10/5/06
Lab 3. Protein Determination
Lab 3.
PROTEIN DETERMINATION
I.
INTRODUCTION
Reading in Biology, 6th ed. By Campbell:
“PROTEINS-MANY STRUCTURES, MANY FUNCTIONS” p. 71-80
“Blood is a connective tissue with cells suspended in plasma” p. 882-884.
Protein is a major and indispensable class of cellular macromolecules. Accordingly,
measuring protein concentrations in an amazingly diverse array of experimental
contexts (How many grams of protein are there in a 27 gram serving of Fruit Loops?)
continually engages the attention, and taxes the resources, of many investigators.
The two essential and frequently cited basic references on methods for general
protein determination are: Layne, (1957) and Peterson, (1983). These articles are on
hard copy reserve for the class at the Science Library in the folder for this exercise.
Two things impressed me as I read these papers.
First, the methods described are all optical methods. That is, measurement of
protein concentration relies on optical properties (absorption or turbidity) of protein
solutions and these optical properties are measured with a device called a
spectrophotometer (or colorimeter). Other types of methods (gravimetric, volumetric,
etc.) for protein determination exist, such as the classic chemical method of Kjeldahl,
but other such methods were not even discussed in these papers. Optical methods
of biochemical analysis are convenient, sensitive and versatile. We will use optical
methods for several additional exercises later in this course.
Second, these papers described no fewer than 11 different methods for protein
determination, even though they limited themselves to a presentation of optical
methods rather than chemical methods.
The existence of so many different methods must mean, I'm very, very sorry to say,
that no single method is appropriate for all experimental needs in all laboratory
contexts. Thus, before tackling the job of conducting protein assays an investigator
is faced with the rather more challenging prospect of choosing the most appropriate
method for a specific application.
In this exercise you perform 2 different protein assays (the Biuret Assay and the UV
absorbance assay) which, if nothing else, should demonstrate the versatility of
optical assays. There will also be food for thought regarding the issues that you must
consider when selecting an assay method for a specific application.
3.1
Lab 3. Protein Determination
10/5/06
You will use these assays to determine the protein concentration in an artificial
“unknown” and in serum.
3.2
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Lab 3. Protein Determination
I.A. General Features of Biochemical Assays
I.A.1.
Dynamic Response Range and Sensitivity
All assay methods have a characteristic dynamic response range. This is the
range of sample concentrations over which the assay produces a differential
response. Consider the situation shown in the graph below:
.
A
B
assay response
100%
0%
amount of protein in sample
dynresp
Assay method B exhibits a differential response over a wider range of values.
However, method A responds to lower protein concentrations; concentrations
that could not be measured by method B. We say that method A is more
sensitive than method B. Therefore, method A may be the method of choice
when samples contain very little protein. It is relatively easy to bring samples
with too much protein into the response range simply by diluting them.
Conversely, concentrating the protein in very dilute samples would usually not
be practical. Therefore, in choosing an assay you want to use a method
sensitive enough to detect the protein in your most dilute sample.
I.A.2.
Specificity
A specific assay gives a response to the substance being assayed and to no
other substance. Any other substance producing the same response as the
substance being assayed is called an "interfering substance". Very few assays
are 100% specific; virtually all respond to some interfering substance. The
adept investigator will know what substances interfere with their assay and
what interfering substances are possibly present in their samples. This
information is sometimes available in the literature but it must often be
determined by the investigator. Think about possible interfering substances in
the 3 assays presented here.
3.3
Lab 3. Protein Determination
I.A.3.
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Sample Recovery
In some situations the amount of sample available may be very limited. In such
cases one would hope to recover the protein from the assay so that it could be
manipulated subsequently. In this case one would want a "non-destructive"
assay. Which of the 3 assay methods presented here is non-destructive?
I.A.4.
Logistics and Economics
Under the heading of logistics we include a variety of issues related to the cost
of materials and labor. You must consider the cost of equipment, chemical
reagents, and staff time to prepare materials and perform assays. Increasingly,
the cost of disposal of hazardous materials is becoming an important issue.
I can assure you that these issues were carefully considered and are a
significant constraint to the design of this laboratory exercise.
I.A.5.
Safety
Prudent investigators will choose to avoid, whenever possible, methods that
present risks to laboratory personnel or to the environment.
See Section II. for a description of the hazards involved in this exercise.
3.4
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Lab 3. Protein Determination
I.B. Biuret Assay
The Biuret reaction involves a reagent containing copper (cupric) ions in alkaline
solution. Molecules containing 2 or more peptide bonds associate with the cupric
ions to form a coordination complex that imparts a purple color to the solution with
Amax = 540 nm. The purple color of the complex can be measured independently of
the blue color of the reagent itself with a spectrophotometer or colorimeter.
The Tetravalent Coordination Complex Formed by Proteins in the Presence of
Biuret Reagent
Protein assays based on the Biuret reaction were developed and used by numerous
investigators throughout the first half of this Century. Typically each investigator
adapted the basic reaction to suit their own needs, introducing their own variations of
concentrations and assay conditions. Thus there are many versions of Biuret assay
in the earlier literature.
The Biuret Assay conditions normally specified by contemporary sources are based
directly on the work of Gornall et al. (1948), usually without attribution. These
workers conducted an extensive investigation of the effect of reagent concentrations,
pH and incubation conditions to arrive at the recipes and procedures we now employ
without much question. (Likely few people performing the Biuret Assay according to
the procedure of Gornall et al. stop to ask themselves why the reagent contains
potassium tartrate.) We do well to remember that these investigators were studying
hepatic lesions, and therefore specifically optimized the assay conditions for
determining proteins in serum.
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Lab 3. Protein Determination
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Using the Biuret Assay to determine protein concentration in an unknown sample
requires, as a preliminary, construction of a standard curve made by assaying a
series of protein solutions with known concentration. The protein bovine serum
albumin (BSA) is frequently used for constructing standard curves because of its
availability and low price.
The typical Biuret procedure (as implemented in our procedure) is:
•
Combine 0.9 ml Biuret Reagent with 0.3 ml of sample in a microcentrifuge tube
and mix thoroughly by vortexing.
•
Wait 5 minutes.
•
Determine the Absorbance at 540 nm vs. a blank containing Biuret Reagent
and di water, but no protein.
I.C. UV absorbance
Virtually all proteins exhibit a strong UV absorbance maximum near 280 nm. This
characteristic absorbance is due almost entirely to the absorbance by the aromatic
rings in the R-groups of the amino acids tryptophan and tyrosine. (As you will recall,
these are 2 of the 20 amino acids ordinarily found in proteins.)
Samples are assayed directly in a spectrophotometer capable of generating and
measuring UV light at this wavelength. No chemical reagent is involved here, as in
the Biuret assay, because the UV absorbance is an intrinsic property of the protein
itself.
3.6
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II.
Lab 3. Protein Determination
PROCEDURES
We provide a standard stock solution of protein at [20 mg/ml]. The protein is bovine
serum albumin (BSA). The first task will be to dilute the stock protein solution to give
a series of solutions with different, known concentrations (Procedure II.A). This must
be done first.
The protein standard solutions will be used to construct standard curves for the 2
assay procedures (II.B, II.C). These can be completed in any order, though the
availability of UV spectrophotometers is limited. Therefore, some groups should do
the UV absorbance assay first.
In addition to the protein standard solutions prepared in II.A, there will also be a
simple "unknown" solution. The unknown is a pure solution BSA in water. Dilutions
of the unknown should be prepared at dilution factors of 10-1 and of 10-2.
This is summarized as follows:
Finally, you will determine the protein concentration in a “real” sample of serum.
Each pair of students will work with a reagent "Kit" containing the following:
•Biuret Reagent
•Bovine serum Albumin Protein Standard [20 mg/ml]
•Protein Unknown
•Serum
Be sure you do not confuse the standard with the unknown protein solution.
3.7
Lab 3. Protein Determination
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II.A. Protein Standards
To construct reasonable standard curves for each of the 3 assay methods over a
wide range (4 orders of magnitude!), you will need 5.0 ml of each standard solution.
There are innumerable ways you might accomplish this. A suggested method is
diagrammed below.
3.8
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Lab 3. Protein Determination
This scheme produces 15 samples (including the original stock) whose
concentrations are known. They will be used to establish the relationship between
protein concentration and absorbance (i.e. to construct a “Standard Curve”).
1. Before coming to class, determine the protein concentration for each of the 15
tubes in the dilution diagram on the previous page. Record the concentration
value in the diagram so that you can refer to it during lab. If you are not sure that
your calculations are correct, you can check them on the web site.
2. Set up fourteen (14) 16 X 120 mm tubes in a rack.
3. Add the indicated volumes of di H2O to each tube.
4. Proceed with the dilution series by transferring the indicated volumes of protein
solution. Be sure you MIX each tube thoroughly before making the next dilution.
You now have 15 BSA standard solutions (the original stock we provided plus the
14 dilutions you just made) whose concentrations are known.
5. You can also make the 2 dilutions (10-1 and 10-2) of the unknown at this time.
HAZARDS
Biuret reagent is a strong base (pH >12) and toxic due to the copper.
Whenever you work with Biuret Reagent solutions you must:
WEAR GLOVES
WEAR EYE PROTECTION
CLEAN UP ANY SPILLS
FOLLOW DIRECTIONS FOR PROPER DISPOSAL
The protein standard solutions containing ONLY BSA are harmless.
3.9
Lab 3. Protein Determination
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II.B. Biuret Assay
Put on GLOVES and SAFETY GOGGLES
1. Add 0.9 ml Biuret Reagent to the appropriate number of microcentrifuge tubes.
Note that there are 15 protein standards, including the 20 mg/ml stock (see
diagram on p. 4.8). Use one tube per unknown. For example, if there are 3
unknown solutions and one blank (i.e. Biuret Reagent and water, no protein), you
will need 15 + 3 + 1 = 19 microcentrifuge tubes.
This Biuret Reagent can be done with the same pipette tip. Minimizing the # of
tips you use saves money and the time it takes you to change tips.
2. Reset the volume of the P-1000 to 300 µl and transfer each of the protein
solutions (or water, for the blank) to the tubes containing reagent.
In this case the issue of whether or not you would need to change pipette tips for
each transfer is more complicated than it was in step 1. If you begin with the
most dilute sample, and work toward the most concentrated sample, then there
should be no significant contamination of one assay tube by the other. However,
there would be contamination of your standard dilutions with Biuret Reagent.
Because you may need the remaining standard protein solution in the dilution
tubes to repeat the experiment, we advise that you change tips for each transfer
in this step of the procedure.
3. Cap and mix all tubes. Wait 5 minutes.
4. Look at the tubes carefully to see if the results are reasonable.
5. Determine and record the absorbance of each of the 15 standards and the
unknown with your colorimeter. Are the values consistent with your visual
inspection?
Remember to use the Biuret Reagent + di water as a reference, not pure water.
Be sure that the wavelength is set to 540 nm and that the proper filter is selected.
It is probably best to simply decant (= pour) the solutions from the assay tubes to
the cuvette. After each reading, return each sample to the microfuge tube it came
from and save all the tubes until you are certain the experiment is complete and
that the results are correct.
HINT: If you begin with the most dilute sample, and work toward the most
concentrated sample, you will not need to rinse the cuvette between samples.
3.10
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Lab 3. Protein Determination
6. Liquid waste disposal: When you are satisfied that your results are correct,
discard all the assay solutions (not the standards) by pouring the contents of
each microcentrifuge tube into a waste beaker designated for Biuret Reagent.
Rinse each tube with a small amount of di water from a squeeze bottle and add
this rinse to the waste beaker. After rinsing, the empty microfuge tubes are
placed in the receptacle provided.
3.11
Lab 3. Protein Determination
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II.C. UV Absorbance
In this assay we use the intrinsic ability of proteins to absorb ultraviolert (UV) light.
The UV absorbtion of native proteins is due almost entirely to the presence of the
aromatic amino acids tryptophan and tyrosine which have absorbtion maxima near
280 nm. The other 18 amino acids normally found in protein absorb weakly, if at all,
in the UV range.
For this assay you must use one of the Pharmacia/LKB Ultrospec
spectrophotometers on the side bench. The colorimeters at your bench work only in
the visible part of the spectrum; they have no source of UV light. Also, you must use
the special UV-transparent cuvettes because the ordinary plastic cuvettes you use
with your colorimeter are opaque to UV light. The UV-tranparent cuvettes are
marked with small dots of red ink at the top. NOTE: You could use the UVtransparent cuvettes with your colorimeter at visible wavelengths, but you cannot
use the ordinary cuvettes with the LKB instruments in the UV.
There are rather lengthy printed instructions taped to the top of the LKB instruments,
but your instructor may be able to give you a brief demonstration. The wavelength is
set to 280 nm and the instrument is blanked using the "SET REF" button on a
cuvette containing di H2O. (You need to blank the instrument only once.)
You will probably want to use the P 1000 to transfer 1 ml from each standard tube to
the cuvette. Again, work upwards, from lower protein concentration toward higher
protein concentration so that you will not need to rinse the cuvette between samples.
As you read each sample, return it to the tube in case you need to re-read a sample
later.
After you have read all the known protein standards, read the unknown and the
dilutions of the unknown.
3.12
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III.
Lab 3. Protein Determination
DATA ANALYSIS AND DISCUSSION
Assemble your data for the known standard solutions in a spreadsheet. You will
have separate columns for proteins concentration (mg/ml), Biuret absorbance
values, and UV absorbance values. Simply ignore any absorbance values < zero.
A. General Comparison of the Assay Methods
The most straightforward way to compare the assay methods to each other (in terms
of their sensitivity and response range) is to draw 2 standard curves on a the same
log/log plot (log Absorbance values vs. log protein concentration). EXCEL makes
this a snap because it eliminates to need to actually calculate logarithms. We urge
you to take advantage of the opportunity to learn how to use EXCEL on the
classroom computers.
Using log scales rather than linear scales on the graph spreads out the data points
that would otherwise be scrunched together at the lower end of the scales. Try it and
see! Note that there is no log value for a negative numbers or for zero. You can
simply eliminate them from the graph. (But you must note in your report that you
have done this.) If you have many negative absorbance values it probably indicates
that the sample concentration is below the detection limit of the method or that there
was a problem with your blank. Also, dirty cuvettes, improper cuvette filling, and
inserting cuvettes in the wrong orientation can cause this problem.
The log/log plot should be labeled Fig. 1. Have your instructor check the plot. They
will tell you whether you should use your own log/log plot for the discussion, or
whether you should refer to the log/log plot provided on the web site.
B. Determining Concentration of the Unknown
Although the log/log plot can be enlightening with respect to the relative merits of the
assay methods (you can immediately see the sensitivity and dynamic response
ranges) the log scales tend to limit the precision with which you can determine the
unknown concentration. To determine the unknown concentration more precisely
you need a linear/linear plot of Absorbance vs.standard concentration that includes
on the dynamic response range of the assay where Beer’s Law applies.
Plot linear/linear standard curves for the Biuret and UV assays sepatately as Fig. 2
and Fig. 3. Do not simply "connect the dots" in your graphs, but subjectively draw a
best-fit straight line to the points.
When drawing the line you may ignore any points that are obviously erroneous, but
you must explicitly state that you have done so.
When you have the standard curves properly plotted, perform the determinations of
the unknown samples.
3.13
Lab 3. Protein Determination
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Remember that your determined values are valid on when the absorbance value of
an unknown (or its dilution) lies within the linear response range of the assay, i.e.
that portion of the standard curve that follows Beer's law. You might need to use the
absorbance value of the undiluted unknown, or the absorbance of one of the
dilutions, depending on the assay and on the protein concentration. Expect to
operate to some extent on a trial and error basis.
Summarize the final results in a table in your notebook of the form:
3.14
Dilution used
BIURET ASSAY
Absorbance
Concentration
(mg/ml)
Corrected
Concentration
Dilution used
UV ASSAY
Absorbance
Concentration
(mg/ml)
Corrected
Concentration
10/5/06
IV.
Lab 3. Protein Determination
QUESTIONS AND DISCUSSION
Which assay method shows the broadest response range and which assay method
is most sensitive? Use the log/log plot (Fig. 1) to evaluate this.
How might the results be affected if a different protein (other than BSA) were used to
create the standard curve?
Evaluate and discuss the pros and cons of each assay method. Do not confine
yourselves to the features described in section I.A.
Did the two assays give similar results for the protein concentration of the
unknowns? If not, which assay do you think is most reliable, and why? The instructor
can provide the actual value of the unknown.
References on Reserve
Gornall, A.G., Bardawill, C. J. and David, M. M. (1949)
Determination of Serum Proteins by means of the Biuret Reaction.
J. Biol. Chem. 177; 751.
Layne, Ennis
Spectrophotometric and Turbidimetric Methods for Measuring Proteins.
Methods in Enzymology, vol. 3, p. 447 (1957)
Peterson, Gary L.
Determination of Total Protein.
Methods in Enzymology, vol. 91, p. 95 (1983)
3.15