pH relatively constant) at that pH. Buffers are most effective when the pH of
the solution is equal to the pKa of the system, as, at this pH, the concentration of the
acid form equals the concentration of the conjugate base form, so we have maximal
conversion of one form to the other, or buffering. We would be lucky to find both the
above conditions fulfilled at the arbitrary pH of one out of many enzymes. Therefore,
the best choice of a buffer would be one whose pKa is close to the required pH.
Physiological pH is about 7.4 so usually reactions can be run close to pH 7 and we
consequently need a buffer with a pKa near 7. Two commonly used buffers are
tris-hydroxymethyl amino methane, Tris base, with a pKa of 8.07, and one of the three
ionization forms of phosphoric acid, H 2PO 4-, with a pKa of 7.20.

Making the Buffer - Theory

After the appropriate pKa has been chosen, a buffer of the appropriate pH can be made
by adjusting the ratio of the base form to that of the acid form, of that buffer, in one
of three ways:
63

1. A salt of the acid form can be added to a salt of the base form in the correct molar
ratio. This will give the desired pH.
2. The acid form of the buffer can be dissolved and its conjugate base can be
formed by the addition of an appropriate amount of strong base.
HA + OH - = A- + H 2O
3. The salt of the base form can be dissolved, and an appropriate amount of the acid
form can be formed in solution by the reaction with a strong acid.
A- + H + = HA

The Concentration of the Buffer

In making solutions we need to make up a given concentration of the solute in a given
volume of solvent. In Biochemistry the concentration of the buffer is defined as the
concentration of both the acid and the base forms of that buffer. Thus all we need to
know when making a buffer is its Mr, its concentration and its volume. When we adjust
the pH the forms will proportion themselves out spontaneously to give the desired pH.
We will use Tris as a general example of how a buffer is made. First, weigh out the
correct mass, in grams, of the base form. Correct mass means the mass that will give
the desired molar concentration when dissolved in the given volume of solution. We allow
the solute to dissolve in a somewhat smaller volume than the final volume calls for, and
then titrate the solution to the correct pH with hydrochloric acid. After the correct
pH is reached we add more solvent until the volume of the solution is brought to the
desired total volume, mix and check the pH. So this is an example of the third method
of making a buffer.

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Procedure:
The recipe in the appendices in the lab manuals, unless specified otherwise, indicates
concentrations corresponding to their working, or 1X, concentration. As an example of
a buffer preparation, let us make 5L of a 4X solution of Tris HCl buffer with a
working concentration of 0.1M.concentration at pH 7.6. TrisHCl indicates that HCl is
the acid needed to titrate the base form of Tris. You could also titrate with H2SO4
Or CH3COOH to make TrisSO4 or Tris Acetate, if needed.
1. Find all the components of your assigned buffer. In this case the solid Tris
base and the HCl solution. All solids are in the west balance room or by the pH
meters. The Acids are under the hood in the east lab. Look for the formula
weight of Tris, or any solid, on its container. Record the Mr in your lab book.
2.

Calculate the number of grams of each component you will need to weigh out.
(Remember, grams = 4(V x C x M r), where V = volume, C = molar concentration and
M r = molecular weight. This calculation gives the mass of Tris base to be weighed

out.

(4x5X0.1X121)g Tris base = 242g of Tris base.

3. When you get to the balance, first put the weighing container on the pan and tare
the balance. Using a clean spatula, add solid to the container until the correct
weight is obtained. Add the solid to a beaker and rinse the weighing container
with water into the beaker. Repeat this for each solid component. When you are
finished with the balance, clean out any spilled chemicals with the brush
provided, and turn off the balance. Clean the used spatulas and return them
to their containers. Sloppy lab practices are not tolerated.
4. Dissolve solids into somewhat less water than the total volume needed. For our
example the initial volume would be about 4.7L. Use a magnetic stirrer and stir
bar.
5. With the standard buffers provided, calibrate the pH meter according to the
published instructions. For maximum accuracy the calibrations standards should
bracket the desired pH. Place the pH electrode into the buffer solution.
6. Titrate the buffer with strong acid, HCl to pH 7.6, the desired pH. Rinse the
electrode and put the electrode back into the storage buffer. Turn off the pH
meter.
7. Bring the buffer to the desired final volume by adding water and re-check your
pH. Adjust again if necessary.

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8. Transfer the buffer to an appropriate storage container. Label the container

with (a) the buffer name, (b) buffer concentration, (c) date made, and (d) your
initials.
8. To use the buffer dilute it buffer to its working, or 1X concentration. For
example, if you want 200 ml of a 1X solution starting from a 10X solution then, use
the calculation from genchem and insert the X designation for concentration
instead if molarity:
V 1 x C 1 = V 2 x C 2, (200ml)(1X) = (xml)( 10X)
Xml = 200(1X/10X) = 20ml.
9. Add 20 ml 10X to 180ml H2O, mix. Decant into appropriate container
for storage.
10. Clean all glassware used to make solutions. Hang out to dry on the racks.

Reagents

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Agarose Gels
Ampicillin Solution (1000X)

Buffers

Bradford Reagent for Protein

Assay
Column Buffer
Coomassie Blue Stain for Gels
Destaining Solution (SDS)
DNA Denaturing Solution
DNA Loading Solution (10X)

DNA Neutralization Solution

Hoescht Assay Solution
IPTG Solution (100X)
LB (Luria Broth)
Liquid Medium

LB (Luria Broth)
Solid Medium

PNPG Solutions

SDS Loading Buffer (10X)

Add 0.5g agarose to 50 ml 1X TAE in a

Wheaton bottle. Microwave for 1 20. Cool hot
solution under running water, and pour. This
makes a 1% gel
A filter-sterilized solution of 50mg/ml in
distilled water.
50mM appropriate buffer weigh out enough
salt to give a 50mM concentration in desired
volume, dissolve in water, pH and then take to
volume. Check pH.
100mg Serva Blue dissolved in 50ml 95%
ethanol. Add 100ml 85% H3PO4, mix, add water
to one liter. Mix.
20mM TrisHCl, pH 7.4, containing 200mM NaCl
and 1mM EDTA.
4X 100mg Coomassie blue tablets in 1L of
40:10:50/methanol: glacial Acetic Acid: water.
40:10:50/methanol: glacial Acetic Acid: water.
0.4M NaOH containing 1M NaCl.
0.25g Bromophenol Blue, 0.25g Xylene Cyanol in
50ml 1mMTrisHCl, pH 8.0. Add 50ml glycerol
and mix.
1M phosphate pH 6.5.
Add 2L 33528 Hoescht dye to 1 ml 2X TNE in
an eppendorff.
Make 100mM in water and filter-sterilize.
10g bactotryptone, 5g yeast extract and 10g
NaCl in one liter of water. Autoclave after
dissolving.
Add Bacto-agar to 1.5% (w/v) to LB, liquid
medium. Autoclave and pour into Petrie dishes.
If antibiotics are to be added, wait until the
agar solution is about
65 C before adding.
p-nitrophenyl--D-galactose, PNPG, in Z
buffer.
Add 15 ml glycerol to 35ml 0.5M Tris HCl, pH
6.8, containing 4g SDS and 15mg Bromophenol
blue.
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SDS Gel Electrode Buffer

(10X)
Separating Gel Solution

SSC (20X)

Stacking Gel
Substrate Solution for Blots

TAE Buffer (10X)

TE Buffer
Tetracyclin Solution (1000X)
TNE Solution (2X)
Towbin Buffer

TSS Solution

Western Blocking solution

Western Salt Solution

Western Wash Solution

250mM Tris base containing 1.92mM glycine

and 1g SDS, pH 8.3.
Add 120ml 30% Acrylamide/bis-acrylamide to
280 ml of 0.54M TrisHCl, pH 8.8, containing
300L of TEMED, mix. This will give a 9% gel
88.2g sodium citrate and 175.3g of NaCl per
liter. Adjust pH to 7.0 with glacial acetic acid
and autoclave before use.
13.6 ml of 30% acrylamide/bis-acrylamide to
87ml of 0.145M Tris HCl, pH 6.5, containing
100L TEMED. This will give a 4% gel.
0.1M TrisHCl, pH 9.5, containing 50mM Mg+2
and 100mM NaCl.
TAE is an acronym for Tris-acetate-EDTA. We
buy a 50X TAE solution and dilute it with water
to make a 1X running buffer. The 50X contains
2M Tris acetate and 50mM EDTA, pH 8.3,
10mM TrisHCl, pH 8.0, containing 0.15M NaCl
and 1mM EDTA.
15mg tetracycline in 70% ethanol-water.
20mM TrisHCl, pH 7.4, containing 2mM EDTA
and 0.2M NaCl.
0.192M glycine, 0.025M Tris base, 0.0013M
SDS, pH 8.3. Make to 15% in methanol.
Add 10g of PEG 3350 or PEG 8000 to 100 ml of
sterile LB. Add filter-sterilized Mg+2 to a final
concentration of 50mM; adjust the pH to 6.66.8 and add enough DMSO to give a final
concentration of 5%(w/v) and filter-sterilize.
1g Blocker in 200ml Western Salt solution
containing 0.2ml Tween 20.

48.6g Tris base, 47gNaCl in 4L of water. Take

the pH to 9.5 with HCl.
0.2ml Tween 20 per liter Western salt solution.

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Xgal Stock Solution

Xgal Agar Plates

YT Medium

Z Buffer

25mg Xgal/ml in 50%mDMSO/water.

Dilute the stock Xgal by a factor of 1000, to
give a final concentration of 25g/ml.
Dissolve 0.8g bactotryptone, 0.5g yeast extract
and 0.5g NaCl in 100 ml of water. Adjust pH to
7.4 and autoclave.
50mM TrisHCl, pH 7.5, containing 50mM Mg+2
and 2mM DTT, dithiotreitol.

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