ABSORPTION SPECTROSCOPY
I.
Colorimetric biuret assay of total protein concentration.
1. Preparation of a calibrator stock solution and the calibration curve.
Pipette protein calibrators and physiological salt to the test tubes as follows:
Test tube
number
1
Protein
concentration
[mg/ml]
0.0
(0.9% NaCl)
2
4
6
8
10
2
3
4
5
6
Volume of
calibrator solution
[ml]
Volume of copper
reagent
1ml
4 ml
1 ml
1 ml
1 ml
1 ml
1 ml
4 ml
4 ml
4 ml
4 ml
4 ml
Absorbance
A540 nm
0.0
‘blank probe’
2. Preparation of the sample for analysis
Take two test tubes and prepare two protein dilutions (20 times and 10 times) for the assay by
pipetting respective volumes of the sample given by the Assistant and NaCl solution,
according to the table:
Test
tube
number
Volume
of the
sample
[µl]
Volume
of 0.9%
NaCl
[µl]
Dilution
Copper
reagent
7
8
50
100
950
900
20x
10x
4 ml
4 ml
A540 nm
Protein
concentration
from the
curve
[mg/ml]
Protein
concentration
in the serum
sample
[mg/ml]
3. Development of the colour reaction
Add 4ml of the biuret (copper) reagent to each test tube (blank, calibrators and samples for
analysis). Mix carefully and let the probes stand for appr. 30 min. at room temperature.
4. Absorbance measurement – at wavelength λ(lambda) = 540 nm on the VISspectrophotometer.
5. Calibration curve.
Plot the absorbance versus concentration curve on the graph paper using values from
table 1, obtained by measuring the absorbance (A540) of calibrator solutions. Plot the
calibration curve and read protein concentration in the examined samples.
6. Calculate the protein concentration in the examined serum sample, considering its
dilution (20x, 10x).
ABSORPTION SPECTRCOSCOPY 1
7. Draw a scheme of a copper-nitrogen of peptide bonds complex and determine the type
of chemical bonds.
II.
Spectrophotometric protein determination - solution of purified protein
1. Measure the absorbance of bovine serum albumin solution at =280 nm with an UV
spectrophotometer and write down the value.
2. Calculate the protein concentration, knowing that kBSA= 0.66 L/g*cm
III.
Answer the questions and solve given problems.
1. Define the absorbance according to the Lambert-Beer law, write the appropriate
equation and describe its components.
2.
Can proteins absorb the electromagnetic radiation at visible range? If yes, give an example
3. Which chemical groups are responsible for the UV light absorption in proteins? What
is the maximum wavelength (λmax) of this absorption?
4. Which chemical groups absorb UV light in nucleic acids? What is λmax of this
absorption?
5. The A280 of a 1% solution of bovine serum albumin (BSA) equals 0.66. What is the
absorbance of 2.5 mg/ml BSA solution (the cuvette thickness is 1cm)?
ABSORPTION SPECTRCOSCOPY 2
6. 0.3ml of a patient’s serum was diluted by adding 2.7ml of physiological salt solution.
The biuret reaction was carried out and the absorbance was equal to 0.4. The
absorbance of a 0.5 mg/ml standard solution was 0.25. Calculate the protein
concentration in the patient’s serum in grams per 100ml and g/l.
7. A serum sample was diluted 20x and the protein content was determined by the biuret
method. The absorbance was equal to 0.3. Using the calibration curve below, calculate
the protein concentration in the serum in g/100ml and g/l.
Protein concentration [mg/ml]
8. The absorptivity (k) at 280nm (A280) of a 1% solution of the enzyme trypsin in a 1cm
cuvette is 1.43. What is the concentration of a trypsine solution that has a A280 of 1.5
in a 0.5cm cuvette?
ABSORPTION SPECTRCOSCOPY 3