REPORT

‘Spectrophotometric determination of protein’

Abstract:
Measuring the concentration of proteins is an essential part of enzyme evaluations or to monitor protein
yields during protein isolation procedures. Decisions on the usefulness of any isolation procedure depend
on knowing the relative concentrations of a particular protein or enzyme in relation to the concentrations
of all the proteins present. Protein concentration in solution is generally measured with
spectrophotometry in the UV range or in the presence of dyes or copper interacting with the protein.

Objective:
1. Introduction:
1.1. protein quantitation:
1.2 spectrophotometry:
2. Main body:
2.1 Protein Applications of Spectrophotometry:
2.2 Understanding the Beer-Lambert Law:
2.3 Detecting Proteins with Absorbance at 280 nm:
2.4 Materials:
2.5 methodology.
1. Introduction:
1.1. protein quantitation:
During protein isolation procedures from plant, animal, insect, yeast, or bacterial material it is important
to determine the concentration of protein at each step in the purify cation procedure. Measuring protein
concentration is also essential for enzyme activity assays. Accurate protein quantitation is essential to all
experiments related to proteins in a multitude of research topics. Different methods have been developed
to quantitate proteins in a given assay for total protein content and for a single protein. Total protein
quantitation methods comprise traditional methods such as the measurement of UV absorbance at 280
nm.

1.2 spectrophotometry:
is a means for determining the concentration of a substance in solution. A dissolved substance will absorb
light of specific wavelengths characteristic of that substance. When light of those wavelengths is passed
through the sample, some of the light is absorbed by the solute, decreasing the amount of light that
passes through the sample. A spectrophotometer is used to measure the difference in the amount of light
entering and leaving the sample. The light that passes through the sample (not absorbed) is called
transmitted light. This difference in the original and transmitted light is called the absorbance.
Absorption spectrophotometry can be used to study the properties of many types of biological molecules,
such as pigments, enzymes, DNA, and many small organic molecules. This methodology can also be used
to measure biological activities of living cells, such as enzyme activities and rates of photosynthesis.
Basically, spectrophotometry is one of the most widely used analytical procedures in biochemistry. It is
commonly used to estimate the level of an analyte in solution and is ideal for simple routine
determination of small quantities of materials. This method is based on the two laws of light absorption
by solutions, namely Lambert's Law and Beer's Law.

2. Main body:
2.1 Protein Applications of Spectrophotometry:
Since proteins absorb light at a specific wavelength, a spectrophotometer can be used to directly measure
the concentration of a purified protein in solution. It is important to note that direct UV measurement at
280 nm yields highly reproducible measurements since no reagents are added to the protein solution and
the protein of interest was not modified or inactivated during the process. It also produces quick results
since the sample does not need to be incubated in order to complete the process. The chemical
composition of the protein (i.e. the number and type of amino acids present) will affect its absorption.
Since a sample protein's absorption at 280 nm will depend on the amount of the amino acids’ tyrosine
and tryptophan, it is very much possible that proteins of similar molecular weight will have different
absorbance values due to their different tyrosine and tryptophan content. In addition, the structure of
protein of interest may also affect the UV absorbance of aromatic side chains. As such, the temperature,
pH, ionic strength, and/or the presence of detergents can affect the ability of aromatic residues to absorb
light at 280 nm, and change the value of the protein’s extinction coefficient. These methods are most
appropriate for purified proteins. Some food components may interfere (or absorb) at these
wavelengths.
2.2 Understanding the Beer-Lambert Law:
Lambert's Law states that "the proportion of light absorbed by a medium is independent of the intensity
of incident light" while Beer's Law maintains that "the absorbance of light is directly proportional to the
concentration of the absorbing medium and the thickness or path length of the medium".
Lambert's Law is expressed as:
Transmittance (T) = P/P0
P= radiant power of beam Exiting the absorption cell.
P0= radiant power of beam exiting the absorption cell.
where transmittance is the ratio of the amount of light transmitted to the amount of light that initially fell
on the surface.
On the other hand, Beer's Law is expressed as:
Absorbance (A) = Extinction coefficient or Molar absorptivity (e) x Concentration (C) x Path length (b) 
where absorbance is the negative logarithm of transmittance. 
Taken together, these laws allow us to use different spectrophotometers with different light sources and
still produce comparable absorption readings. In addition, these laws also give us the ability to measure
samples of different path length and compare the results directly with each other.

2.3 Detecting Proteins with Absorbance at 280 nm.

Proteins comprising aromatic rings in their primary sequence absorb light at 280 nm. The absorbance at
280 nm is primarily due to the presence of the amino acids’ tryptophan (max 279.8 nm) and tyrosine (max
274.6 nm). The absorbance measured is directly proportional to the concentration of the protein solution
and the pathlength, i.e., follows the Beer-Lambert law. Since the method is based on absorbance by
tryptophan and tyrosine residues in a protein, the absorbance values will differ in relation to the number
of tryptophan and tyrosine residues present. The method can be used to detect protein in the 20–3,000
μg range. The method is particularly useful for the detection of proteins eluting from gel fi literation, ion-
exchange, affinity, hydrophobic, and chromatofocusing chromatography columns as there is no loss of
protein. Nucleic acids are often present in protein solutions and contribute to absorbance values at 280
nm. A compensation for the presence of nucleic acids should be made.

the protein sample must not contain any non-protein components with substantial absorption at 280 nm,
such as nucleic acids contaminants. For pure protein samples, the exact amino acid sequence of the
analyzed protein must be known and an absorption coefficient specific to the particular protein must be
calculated prior to determining the concentration of solution. This method is quickest, but error-prone
and is incompatible with a broad array of protein extraction methods which frequently employ detergents
and denaturing agents.

2.4 Materials.
1. One milliliter Quartz cuvettes are recommended. Some plastic cuvettes are now available where the
plastic does not absorb light at 280 nm.
2. A UV spectrophotometer.
2.5 methodology.
1. For calibrating with standards, use the 3 mg/ml standard protein solution to prepare dilutions of 20,
50, 100, 250, 500, 1000, 2000, and 3000 µg/ml in the same solvent as used to prepare the sample
protein. Prepare a blank consisting of solvent alone.

2. Turn on the UV lamp of the spectrophotometer and set the wavelength to 280 nm. Allow the
instrument to warm up 30 min before taking measurements.
3. Zero the spectrophotometer with the solvent blank.
4. Measure the absorbance of the protein standard and unknown solutions.
5a. If the a280 (absorptivity) of the protein is known: Calculate the unknown sample concentration from
its absorbance value using following Equation.
In this equation a280 has units of ml/mg cm and b is the path length in cm.

A
concentration=
a∗b

5b. I f standard solutions are used for quantitation: Create a calibration curve by either plotting or
performing regression analysis of the A280 versus concentration of the standards. Use the absorbance
of the sample protein to determine the concentration from the calibration curve.

3. CONCLUSION:
spectrophotometric methods measuring the concentration of a sample protein in solution. These
methods are most appropriate for purified proteins. Some food components may interfere (or absorb)
at these wavelengths. Determination of protein concentration by measuring absorbance at 280 nm (is
based on the absorbance of UV light by the aromatic amino acids tryptophan and tyrosine, and by
cystine, disulfide bonded cysteine residues, in protein solutions. The measured absorbance of a protein
sample solution is used to calculate the concentration either from its published absorptivity at 280 nm
or by comparison with a calibration curve prepared from measurements with standard protein
solutions. This assay can be used to quantitate solutions with protein concentrations of 20 to 3000
µg/ml.

4. references
1.HANDBOOOKF FOODA NALYTICAL CHEMISTRY Edited by Ronald E. Wrolstad.
2.Western Blotting Methods and Protocols Edited by Biji T. Kurien.
3. Spectrophotometry and Its Application in Protein Estimation. Available at:
https://info.gbiosciences.com/