|
|
PROTEIN
DETERMINATION |
|
|
|
|
Introduction Biochemical research involves the
detection and analysis of various classes of biochemicals. From the readings in your text you may
recognize the four major classes, proteins, lipids, nucleic acids and
carbohydrates. In this exercise we
will examine a method that is used to detect and measure the amount of
protein in a sample. This is important
not only from the standpoint of being able to determine the nutritional value
of food but also from the standpoint of research where it is often necessary
to quantify the amount of protein in a sample (for example during the
purification of an enzyme). In particular in this exercise we will be
measuring protein content of an egg.
We will assume that all of the protein is found in the white of the
egg. To determine the protein content
we will separate the egg white from the yolk of the egg, dilute the egg
white, and then determine the protein content using the biuret
method relative to a protein standard curve. The biuret
method is based upon the fact that alkaline copper ions will form a complex
with a reagent called "biuret". When the complex forms, there is a color
change in the
solution. This change can be detected in an instrument that
measures the amount of light passing through a solution at a given wavelength
of light (called a spectrophotometer).
This procedure will also detect proteins because "biuret" is a chemical very similar in structure to
the basic structure of proteins. |
|
|
|
|
Materials Protein standard (20 mg/mL) 0.1 M NaOH Spectrophotometer 1000
mL Graduated Cylinder Biuret reagent (about 100 mL
per assay)* Balance (to weigh egg) Egg 1000
mL beaker Pipettes and pipette bulbs Test
tubes *Dissolve 2.25 g NaK Tartrate
(tartaric acid can be substituted) in 100 mL of 0.2
M NaOH. Add
0.75 g CuSO4-5H2O and dissolve. Add 1.25 g KI and dissolve. Dilute to 250 ml. Works best with distilled water. |
|
|
|
|
Procedure Initial preparation of sample: 1. Collect the necessary materials. Weigh the egg and record the information. 2. Crack the egg and separate the white
into a previously weighed (tared) 1000 mL beaker. Weigh
the beaker and the egg white and record the weight. Add approximately 250 mL
of the 0.1 M NaOH. 3. Transfer the diluted egg white to the
1000 mL graduated cylinder and dilute the egg white
to exactly 1000 mL using 0.1 M NaOH
(be careful because NaOH is caustic - like Draino). Stir
until the protein dissolves into a clear solution. Preparation of standard curve: 1. Obtain and clean six test tubes. Label one test tube the blank and the
remainder one through five. 2. Add one ml of water to the
blank. Then to each of the tubes
numbered one through five add 0.2, 0.4, 0.6, 0.8 and 1.0 mL
of the protein standard solution (10 mg/mL protein)
to each tube respectively. 3. Make the volume in tubes one through
five up to 1.0 mL by adding 0.8, 0.6, 0.4 and 0.2 mL of water to tubes one though four. 4. Add 5.0 mL
of the biuret reagent to each test tube and mix. 5. Allow tube to stand for 10 minutes at
room temperature and then read result in spectrophotometer 6. Graph results of concentration
(horizontal) vs. absorbance (vertical). Treatment of sample: 1. Obtain and clean four test tubes and
label A1, A2, A3, and B1, B2, B3.
Place 0.5 mL of sample in the "A"
tubes and 1.0 mL of sample in the "B"
tubes. Make up the volume in the
"A" tubes to 1.0 mL with water. 2. Add 5.0 mL
of the biuret reagent supplied by your teacher to
each test tube and mix carefully by tapping the bottom of the test tube. 3. Allow tube to stand for 10 minutes at
room temperature and then read result in spectrophotometer. 4. Calculate the amount of protein based
upon the absorbance of the unknown compared to the absorbance of the standard
curve. Then multiply times the
dilution factor to obtain the value of the protein in the egg white (this is
intentionally vague so that you will attempt to figure it out on your own
first). |
|
|
|
|
Questions 1. In drawing the graph of the standard
curve are the data points to be connected or is a line to be drawn through
them? Justify your answer! 2. What is the purpose of the
"blank".
What is the concentration of protein in each of the tubes of the
standard curve? 3. What is "biuret"?
How would you go about finding the structure of biuret?
Compare the structure of biuret to that of an amino
acid. 4. What is the control in this
experiment? 5. What is the dilution factor and how
do determine it? 6. Is there a hypothesis in this
experiment? If so, what is it? 7. From a nutrition or health book (or
some other identified source) find out how much protein is reported to be in
an egg. Is this similar to your
result? 8. The standard curve for this
experiment was prepared using casein, a proteolytic
digest of milk proteins. Do you think
this is appropriate to use as the standard?
Why or why not? 9. Precision is a measure of how
reproducible a scientific measurement is.
Accuracy is a measure of how close to the actual or truth a
measurement is. Discuss your results
in terms of precision and accuracy. 10. If you were using this method to
measure proteins in blood would it be better to use casein, albumin or
collagen for the standard? Why? |
|
|
|
|
|
|