U V - V i s S p e c t r o s c o p y
Introduction
Proteins are the most important molecules in living material next to nucleic acids. They are produced by transcription of
DNA.
Proteins are very important in regulating metabolic mechanisms, for example as hormones or enzymes, apart from their structural role.
The recent increase in interest in proteomics has made the measurement and characterization of proteins ever more important.
Many techniques exist for determination of protein concentration, offering choices of sensitivity, specificity and speed.
This application will outline the methods available, and show how easily these can be carried out using downloadable SOP’s, methods and report templates for the UV WinLab V4.00
software package.
Methods
Methods most commonly used include direct measurement at
280nm, corrected ratio of
230nm/260nm, and reactive methods using reagents such as
Biuret, Folin Ciocalteau and
Coomassie blue. These methods are described in more detail in
Table 1.

Table 1. Features of Common Protein Assays
Application Principle Detection
Range
Lowry
Bradford
Biuret
BCA
Absorbance at 280nm
Corrected ratio
230/260nm
Warburg-
Christian
Determination of aromatic amino acids
Dye binding with
Coomassie Brilliant
Blue
Determination of number of peptide bonds
Reduction of copper by protein, formation of Cu(I)–BCA complex
Determination of tyrosine and tryptophan in protein
Determination of peptide bonds with baseline correction
Determination of aromatic amino acids with compensation for nucleic acids
5-200 µ g/mL
Comments
Assay offers good sensitivity, but depends on amino acid composition of the protein.
Susceptible to interference by phenols, aromatic amino acids
Assay offers good sensitivity, interference by detergents
10-200 µ g/mL
(1-10 µ g/mL)
(
200-
5000
200-
1200
>50 µ
µ
µ
5-250 g/mL g/mL
µ g/mL) g/mL
Assay is relatively insensitive, but is easy to carry out, has little dependency on the protein composition. Susceptible to interference from amines and ammonium salts
Assay offers good sensitivity, has little dependency on amino acid composition of the protein. Compatible with ionic and non-ionic detergents. Susceptible to interference by high metal concentration, strong reducing agents and chelating substances such as EGTA
Offers relatively poor sensitivity and selectivity, but is easy to carry out and non-destructive.
Depends on amino acid composition of protein, and is susceptible to interference by nucleic acids, phenols and aromatic hydrocarbons
>5 µ g/mL Assay is normally used as a purity check.
Interferences by many organic compounds, nitrate, nitrite. Baseline correction carried out at
320nm.
50-3000 µ g/mL Assay is normally used as a purity check. Offers better sensitivity and specificity than direct absorbance at 280nm. Interference from phenols and aromatics. interference from nucleic acids can be compensated for.
Effect of Protein
Composition
Different proteins have different amino acid compositions. The ratio of peptide bonds to mass and the relative amount of aromatic amino acids vary for different protein types.
This results in varying response for some of the less specific protein assays such as direct absorbance and the
Lowry method.
Choosing the Appropriate
Method
Correct choice of method for determination of proteins depends on four criteria:
• Speed and ease of assay
• Sensitivity
• Dependence on protein composition
• Rejection of interferences
The most rapid assays are based on direct absorbance. Simple measurement at 280nm is commonly used as a rapid means of determining protein concentration: other direct absorbance methods using more than one wavelength can reject interferences for use as purity checks.
The Lowry method offers high sensitivity but is prone to interferences and can be strongly dependent on the amino acid composition of the protein under analysis.

The Biuret and BCA (Figure 1) methods have little dependency on the amino acid content of proteins under analysis, but are susceptible to interferences.
Calibration with Secondary
References
Optimally, calibration should be carried out with the same protein that will be assayed.
The use of a correction factor can be employed, however, to allow calibration with less expensive proteins, by using them as secondary reference materials:
• Calibrate the method with the protein predominant in the samples to be assayed to generate the real protein concentration value [P]
1
• Calibrate with the secondary Reference
Material to generate the observed protein concentration value [P]
2
• Determine the correction factor ( F ) as
[P]
2
/ [P]
1
For example, using immunoglobulin IgG as primary reference produced a concentration of 1000 mg/mL.
Using bovine serum albumin
(BSA) as secondary reference produced a value of 800 mg/mL. Therefore:
F =1000/800=1.25.
After calibration against BSA, results must be multiplied by a factor of 1.25.
Figure 1. Effect of different proteins on the BCA Assay

The Lowry Method
Color development in the
Lowry method is caused by a two-step reaction:
1.Biuret reaction of protein with copper ions in alkaline medium
2.Reduction of phosphomolybdic and phosphotungstic reagent by tyrosine and tryptophan residues in the reacted protein.
Tyrosine, tryptophan, most phenols (except nitrophenol), uric acid, guanine and xanthine react with Folin’s reagent to produce color. Minor interference is observed with adenosine, adenine, guanosine, hypoxanthine, cytosine, uracil, thymine and thymidine.
Glycine, Ammonium sulfate and hydrazine all decrease color development.
Table 2. Reagents and Procedures for the Lowry Assay
Reagents
Procedure
Stock Reagent A : Dissolve 10g sodium carbonate in
500mL of 0.1N sodium hydroxide solution
Stock Reagent B : Dissolve 0.5g potassium sodium tartrate in 30mL of deionised water in a 50mL volumetric flask.
Dissolve 0.25g of copper sulfate pentahydrate in this solution and make up to 500mL with deionised water.
Working Reagent A : (Alkaline copper solution) mix 50mL of
Stock Reagent A with 1mL of Stock Reagent B
Working Reagent B : (Folin’s Reagent) Dilute 50mL of
Folin-Ciocalteu’s phenol reagent (density 1.22 kg/L) with deionised water to 100mL
Protein Reference Stock Suspension: 10mg/ml protein.
Dissolve 500mg of protein in 0.9% sodium chloride solution and dilute to 50mL. Take suitable aliquots and dilute with
0.9% sodium chloride solution to produce reference protein samples which cover the expected concentration range of analytical samples. Reference samples must be prepared daily; the Reference stock is stable for three days at 5C.
Pipette 1mL of Working Reagent 1 into a labeled test tube.
Add 0.2mL of sample. Mix thoroughly.
Incubate for at least 10 minutes at room temperature.
Add 0.1mL of Working Reagent B, mix thoroughly.
Incubate for at least 30 minutes at room temperature.
Measure the absorbance at 750nm for [protein] below
40ug/ml, above this concentration measure absorbance at
500nm.
Figure 3. Typical Calibration Plot for Lowry Protein Assay
Figure 2. Absorbance
Spectrum of the Lowry
Color Product

The Bradford Method
Color development in the
Bradford method is based on the fact that Coomassie Brilliant
Blue G-250 exists in two color forms, red and blue. The red form is converted to the blue form on binding to proteins.
This causes a shift in the absorption maximum from
465nm to 590nm. The increase in absorption at 590nm is monitored. The protein-dye complex has a high extinction coefficient, leading to high sensitivity for measurement of protein.
Binding of the dye to protein is a very rapid process and the protein-dye complex remains dispersed in solution for a relatively long time.
Table 3. Reagents and Procedures for the Bradford Assay
Reagents
Procedure
Coomassie Brilliant blue Reagent : Dissolve 100mg
Coomassire Brilliant Blue in 50mL ethanol, add 100mL of phosphoric acid and dilute with deionised water to 100mL.
Stock Solution (10mg/L protein) : Suspend 500mg of protein in 0.9% sodium chloride solution and dilute to 50mL.
Take suitable aliquots and dilute with 0.9% sodium chloride solution to produce references which cover the expected concentration range of analytical samples. Reference samples must be prepared daily; the Reference stock is stable for three days at 5C.
For [protein] 10 to 200mg/ml:
Pipette 1mL of reagent into a labeled test tube.
Add 1.0mL of sample and mix thoroughly.
Measure between 2 and 60 minutes after mixing at 590nm.
For [protein] 1 to 10mg/ml:
Pipette 1mL of reagent into a labeled test tube.
Add 0.5mL of sample and mix thoroughly.
Measure between 2 and 60 minutes after mixing at 590nm.
For [protein] 100 to 1000mg/ml:
Pipette 5mL of reagent into a labeled test tube.
Add 0.1mL of sample and mix thoroughly.
Measure between 2 and 60 minutes after mixing at 590nm.
Figure 4. Absorbance
Spectrum of the Bradford
Color Product
Figure 5. Typical Calibration Plot for Bradford Protein Assay

The Biuret Method
Substances containing two or more peptide links form a purple colored complex with copper salts under alkaline conditions.
The structure of a part of the copper (II)-inner complex is shown in Figure 6.
The reaction is very specific for proteins, there are virtually no biological materials that react to any significant extent with the
Biuret reagent. Lipids can cause turbidity in the reaction mixture, to avoid this the sample should be shaken with diethyl ether or petroleum ether and the organic phase removed by centrifugation.
The Biuret reaction cannot be used with ammonium salts. In such cases, proteins must be separated before analysis.
Color yields of different proteins are not identical, but differences are much smaller than for
Foiln/Phenol and direct absorbance methods.
Figure 6. The Copper(II)-Inner Complex
Table 4. Reagents and Procedures for the Biuret Assay
Reagents
Procedure
Working Reagent : Dissolve 1.5g copper sulfate and 6g potassium sodium tartrate in about 500mL deionised water in a 1 liter volumetric flask. While stirring, add
300mL of 10% aqueous sodium hydroxide solution and make up to 1 liter with deionised water
Stock Solution (50mg/L protein) : Suspend 1000mg of protein in 0.9% sodium chloride solution and dilute to
20mL. Take suitable aliquots and dilute with 0.9% sodium chloride solution to produce references which cover the expected concentration range of analytical samples. Reference samples must be prepared daily; the Reference stock is stable for three days at 5C.
Pipette 1mL of reagent into a labeled test tube.
Add 0.2mL of sample and mix thoroughly
Incubate for 30 minutes at room temperature Measure the absorbance at 549nm.
Figure 7. Absorbance Spectrum of the Biuret Color Product
Figure 8. Typical Calibration Plot for Biuret Protein Assay

The BCA Method
Substances containing two or more peptide links form a purple colored complex with copper salts under alkaline conditions.
The structure of a part of the copper (II)-inner complex is shown in Figure 7.
Optimum pH for the BCA assay is 11.25. Substances which lower or raise the pH interfere.
EDTA and strong chelating agents reduce the amount of free copper ions necessary for the reaction.
Reducing sugars can cause interference at elevated temperatures and reaction times.
Reducing agents generally cause high blank values, although low concentrations can be corrected in the blank. Other interferences include phenol red, uric acid, lipids, amino acids with reducing properties, iron and high concentrations of metals.
Figure 9. The BCA – Cu
++
Complex
Table 5. Reagents and Procedures for the BCA Assay
Reagents
Procedure
Working Reagent : Dissolve 1.5g copper sulfate and 6g potassium sodium tartrate in about 500mL deionised water in a 1 liter volumetric flask.
While stirring, add 300mL of 10% aqueous sodium hydroxide solution and make up to 1 liter with deionised water
Stock Solution (50mg/L protein) : Suspend 1000mg of protein in 0.9% sodium chloride solution and dilute to
20mL.
Take suitable aliquots and dilute with 0.9% sodium chloride solution to produce references which cover the expected concentration range of analytical samples.
Reference samples must be prepared daily; the
Reference stock is stable for three days at 5C.
Pipette 1mL of reagent into a labeled test tube.
Add 0.2mL of sample and mix thoroughly
Incubate for 30 minutes at room temperature Measure the absorbance at 549nm.
Figure 10. Absorbance Spectrum of the BCA Color Product
Figure 11. Typical Calibration Plot for BCA Protein Assay

Estimation by Direct
Absorbance
Aromatic amino acid residues in proteins (tyrosine and tryptophan) have a strong characteristic absorbance at 280nm, which can be used for protein estimation
(Figure xx). Since the aromatic amino acid content of proteins varies within reasonable limits, then direct estimation (not determination) of protein concentration can be carried out by measurement of absorbance at
280nm.
Protein concentration can be calculated if the molar extinction coefficient ε at 280nm is known:
Figure 12. Absorption
Spectrum of BSA
Interferences are any compounds which absorb at 280nm, this includes nucleic acids, phenols and aromatic compounds (Table xx). Sample turbidity can also cause interference, so correction is often carried out by subtraction of the absorbance at 320nm, where proteins and nucleic acids do not absorb. Important analytical wavelengths are shown in Table 6.
Molar coefficients vary between proteins. For example, human serum proteins have coefficients ranging from 0.4 to 2.0 L/g.cm
(Figure xx).
If the molar extinction coefficient is unknown, then a mean coefficient of 1.05 (according to
Richterich) or 0.8951 (according to Warburg and Christian) can be used.
Table 6. Important Wavelengths of Proteins and Nucleic Acids
230nm Peptide bonds of proteins.
Baseline minimum of nucleic acid spectra.
260nm
280nm
320nm
Nucleic acids.
Aromatics, aromatic amino acid residues of proteins and peptides
Neither proteins nor nucleic acids absorb.
Table 7. Reagents and Procedures for the BCA Assay
Protein
280
(L/g.cm)
Tyrosine content (%)
Albumin 0.58
4.07
Biuret
(%)
100
7.29
112 α 1-acid glycoprotein
α 1-antitrypsin haptoglobulin caeruloplasmin
α 2-macroglobulin transferrin hemopexin
0.89
0.53
1.20
1.49
0.81
1.12
1.69
G immunoglobulin (IgG) 1.38
M immunoglobulin (IgM) 1.33
γ A immunoglobulin (IgA) 1.34
2.55
6.78
8.90
5.71
4.84
5.30
6.11
4.89
4.95
116
98
104
107
99
112
100
Table 8.Procedure for Direct Measurement at 280nm.
Procedure Use a blank suspension to perform an autozero
Measure the absorbance at 280nm
If necessary, dilute the samples with 0.9% sodium chloride solution

Refinements to the Direct
Absorbance method
Measurement of several wavelengths can be carried out to negate interference from nucleic acids and aromatics.
These methods can be used to determine purity or absolute concentration. An example is direct measurement at 230nm and 260nm using the Corrected
Ratio method: peptide bonds of proteins absorb strongly at
230nm and very weakly at
260nm. Nucleic acids, conversely, absorb strongly at
260nm and weakly at 230nm, so measurement of the 230/260 ratio gives a good measure of protein purity, with respect to contamination by nucleic acids.
Sometimes the samples are
Table 9. Downloadable Materials.
contaminated by light-scattering particulates. This causes turbidity and high background signals. Measurement of the absorption at 320nm, where proteins and nucleic acids have weak absorbance, is used to correct for turbidity-dependent baseline.
The Warburg-Christian method also employs direct absorbance measurement to eliminate interferences from nucleic acids.
Conclusion
UV WinLab v.4.00 is a flexible, programmable software package which simplifies the construction of routine methods. Custom sample information, data processing and manipulation and results reporting can all be easily
Application SOP
Lowry Lowry SOP
Bradford
Biuret
Bradford SOP
Biuret SOP
BCA SOP
Protein by A280 SOP
BCA
Absorbance at 280nm
Corrected ratio
230/260nm
Warburg-
Christian
Protein Purity SOP
Warburg-Christian SOP
Method
Protein by Lowry
Protein by Bradford
Protein by Biuret
Protein by BCA
Protein by A280nm programmed and saved as turnkey methods.
For all of the methods listed in this Technical Note, downloads are available from the
PerkinElmer Website to assist in setting up the assay. These downloads include a pre-prepared method, detailed SOP and report template with all calculations, graphics and results.
Downloadable items are listed in
Table 9.
Protein Purity 230/260nm
Report Template
LowryReport
BradfordReport
BiuretReport
BCAReport
A280Report
ProteinPurityReport
Protein by Warburg-Christian WarburgReport
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