Practical 1

QUANTITATIVE DETERMINATION OF PROTEIN

CONCENTRATION USING THE BIURET TEST

Specific learning objectives

 To introduce general techniques for assaying biochemical compounds
using as an example, protein, one of the major chemical constituents of
living cells.

 To use these techniques to develop and test a hypothesis related to a

biological question.

 To develop skills in graphical presentation of experimental results.

Note: Before arriving to class, make sure you read the manual
and understand the experiments. Draft the experiment flow
together with the objectives in the practical notebook. The
information in the first few pages will be assessed via a quiz at
the beginning of the practical session.

SPECIAL EQUIPMENT: You will need a sharp HB pencil, a

ruler, an eraser, a permanent marker pen and two sheets of 1-2
mm graph paper for the practical class and post-lab test.

1.1
PRE-LABORATORY PREPARATION
1. Read the following concepts from Campbell Biology (10th Ed, Australian version).
 Concept 2.3
 Concept 5.4
 Concept 10.1

2. Before coming to the practical, using the textbook and these notes, make sure you
are able to define the following terms.

absorbance peptide bond protein

amino acid pH reagent blank
buffer photosynthesis rubisco
control polypeptide standard curve
covalent bond

3. Please view the video ‘How to use a spectrophotometer pre-lab video’. This video is
available in the ‘Laboratory Information’ block of Moodle under ‘Technical
Resources’.
4. Read and understand the introduction to this practical. You will be assessed on this
information in the pre-lab quiz.
5. It is essential you can answer pre-lab questions 1-3 (found on p1.4) before attending
this practical. The links and information within the Introduction will help you
answer question 3. Your TA will assess your understanding of these questions in a
pre-lab discussion prior to beginning the ‘wet’ practical.
6. Bring along your lab notebook with the layout of the objectives and experiment flow
chart of Practical 1.

LINKS
For further information on proteins and protein structure, see Kimball’s Biology Pages at:
http://www.biology-pages.info/P/Proteins.html

INTRODUCTION
Proteins are long chains constructed from amino acid building blocks linked together by
covalent peptide bonds. Peptide bonds are formed by a dehydration reaction (i.e. the formation
of a larger molecule from smaller units accompanied by the elimination of water) between two
amino acids.

amino acid amino acid

peptide bond

1.2
(For more detail see Figure 5.15 in Campbell Biology 10th Ed’)
A solution of alkaline copper sulphate (called biuret reagent) reacts with compounds containing
peptide bonds to give a violet-coloured complex. The intensity of the colour is a measure of
the number of peptide bonds, and therefore an indication of the amount of protein in the
sample.

The procedure for determining the concentration of protein in an unknown solution is to first
construct a standard curve (Fig. 1) by reacting biuret reagent with standard protein solutions of
known concentration, and then plotting the intensity of colour of the solution against protein
concentration. Intensity of colour is most easily determined by measuring the absorbance of
the solution using a spectrophotometer. The absorbance is the amount of light absorbed by the
coloured solution and is typically proportional to the concentration of pigment in that solution.

0.06

0.04

0.02

0
0 5 10 15 20 25
Protein concentration (mg ml-1)

Figure 1. An example of a standard curve of absorbance plotted against

protein concentration for solutions of known protein concentration.
Once a standard curve has been constructed, it can be used to calculate the protein
concentration of an unknown solution by finding the protein concentration that corresponds to
the absorbance reading obtained for that unknown solution (Fig. 2).

0.06

absorbance of unknown solution

0.04

0.02
protein concentration of
unknown solution

0
0 5 10 15 20 25
-1
Protein concentration (mg ml )

Figure 2. Determination of the protein concentration of a solution using

the standard curve in Figure 1.

1.3
EXPERIMENTAL QUESTION
Photosynthesis is the process by which atmospheric CO2 is fixed by plants. In the Calvin
cycle, each molecule of CO2 is added to a molecule of ribulose bisphosphate (RuBP), a five-
carbon sugar with a phosphate group on each end. The enzyme that catalyzes this step is RuBP
carboxylase (rubisco for short). This enzyme is by far the most abundant protein in
chloroplasts, often forming about 80% of total leaf protein. When a plant is photosynthesising
rapidly, rubisco levels are usually high.

Today you will use a technique commonly used by biologists to measure the protein
concentration in plants and animals. You will first prepare a series of standards and construct a
standard curve. You will then use this standard curve to determine the concentration of protein
in two silverbeet leaf extract solutions grown under different growth conditions. As rubisco is
the most abundant protein in plants, we will assume (for this experiment) that any difference in
rubisco concentration between the leaf extracts will also be evident in the total leaf protein, i.e.
that leaves with decreased concentrations of rubisco will have decreased levels of total protein.

Consider your answers to pre-lab questions 1-3. Your answer to Q 3. will serve as your
hypothesis for the remainder of this practical.

SAFETY NOTES

1. Lab coats must be worn for the duration of this practical class.
2. Safety glasses must be worn for the duration of the class.
3. Biuret is caustic. Please take care when using it.

PRE-LAB QUESTIONS
Q 1. In this practical exercise you will be using biuret reagent. Why is biuret
reagent used in this experiment?
Q 2. What is the experimental reason for constructing a standard curve?
Q 3. Based on the above information and your own thinking, which plant do you
hypothesize will have more rubisco, one raised in the sun or one raised in shade?

1.4
EXPERIMENTAL PROCEDURE

Work in groups

Part 1. Constructing the standard curve

You have been given a standard protein solution in a phosphate buffer (tube labelled "BSA" for
bovine serum albumen) with a protein concentration of 10 mg ml -1. The buffer acts to stabilise
the pH of the solution in order to prevent deterioration of the protein.

Table 1. Experimental protocol for construction of the protein standard curve.

Tube
1 2 3 4 5 6

Buffer (ml) 1.0 0.8 0.6 0.4 0.2 0.0

BSA Protein solution 0.0 0.2 0.4 0.6 0.8 1.0

(10 mg ml-1) (ml)

Biuret reagent (ml) 4.0 4.0 4.0 4.0 4.0 4.0

Total volume (ml) 5.0 5.0 5.0 5.0 5.0 5.0

Final protein concentration

(mg ml-1)

Absorbance

1. Take 8 colorimeter tubes and label them 1 – 6 (as per above table), and ‘Sun’ and ‘Sh’. The
labels must be written within the top 1 cm of the tube to avoid interfering with the
absorbance measurement.
2. Carefully insert the pipette labelled "buffer" into the "pi pump" supplied. See Appendix 1 on
the whiteboard for a demonstration on how to do this. Hold the pipette close to the label at
the top of the pipette. Insert the pipette firmly into the pi pump as far as it will go without
forcing it.

Warning: Do not hold the pipette at a distance from the end you are inserting into the pi
pump because it might flex and break!

3. Practice using the pi pump with a little buffer by drawing it up and releasing it back into the
buffer tube a few times.

4. Pipette into tubes 1 - 6 the volumes of buffer indicated in Table 1. Note: the pipettes on
your bench are 1 ml pipettes. The “0” level on the pipette equals 1ml in volume - if
you are not sure, ask a TA.

1.5
 5. Change the pipette in the pi pump to the one labelled "BSA". Pipette into tubes 1 - 6 the
volumes of protein solution indicated in Table 1.

6. When all the tubes are prepared, add 4 ml of biuret reagent to each tube from the auto-
dispenser in the fume cupboard. The auto-dispenser is set at 4 ml. To dispense the biuret,
simply pull the piston up as far as it will go and then release it.
Note: biuret is caustic so wash your hands with water if spillage occurs.

7. The final volume of each tube should be identical (5.0 ml). Cap the six tubes using the
parafilms provided and mix the contents by inverting them several times. Record the time.
Leave at room temperature for 25 minutes to fully develop the colour. The colour is stable
for up to two hours. Absorbance should be measured within this time.

** To save time, commence Part 2 preparations of Sun and Shade solutions before
proceeding to the calculations below. **

Calculation of the protein concentration in tubes 2 - 6 (mg protein/tube)

While you are waiting for the colour to develop in your samples calculate the protein
concentration in tubes 1 - 6 and enter the results in Table 1.

The general equation for calculating concentrations in samples with different volumes is:

Concentration (pre-dilution) x Volume (pre-dilution) = Concentration (post-dilution) x Volume (post-dilution)

or C1V1 = C2V2

For example, the protein concentration in tube 2 is calculated as follows:

Tube 2 contains 0.2 ml of a solution with a protein concentration of 10 mg ml-1,
i.e. C1V1 = 10 mg ml-1 x 0.2 ml. The solution is then diluted to a final volume of 1.0 ml (V 2)
with an unknown concentration (C2). Using the formula above

10 mg ml-1 x 0.2 ml = C2 x 1.0 ml

10 mg ml-1 x 0.2 ml
Concentration of tube 2 (C2) = ---------------------------------------

1.0 ml

= 2 mg ml-1

1.6
Measuring the Absorbance (refer to pre-lab video on this procedure in Moodle!)
The amount of colour in a solution can be measured by measuring its absorbance, using a
spectrophotometer. When light passes through a coloured solution, the amount of light
absorbed is proportional to the number of molecules in the light path and to the length of the
path the light has to travel, because the light becomes more scattered the further it travels. In a
spectrophotometer, light passes through a solution in a tube of standard diameter. The
transmitted light falls on a photoelectric cell and the current produced is a direct measure of the
light transmitted through the solution. The absorbance of the solution (i.e. the light absorbed
by the solution) is read directly from the instrument. Since the amount of protein in the
experimental solutions is proportional to the amount of colour after adding biuret reagent, we
can use the absorbance measurement to determine protein concentration. Note that absorbance
has no units. It is the amount of light absorbed across selected wavelengths, calculated as the
logarithm of the reciprocal of transmittance (the fraction of light passing through the solution,
not absorbed by it).

Tube 1 is called the "reagent blank" as it is used to correct for any colour produced by the
reagents themselves in the absence of any protein. By using this tube to zero the
spectrophotometer, the amount of colour produced by the reagents alone is automatically
subtracted from all the reaction tubes. We have set the wavelength of light to 520 nm in the
spectrophotometer, which is optimal for absorption by the violet-coloured biuret-protein
complex. There is little absorbance by chlorophyll at this wavelength, so the green colour of
your plant solutions will not be detected (and will therefore not interfere with the measurement
of protein).

Read the instructions next to the spectrophotometer. After setting the spectrophotometer to
zero with tube 1, read and record in Table 1 the absorbance of tubes 2 - 6. (Note: Use the same
spectrophotometer in subsequent experiments).

F 1. Using 1 or 2 mm graph paper, plot absorbance on the y-axis against protein concentration
on the x-axis to obtain the standard curve, as in the example in Figure 1. Make sure that your
figure has a fully descriptive caption. (See the whiteboard for guidelines).

1.7
Part 2. Determining the protein concentrations of silverbeet tissues

Preparation of your silverbeet samples

Now that you have some experience in constructing and using a standard curve to measure the
protein concentration of a manufactured sample, you can apply your skills to a real biological
question. Do silverbeet leaves that are grown in the sun contain more rubisco than those
growing in shade? You are provided with two suspensions of silverbeet leaf, one from plants
grown in full sun and the other from plants grown in shade.

1. Using the appropriately labelled pipette, dispense 1 ml of the ‘Sun’ suspension into your
“Sun” labelled tube, and 1 ml of the ‘Shade’ suspension into your “Sh” labelled tube.
2. Add 4 ml of biuret solution to each tube. Stopper the tubes and mix them by inverting them
several times. Wait 25 minutes for the colour to develop.

Table 2. Experimental protocol for determining the protein concentrations of silverbeet grown
in the sun and shade.

Sun Shade
Columns for modified
Procedure procedure(s)

Protein solution (ml) 1.0 1.0

Biuret reagent (ml) 4.0 4.0

Absorbance

Protein concentration derived

from standard curve (mg ml-1)

Protein concentration of original,

unmodified solution (mg ml-1)

Determining the protein concentration in each silverbeet sample

Measure the absorbance of the tubes labelled "Sun" and "Sh", using Tube 1 to zero the
spectrophotometer. DO NOT invert your tubes before reading (because the resuspended
chloroplasts may interfere with your result). Mark the absorbance readings on the y-axis of
your graph. Determine the protein concentrations of each of the silverbeet samples using the
standard curve, indicating with a dashed line how you obtained your protein concentrations (as
in Fig. 2). You may need to carry out a modified procedure before being able to estimate the
original protein concentration.

Record your methods and results in Table 2.

Repeat the procedure to accurately determine the protein concentration, but it’s tricky, so
check with a TA first that your modified procedure is correct. Show the details of your
methods and results including the final determination of the protein concentration of the
original solution by completing Table 2.

1.8
QUESTIONS FOR REVISION
These questions should be considered in preparation for your practical test.

1. In your Figure 1, should the line connect all the data points or should it be a line of best fit?
Why?
2. Is it possible to determine the protein concentration of a solution from the standard curve if
the measured absorbance is off the scale of the y-axis, i.e. higher than the highest y-value on
the standard curve? Why/why not? Explain your reasons.

3. If you have a solution where the measured absorbance is off the scale of the y-axis (as in
question 2, above) describe two ways in which you would modify the experimental
procedure to determine the protein concentration of this solution. Ensure to use the correct
biological/technical terms in your answers.
4. Do your results support your hypothesis regarding these tissue samples?
5. What is experimental error? List three examples of possible sources of error that may have
occurred in your work in this practical class.
6. What is a negative result? What are 3 possible biological reasons for a negative result in this
experiment?
7. What is the single major design problem in this experiment in drawing conclusions about
different concentrations of rubisco in response to different growth light conditions?

FURTHER POINTS TO CONSIDER

Biologists often need to know the concentrations of various compounds, as you have
investigated in this practical session, in order to understand broader biological questions.
Some examples relevant to this week's practical are listed below.

(1) Do plants living under shaded conditions, e.g. on the floor of a rainforest, have
mechanisms for maximising the amount of intercepted light? Shade-adapted plants often
have thinner leaves, creating a greater surface area for light absorption per unit leaf dry
weight. Most plants can also respond by increasing the concentration of chlorophyll per
unit leaf area.

(2) Do plants raised under different growth conditions provide different levels of food
resource for herbivores? Leaves grown in higher light regimes may be more nutritious for
herbivores than shade leaves due to potentially higher concentrations of protein and
carbohydrate.

1.9
Important
Before leaving the lab please empty all tubes into the labelled chemical waste container on the
sink and then place them in the tubs provided. Place pipettes tip-side-up in the “Pipette
discard” container on the sink and wipe your bench clean.

ASSESSMENT SUMMARY

This practical will be assessed via a pre-lab quiz (at the start of your practical session), your
participation in class including the production of Figure 1 and a post-lab in-class test, held in
the last portion of the class. You will have 20 minutes to complete the test which will be held
under exam conditions. You may use your completed Figure 1 as a resource but no other notes
are allowed. Computers, phones, calculators, textbooks and non-approved notes are forbidden
in this test. Tests will be marked as hard copies and will be returned to you in your practical 2
class.

This practical is worth 6% of your overall grade for BIO1011.

Activity Marks
Pre-lab quiz /4
Good laboratory practice (including creating an appropriate graph in
/2
class)
Post-lab test /14
Total /20

1.10