Practical No : 03

Determination of Protein concentration by Ultraviolet

absorption at 280nm

Name : H.M.A. Bandara.

Reg. No : PGIS/CH/MSc/CHN/17/02
Practical No : 03

Title : Determination of Protein concentration by Ultraviolet absorption at 280nm

Objectives : To gain experience in UV spectrophotometric analysis of proteins

Introduction :

The need to determine protein concentration in a solution is a routine requirement during protein and enzyme
studies. In this experiment, you will determine protein concentrations of given solution using
spectrophotometric methods. You will use a method of determining protein concentration, which involves
measuring the optical absorption by protein themselves.

The optical absorption of a solution is given by the following equation:

A=- log { I/Io}

A=ξIC

A – Absorbance

I – Intensity of Transmitted radiation

Io – Intensity of Incident coefficient

ξ – Molar extinction coefficient

I – Path length

C – Concentration

The aromatic amino acids tyrosine and tryptophan in a protein exhibit an absorption maximum at a wavelength
of 280nm. The proportions of these amino acids and therefore the absorption vary from one protein to another.
However for most proteins the extinction coefficient lies in the range of 0.4-1.5. Therefore absorption at 280nm
gives only rough idea of the protein concentration in a mixture of proteins. More accurate measurements are
obtained with purified proteins.
Materials & Method:

 UV/Visible spectrophotometer
 Casine from Bovine milk
 Unknown Protein solution
 0.1M NaOH
 Essential glassware and tubes

0.05g of casine was dissolved in 10ml of 0.1M NaOH, to obtain 0.5% casine solution. Then total volume 3ml,
dilution series of 0.25%, 0.125%, 0.0625%, 0.03125%, and 0.015625% were prepared. The UV absorptions were
measured and constructed a calibration curve using obtained data. Then absorption of provided unknown
solution was measured and the concentration of unknown solution was found using the calibration curve.

Observations

UV-Visible Spectral Data for the given solutions were observed. The concentrations (%) and respective UV
absorption measurements are tabulated in table 01.

% Concentration 0.25 0.125 0.0625 0.03125 0.01562 Unknown

Total Volume (ml) 3 3 3 3 3 3
UV Absorption value 1.9840 0.9689 0.4866 0.2461 0.1296 0.8913
Table 01
Calculations & Results

The concentration of the unknown solution is determined by Ultraviolet absorption at 280nm.the calibration
curve for observed data are given in Graph 01.

Calibration Curve (% Concentration Vs. Absorbance)

2.5

2 y = 7.9179x - 0.004
Absorbance

1.5

0.5

0
0 0.05 0.1 0.15 0.2 0.25 0.3
% Concentration

Graph 01

Calibration curve equation: y=7.917x-0.004

UV Absorbance measurement of the Unknown solution = 0.8913

0.8913+0.004
Therefore the % Concentration =
7.917

=0.1131

0.113086∗1000
The concentration of the Unknown solution =
100

= 1.131 gL-1
Conclusion

The method is simple, rapid, and non-destructive. The relationship between protein concentration and
absorbance is linear. Despite its technical simplicity, this method is rather insensitive (0.2 to 2 mg/ml).Protein-
to-protein variation is high. As the A280 is due to the aromatic rings, the sensitivity of the assay is dependent
on the number of aromatic rings containing amino acids. Low amounts of nucleic acids (1 µg) interfere with the
A280.the concentration of unknown protein sample obtained from this method is 1.131 mg/ml.

Discussion

Structurally, proteins are the most complex bio macromolecules. This structural complexity is due to the
composition and sequence of the amino acids that make up proteins. The composition and sequence of amino
acids is different for every protein. As such, the unique chemical and physical characteristics of a protein can
be used to isolate it from other cellular components using basic chemical techniques. Once a protein has been
isolated, one can initiate characterization studies, information such as pH- and heat-stability, that could prove
helpful in ascertaining the protein’s structure and/or function without knowing its amino acid sequence.
Proteins are denatured (lose their natural structure and solubility) by a variety of denaturing agents, such as
organic solvents, acids, bases, heavy metals (e.g., mercury or lead) and high salt concentration.

Many different methods have been devised to separate proteins from other proteins or biomolecules. Several
of these separation methods are based on the solubility of proteins under various conditions. Casein is a
heterogeneous mixture of proteins found in milk. Casein is a nutritionally-adequate protein. This means that it
has all the essential amino acids required for normal growth and development. Casein is a phosphoprotein
produced in mammary tissue, and is 3 - 5% of bovine and human milk. In this practical we studied the proteins
is casein (milk protein). It is isolated from milk by removing the cream (lipid) and acidifying the resulting skim
milk, which causes the casein to precipitate.

Casein is soluble in water in its native or natural state. However, it may precipitate from solution in the presence
of acids, alkali, heavy metals and other denaturing agents. Many of the chemical properties of proteins are due
to the characteristics of the R groups of the amino acids that make up the protein. For example, acidification of
ionized groups in proteins may neutralize their ionic charges resulting in decreased solubility in water or
alterations in their interactions with other ionic and polar groups, causing them to precipitate. Heavy metals
may bind to ionic or polar groups on the surface of proteins, causing them to aggregate and diminishing their
interactions with water molecules and rendering them insoluble in water.

The molar absorption coefficient, molar extinction coefficient, or molar absorptivity, is a measurement of how
strongly a chemical species absorbs light at a given wavelength.

UV-Visible Spectroscopy

Spectroscopy is the study of the interaction of molecules with light. It has long been known that atoms and
molecules absorb light. The light is absorbed because it interacts with the various types of motion that a
molecule may undergo. These motions occur in different ranges of frequency, so they interact with light in
different wavelength (frequency) ranges. For a molecule to absorb a photon of light, one of its motions must
occur with the same frequency that the light wave oscillates with. By measuring the frequencies of light
absorbed by the molecules of a substance, we obtain information about the molecular motions and often about
the molecular structure.

Table 1 shows the molecular motions that interact with light in the various wavelength regions.

Wavelength range of
Molecular Motion Type of Spectroscopy
Electromagnetic Radiation

spinning of an atomic nucleus radio Nuclear Magnetic Resonance

rotation of the molecule in space microwave Rotational Spectroscopy

vibrations of the molecule

infrared Vibrational (Infrared) spectroscopy
(bond stretches and bends)

motions of the electrons from

ultraviolet, visible UV-Visible Spectroscopy
one energy level to another

The principles of UV-Vis spectrophotometer are light intensity changes by Absorbance or Transmittance and
Quantity measurement using Absorbance.
Transmittance, t : It/Io= t

Absorbance, A : log I/ t = A

In UV-visible spectroscopy, a prepare sample, place it in a UV-visible spectrometer (an instrument), and obtain
the UV-visible (UV-vis) spectrum. The instrument automatically exposes the sample to all of the wavelengths of
light in the infrared region, and measures how much of the light is absorbed by the sample at each wavelength.
It then produces a plot of the percentage of the absorbance by the sample at each wavelength. The absorbance,
A, is 0 at wavelengths where the sample does not absorb light, and is greater than 0 at wavelengths where the
sample absorbs. A particular substance absorbs UV-vis radiation at characteristic wavelengths; we can use the
UV-vis spectrum to help identify the substance. For example, the substance with this UV-vis spectrum absorbs
UV-vis radiation in the wavelength regions 200-300, 400, and 500 nm. Normally we report only the wavelengths
at which the absorbance maximizes. These wavelengths are called the λmax values for the substance. This can be
calculated as follows.

 A=alC
 A= ξlC

l- Sample path length

Absorbance Constant

ξ- Molecular absorbance constant

C-Sample Concentration

This equation is called Beer's Law. Substance is the molarity of the substance that is absorbing the light; l is the
length of sample through which the light passes, almost always 1 cm; and ξ is called the molar absorptivity of
the substance at the particular wavelength and has units of M -1cm-1.
There are some limitations of the Beer-Lambert law, Deviations in absorptivity coefficients at high
concentrations (>0.01M) due to electrostatic interactions between molecules in close proximity, Scattering of
light due to particulates in the sample, Fluorescence or Phosphorescence of the sample, Changes in refractive
index at high analysis concentration, Shifts in chemical equilibria as a function of concentration and Stray light
scattering.

Sample Preparation

UV-visible spectra are most easily obtained from solutions of the substance of interest in a suitable solvent. A
solvent is suitable if a) the substance dissolved in it without reaction, and b) the solvent has no absorbance in
the wavelength range of interest. Water is a good solvent for UV-visible spectroscopy because it has no
absorbance over the entire wavelength range from 1100 to 200 nm.

The cuvettes are precision made, highly polished, expensive and polished faces should not be touched for the
best results. An identical cuvet may be filled with solvent only. The cuvets are placed in the instrument and the
spectrum is scanned. If absorbance bands go "off scale," appropriate dilution of the original solution should
enable to get them "on scale." A series of dilutions (called serial dilution) may be necessary to bring all
absorbance bands on scale. When finished, empty the cell into a waste container. Rinse it several times with
distilled water, then a couple of times with small amounts of acetone.

Spectral properties of amino acids

Trp, Tyr, and Phe contain conjugated aromatic rings. Consequently, they absorb light in the ultraviolet range
(UV). The extinction coefficients (or molar absorption coefficients) of these three amino acids are:

Amino acid Extinction Coefficient λ (λmax)

Trp 5,050 M-1cm-1 (280 nm)

Tyr 1,440 M-1cm-1 (274 nm)

Phe 220 M-1cm-1 (257 nm)

The above table shows that Trp absorbs UV light the strongest. Furthermore, since both Trp and Tyr show the
maximum light absorbance at approximately 280 nm the absorption maximum of most proteins is around 280
nm. In contrast, the absorption maximum for nucleic acids is approximately 260 nm.

Aromatic amino acids are relatively nonpolar. To different degrees, all aromatic amino acids absorb ultraviolet light.
Tyrosine and tryptophan absorb more than do phenylalanine; tryptophan is responsible for most of the absorbance
of ultraviolet light (ca. 280 nm) by proteins. Tyrosine is the only one of the aromatic amino acids with an ionizable
side chain. Tyrosine is one of three hydroxyl containing amino acids.

Least hydrophobic Very hydrophobic

Tryptophan has much stronger fluorescence and higher quantum yield than the other two aromatic amino acids. The intensity,
quantum yield, and wavelength of maximum fluorescence emission of tryptophane is very solvent dependent. The
fluorescence spectrum shifts to shorter wavelength and the intensity of the fluorescence increases as the polarity of the
solvent surrounding the tryptophane residue decreases. Tryptophan residues which are buried in the hydrophobic core of
proteins can have spectra which are shifted by 10 to 20 nm compared to tryptophans on the surface of the protein. Tryptophan
fluorescence can be quenched by neighbouring protonated acidic groups such as Asp or Glu.

Tyrosine, like tryptophan, has strong absorption bands at 280 nm, and when excited by light at this wavelength, has
characteristic emission profile. Tyrosine is a weaker emitter than tryptophan, but it may still contribute significantly to protein
fluorescence because it usually present in larger numbers. The fluorescence from tyrosine can be easily quenched by nearby
tryptophan residues because of energy transfer effects. Also, tyrosine can undergo an excited state ionization which may result
in the loss of the proton on the aromatic hydroxyl group that leads to quenching of tyrosine fluorescence.

Phenylalanine with only a benzene ring and a methylene group is weakly fluorescent. The experimental sensitivity (the product
of quantum yield and molar absorbtivity maximum) is especially low for this residue. Phenylalanine fluorescence is observed
only in the absence of both tyrosine and tryptophane. The simple structure of phenylalanine may preeminently demonstrate
the effect of structure on fluorescence. Adding a hydroxyl group, as in tyrosine, causes a 20 fold increase in fluorescence. If an
indole ring is added as in tryptophan, the relative fluorescence increases to 200 times that of phenylalanine.

Ultraviolet Absorption methods

In ultraviolet (UV) absorption methods, proteins are measured directly without the addition of any reagents.
Proteins have two absorption maxima: 280 nm and 200 nm.In absorption spectroscopy, an electron absorbs
photons. Photons with lower energy levels have longer wavelengths, and thus electrons that are excited at 280
nm have absorbed less energy than those at 200 nm. Electrons that are excited at 280 nm require less energy
because they lie within the aromatic rings, which stabilize the excited state due to resonance.

Absorbance at 280 nm (A280)

The quantitation of protein by this method can only be applied to pure protein. Nonetheless, absorbance is
widely used for monitoring purification progress and for generating a protein elution profile during column
chromatography. The elution profile provides a guide for pooling fractions containing proteins. For A280, the
amino acids containing aromatic rings, such as tryptophan, tyrosine, phenylamine, and histidine, are involved.

The method is relatively sensitive, being able to measure protein concentrations as low as 10 mgcm 3, and, unlike
colorimetric methods, is non-destructive, i.e. having made the measurement, the sample in the cuvette can be
recovered and used further. This is particularly useful when one is working with small amounts of protein and
cannot afford to waste any. However, the method is subject to interference by the presence of other compounds
that absorb at 280 nm. Nucleic acids fall into this category having an absorbance as much as 10 times that of
protein at this wavelength. Hence the presence of only a small percentage of nucleic acid can greatly influence
the absorbance at this wave- length. However, if the absorbances (A) at 280 and 260 nm wavelengths are
measured it is possible to apply a correction factor:

The great advantage of this protein assay is that it is non-destructive and can be measured continuously, for
example in chromatographic column effluents. Even greater sensitivity can be obtained by measuring the
absorbance of ultraviolet light by peptide bonds. The peptide bond absorbs strongly in the far ultraviolet, with
a maximum at about 190 nm. However, because of the difficulties caused by the absorption by oxygen and the
low output of conventional spectrophotometers at this wavelength measurements are usually made at 205 or
210 nm. Most proteins have an extinction coefficient for a 1 mgcm 3 solution of about 30 at 205 nm and about
20 at 210 nm. Clearly therefore measuring at these wavelengths is 20 to 30 times more sensitive than measuring
at 280 nm, and protein concentration can be measured to less than 1 mgcm 3 . However, one disadvantage of
working at these lower wavelengths is that a number of buffers and other buffer components commonly used
in protein studies also absorb strongly at this wavelength, so it is not always practical to work at this lower
wavelength. Nowadays all purpose-built column chromatography systems (e.g. fast protein liquid
chromatography and high-performance liquid chromatography (HPLC)) have in-line variable wavelength
ultraviolet light detectors that monitor protein elution from columns.

Absorbance at 205 nm (A205)

In the far UV region (190 nm), the peptide bond absorbs photons very strongly(about 50-fold more sensitive
than at 280 nm). Thus, protein can be measured at this wavelength because of the large number of peptide
bonds in a protein. But it is not possible to measure the peptide peak at this wavelength using routine
spectrophotometers, because oxygen is absorbed in this region. Measurements at 205 nm give sufficiently
accurate results, although the absorbance of the protein is about half at 205 nm than at 190 nm. Advantages of
this method are Sensitivity is higher at 205 nm compared to 280 nm. There is little variation between proteins
at 205 nm, because peptide bonds are measured. Like A280, A205 is linear with the protein concentration. The
A205 is also rapid and non-destructive. The disadvantage of the A205 is that the compatibility of most chemicals
is relatively low compared to A280.

Determination of protein concentration using A280

For an unknown protein or protein mixture, the following formula can be used to obtain a rough estimate of
protein concentration. Using this procedure, a protein of 20 µg/ml to 3 mg/ml can be measured.

Concentration (mg/ml) = A280/path length in cm

Since nucleic acids absorb strongly at 280 nm, crude cell extracts containing RNA and DNA produce erroneously
high protein estimates. In that case, the absorbance of the extracts at 260 nm (A260) is measured as the
procedure outlined above. The protein concentration of the solution is corrected when nucleic acid is present
using the following formula:

Protein concentration (mg/ml) = 1.55 A280 - 0.76 A260

For a protein whose absorbance coefficient is known, protein concentration is determined by any of the
following formulas, assuming the protein is measured with a cuvette of 1 cm path length. The absorption
(extinction) coefficient is expressed as E280 1 mg/ml, E280 1%, or E280 M.

Protein content in mg/ml can easily be converted from the percentage protein and the molarity as follows:

Buffers used to dissolve for spectroscopic analysis should not absorb strongly at 280nm or 235nm. A high A280
reading for the buffer, relative to H2O, indicates the presence of an interfering substance. Although moderate
absorbance at 280nm can be balanced by readjusting the zero setting of the spectrophotometer, most
instruments have a limited range of sensitivity at high absorbencies (A280>1.0) due to interference by stray
light. It is advisable to avoid using solutions whose total absorbance (buffer plus protein) at 280nm is > 1.5. It
should be stressed that setting the A 280 of the buffer blank to zero does not eliminate the concentration of the
buffer to the total A280 of the protein solution.

The simplest, most straightforward procedure for establishing the concentration of protein in solution is the
measurement of absorbance (A) at 280nm. Photons with higher energy have shorter wavelengths, hence,
electrons that are excited at 280nm (e.g. electrons that lie within aromatic rings) absorb less energy than those
at 200nm. This is due to the excited state of such electrons being stabilized by resonance with other electrons
within the aromatic ring. Amino acids containing aromatic side chains include tyrosine,phenylalanine,
tryptophan and histidine.
References

1. Alastair Aitken & Michele Learmonth , ‘Protein Determination by UV Absorption’ ,in John M. Walker
(ed) 1996, The protein protocols handbook, Humana Press, New Jersey, pp.3-7.
2. Walker,J, ‘Protein structure, purification, characterisation and function analysis’, in Keith Wilson & John
Walker (ed.) 1997, Principles and techniques of biochemistry and molecular biology,7th edn, Cambridge
University Press, pp. 300-51.
3. Frank R Milio & William M Loffredo, Quantitative testing for amino acids and protein,Chemical
Education Resources.Inc, Pennsylvania.