Experiment 1 Estimation of Protein using Spectroscopy Method
EXPERIMENT 1
TO DETERMINE THE
CONCENTRATION OF A
PROTEIN SAMPLE BY
USING ULTRAVIOLET (UV)
SPECTROSCOPY METHOD
Structure
1.1 Introduction
1.4 Procedure
Expected Learning Outcomes
1.5 Results
1.2 Principle
1.6 Precautions
1.3 Materials Required
1.1 INTRODUCTION
Protein concentration estimation is one of the most commonly performed tasks
in a biochemistry lab. The methods available for estimating the protein
concentration are i. amino acid analysis followed by acid hydrolysis of the
protein ii. analyzing the changes in the spectral properties of certain dyes in
the presence of proteins and iii. spectrophotometric estimation of the proteins
in UV regions. The last method can be used for estimating the protein
concentration for both pure as well as an unknown mixture of proteins; UV
spectroscopic quantization is popularly known for the estimation of purity of
proteins. For purified protein samples UV spectroscopic quantization is the
best method of choice. This method is easy, less time consuming and the
protein sample used can be recovered back for future use.
Electromagnetic radiation:
Absorption of ultraviolet radiation is a general method used for estimating a
large number of bioanalytes. The region of the electromagnetic radiation
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BBCCL-106 Proteins
ranging from ~10 – 400 nm is identified as the ultraviolet region and different
energies of UV region; can be divided into three regions (Fig. 1.1):
i. Near UV region (UV region nearest to the visible region; λ ~ 250 – 400
nm)
ii. Far UV region (UV region farther to the visible region; λ ~ 190 – 250
nm)
iii. Vacuum UV region (λ < 190 nm)
Fig. 1.1: Diagrammatic representation of the energy levels of Electromagnetic
spectrum/radiation.
Expected Learning Outcomes:
After performing this experiment, you shall be able to
explain the relationship between wavelength and absorbance;
describe the working principle of U.V and Visible spectrophotometer; and
enlist the limitations of U.V absorption method for estimation of protein
concentration.
1.2 PRINCIPLE
Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and
200 nm. Aromatic rings present in the amino acids are the primary reason for
the absorbance peak at 280 nm and at 200 nm peptide bonds has its peak.
Secondary, tertiary, and quaternary structure all affect absorbance, therefore
factors such as pH, ionic strength, etc. can alter the absorbance spectrum.
Absorption spectrum of a protein (Fig. 1.2) suggests that the proteins can
absorb both in near UV and far UV regions.
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Experiment 1 Estimation of Protein using Spectroscopy Method
Fig. 1.2: Absorption spectrum of a peptide in the near and far UV regions.
Absorption of UV radiation is usually represented in terms of absorbance and
%transmittance:
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 (𝐴)=−𝑙𝑜𝑔 (I / 𝐼0) ----------------------------- equation 1
%𝑇𝑟𝑎𝑛𝑠𝑚𝑖𝑡𝑡𝑎𝑛𝑐𝑒 (%𝑇) = I / 𝐼0×100 ----------------------------- equation 2
where, ‘𝐼0’ and ‘I’ represent the intensities of light entering and exiting the
sample, respectively.
Absorbance of an analyte depends on the concentration of the analyte and the
path length of the solution (Beer-Lambert Law):
A = εcl-------------------------------------- equation 3
Where, ε is the molar absorption coefficient, c is the molar concentration of the
analyte and l is the path length of the cell containing the analyte solution. If
molar absorption coefficient of the analyte and the path length of sample cell
are known, concentration can directly be determined using Beer-Lambert law.
This method is recommended for calibrating bovine serum albumin or other
pure protein solutions for use as standards in other methods.
1.3 MATERIALS REQUIRED
Glassware and Equipment’s: Pipettes, Pipette tips, Disposable microfuge
tubes, Quartz cuvettes (suitable for wavelengths smaller than 205 nm) and
UV/Visible spectrophotometer.
Chemicals and Reagents: Dissolve 1 mg of Bovine serum albumin (BSA) in
100 ml buffer (or in water), buffer alone will act as the blank.
Preparation of Tris buffer (0.1 M, pH 7): Prepare a 0.1M Tris buffer by
dissolving 12.1 gm of Tris Base to a total volume of 1 litre of distilled water.
Obtain the desired pH by adding drop by drop by addition of 0.1M HCl (check
the pH at regular intervals).
1.4 PROCEDURE
Carry out steps 1-4 (280 nm only) for a very rough estimate.
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BBCCL-106 Proteins
1. Warm up the UV lamp (about 15 min or follow the instruction of
spectrophotometer manufacturers).
2. Adjust wavelength to 280 nm
3. Calibrate to zero absorbance with buffer solution only
4. Measure absorbance of the protein solution
5. Adjust wavelength to 260 nm, (if there is nucleic acid contamination)
6. Calibrate to zero absorbance with buffer solution only
7. Measure absorbance of the protein solution
1.5 RESULTS
Unknown proteins or protein mixtures: Use the following formula to roughly
estimate protein concentration. Path length for most spectrometers is 1 cm.
Concentration (mg/ml) = Absorbance at 280 nm divided by path length (cm.)
Pure protein of known absorbance coefficient: Use the following formula
for a path length of 1 cm. Concentration is in mg/ml, %, or molarity depending
on which type coefficient is used.
Concentration = Absorbance at 280 nm divided by absorbance coefficient
To convert units, use these relationships:
mg protein/ml = % protein divided by 10 = molarity divided by protein
molecular weight
Unknowns with possible nucleic acid contamination: Use the following formula
to estimate protein concentration:
Concentration (mg/ml) = (1.55 x A280) - 0.76 x A260)
1.6 PRECAUTIONS
Cold solutions can fog up the cuvette, while warm solutions can release
bubbles and interfere with the readings.
For concentrated solutions (absorbance greater than 2) simply dilute the
solution.
Absorbance coefficients of some common protein standards:
i. Bovine serum albumin (BSA): 63
ii. Bovine, human, or rabbit IgG: 138
iii. Chicken ovalbumin: 70
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Experiment 1 Estimation of Protein using Spectroscopy Method
Advantages:
i. Quantifying protein by directly measuring absorbance is fast and
convenient, since no additional reagents or incubations are
required.
ii. No protein standard need be prepared and the procedure does not
consume the protein.
iii. The relationship of absorbance to protein concentration is linear.
iv. The most common use for this method is to monitor fractions from
chromatography columns, or any time a quick estimation is needed
and error in protein concentration is not a concern.
Disadvantages:
i. Because different proteins and nucleic acids have widely varying
absorption characteristics there may be considerable error,
especially for unknowns or protein mixtures.
ii. Any non-protein component of the solution that absorbs ultraviolet
light willintefere with the measurements.
iii. Cell and tissue fractionation samples often contain insoluble or
colored components that interfere.
SAQ
1. Define electromagnetic radiation?
2. Explain the principle behind U.V. absorption spectrophotometer.
3. What are the units used to express protein concentration?
4. At ________ wavelength proteins show maximum absorption.
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