Determine the concentration of an unknown protein solution using two different spectroscopic methods (the biuret method and UV method) for the measurement of protein concentration.

Instructions for the experiment

Protein determination using spectroscopy
Ensure you have completed the pre-lab associated with this practical before you arrive at the lab.

Practical Session Aims and Objectives

In this practical, you will determine the concentration of an unknown protein solution using two different spectroscopic methods (the biuret method and UV method) for the measurement of protein concentration. The two methods will be compared based on:

• Accuracy – the closeness of the results obtained to the actual value.
• Precision – which method shows the least variation in replicate measurements?
• Sensitivity – which method can reliably detect the smallest amount of protein?
• Specificity – how does each method respond to the presence of other substances i.e. those which are not protein?

Measuring protein concentration using spectroscopy

There are many methods available for determining protein concentration. Most of these methods rely either on colorimetric assays, e.g. biuret method or the use of UV absorbance method. The choice of method depends upon a variety of factors, including the amount of protein present, the specificity of the method, the presence of interfering substances, and the amino acid composition of the protein.

Biuret method
The biuret reaction has been in use since the end of the 19th century and is the method of choice in clinical laboratories because of its simplicity, rapidity and reliability.
The peptide bonds of protein react with the copper II ions in alkaline solution to form a blue-violet complex (the so-called biuret reaction), each copper ion complexing with 5 or 6 peptide bonds. The colour formed is proportional to the protein concentration and is measured at 520-560 nm.

Taken from: https://www.piercenet.com/method/chemistry-protein-assays

UV method
UV absorbance spectroscopy is based on the fact that aromatic amino acids such as tryptophan and tyrosine absorb ultraviolet light strongly at 280 nm. The tryptophan and tyrosine content of many proteins remains fairly constant, and so the absorbance of protein solutions at 280 nm can be used to determine their concentration. The advantages of this method are that the procedure is simple to carry out, and the protein can be used directly, without the addition of reagents, and is not modified or inactivated by the process (as in the colorimetric assay).

As introduced in Intro 2 practical Spectroscopy can be used to determine the concentration of a substance (in this case protein) as, amount of radiation absorbed, i.e. the height of the peaks measured as ABSORBANCE, gives quantitative information that can be used to determine the amount of substance present.

Beer-Lambert Law
For each wavelength of light passing through the spectrometer, the intensity of the light passing through the reference cell (i.e. cuvette) is measured. This is usually referred to as Io (incident light). The length of this reference cell (cuvette) is also important and is represented as l.

The intensity of the light that passes through the sample cell is also measured for that wavelength It, (also known as the transmitted light). If It is less than Io, then the sample has absorbed some of the light.

Water does not absorb much light (apart from weak absorbance in the red part of the visible spectra) and so all the light is transmitted through the sample. Therefore if you have 100 % transmittance of light your absorbance value is zero.

If a sample absorbs visible light, the amount of absorbing particles (e.g. moles of the substance) is related to the absorbance, A.
The number of moles of a substance in solution is measured by Molar concentration, c, therefore:
A = c

The more substances present the greater the absorbance value. This relationship is known as Beer-Lambert’s law.
Using the relationship between absorbance and concentration Beer-Lambert’s law is represented by the following equation:
A =  c l
or
A =  c b

• A is the absorbance (a dimensionless quantity)
• is a constant, molar absorbtivity or molar absorbance coefficient (units: L mol-1cm-1) and the slope of a Beer-Lambert’s law plot
• c is the molar concentration (units: mol/L)
• l or b is the light path in centimetres (units: cm)

The linearity of the Beer-Lambert law can be limited by factors such as:
• 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.
Preparation of a standard curve
A standard curve (also known as a calibration graph), can be used to determine the concentration of an unknown sample. A series of samples with known concentrations are prepared, these are known as standards. By measuring the absorbance of each standard, the absorbance values against each concentration can be drawn on a graph to generate the standard curve.

Usually the concentration is expressed in molar terms (i.e. mol/L). Sometimes other units may be used, for example, concentrations of proteins are usually measured as g/L or as mg/L.

The concentration of the unknown sample can either be extrapolated from the graph (primarily used if your graphs are hand drawn) or determined mathematically from the equation for the line (if your graphs have been prepared using Excel).

Protein concentration is proportional to absorbance

In order to assure that a test run is valid and results are reliable, Quality Control Samples (QC) should be used in the performance of each assay.
The Quality Control Samples should be treated in the exact same manner as the test samples and are used to validate the test run. The assumption is that if the method performs (either during the validation or routine analysis) in an acceptable manner as measured by the QC samples, then the results obtained for the study samples are valid and can be reported with confidence.
Experimental work

In this experiment two methods are used to determine the concentration of protein in a solution. The Biuret method is based on the formation of a coloured complex, whereas the UV method relies on the absorption of light in the UV part of the spectrum by particular amino acids in the protein’s structure.

Practical hypothesis:
Absorbance is directly proportional to protein concentration

Practical objectives:
• prepare a stock solution of Bovine serum albumin (20g/L),
• prepare a series of standards of known concentration,
• use the biuret method of protein determination to prepare a standard curve and determine the concentration of an unknown amount of protein,
• use the UV method of protein determination to prepare a standard curve and determine the concentration of an unknown amount of protein,
• use the QC samples to ensure that your methods are accurate.
Reagents
Reagent Hazard
Biuret reagent
Bovine serum albumin powder No hazard
Unknown sample of albumin, X No hazard
QC low sample of albumin (0.5 g/L) No hazard
QC sample of albumin (4 g/L) No hazard
QC high sample of albumin (30 g/L) No hazard

Additional Material
Absorbance spectra in the ultraviolet and visible range of radiation (200 – 600 nm) for:
• protein (albumin)
• protein (albumin)/copper complex (Biuret method).
Preparation of the stock albumin solution and the standards of known concentration.

• Prepare 30 mL of a 20 g/L stock solution of Albumin. How much albumin will you weigh out to make this solution?
Please record this amount in your lab book

**Bovine serum albumin should be stored on ice at all times. Please return the bottle to the ice bowl provided after use**

• Calculate the final protein concentration in each of the standards to complete the table. Please make a record of this table in your lab book.

Volume (mL)
Stock solution (20 g/ L) 5.0 4.0 3.0 2.0 1.0 0
Distilled water 0 1.0 2.0 3.0 4.0 5.0
Final protein concentration (g/L) 20 BLANK
0

• Using your stock solution (20 g/L albumin) prepare the series of standards in 6 test tubes using the volumes indicated in the table above.

These standards will be used for both the UV and visible methods of protein determination and should not be discarded.

Determining protein concentration using the biuret method
• Pipette 0.5 mL of each standard (0-20 g/L) into clean, labelled test tubes.
• Pipette 0.5 mL of sample X into 5 clean and labelled test tubes.
• Pipette 0.5 mL of each QC sample into 5 clean and labelled test tubes.
• Pipette 4.5 mL of Biuret reagent to ALL tubes (the standards, the sample X replicates and the QC sample replicates).
**which pipette will you use to measure this volume and why?**
• Mix each tube well.
• Incubate the tubes at room temperature for 10 minutes for maximum colour to develop.
• While waiting for the colour to develop, examine the absorbance spectrum of the protein/copper (Biuret) complex provided.
• Determine the wavelength of the absorbance peak ( max) in the visible region of the spectrum and set the spectrophotometer to this wavelength.
The Absorbance maxima for the biuret complex is…………………………….
• Set the spectrophotometer to zero using the BLANK sample.
• Do not fill the cuvette more than 2/3 full and wipe the outside of the cuvette dry with tissue.
• Measure the absorbance of the Biuret/protein complexes of each of the standard solutions of protein, the five replicates of X and the QC samples.
• Record the results for the five dilutions in your lab book. Get your results checked by the lab supervisor.
• Analyse the variability of the class results by writing the absorbance value of your standard dilutions (0 – 20 g/L) on the white board.
• Draw a calibration graph of absorbance (vertical axis) against concentration (horizontal axis). The graph should go through zero for both axes.
• Read off protein concentration for each of the five replicate analyses of sample X. Record these values in your lab book.

Determining protein concentration using the UV method
• Prepare 1 in 10 dilutions of each standard (0-20 g/L). You do not need to dilute the blank.
• Prepare 5, 1 in 10 dilutions of sample X.
• Prepare 5, 1 in 10 dilutions of each QC sample.
• Mix each tube well.
** You will need to use quartz cuvettes to measure absorbances in the UV range, these cuvettes are very expensive so please do not throw them away**
• Determine the wavelength of the absorbance peak ( max) of albumin in the UV region of the spectrum and set the spectrophotometer to this wavelength.
The Absorbance maxima for the albumin is…………………………….
• Set the spectrophotometer to zero using distilled water in a quartz cuvette.
• Do not fill the cuvette more than 2/3 full and wipe the outside of the cuvette dry with tissue.
• Measure the absorbance of the Biuret/protein complexes of each of the standard solutions of protein.
** how many times should you read the absorbance of these standards?**
• Measure the five replicates of X and the QC samples.
• Analyse the variability of the class results by writing the absorbance value of your standard dilutions (0 – 20 g/L) on the white board.
• Draw a calibration graph using the data from the diluted standards and determine the protein concentration in the five replicate dilutions of sample X from the graph.

Analysis of data
From the concentration values determined (not the absorbance values), calculate the mean, Standard deviation (SD) and co-efficient of variation (CV) for the 5 sample X values and the values obtained for the QC samples. The formulae for working out the SD and CV values are in the Glossary of terms at the front of this lab manual.

Why did you do 5 replicates for each sample?
…………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………….

Which method is more accurate? Why?
……………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………..

Which method is more precise? Why?
……………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………..

Which method is more sensitive?
…………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………….

Investigation of the specificity of each method

Additional Reagents
Reagent Hazard
Solution of the amino acid tyrosine No hazard
Solution of the amino acid alanine No hazard

Additional Material
Absorbance spectra in the ultraviolet and visible range of radiation (200 – 600 nm) for: – alanine
– tyrosine

An ideal analytical method is one which will detect only the test substance, in this case, protein. Such a method is said to have high specificity for that substance. However, very frequently some substances other than the one under investigation will be detected, and are said to “interfere” with the analysis. This will give inaccurate results. There are often many contaminating substances in biological samples, two examples of which are the amino acids alanine and tyrosine.

• As a class devise a simple experiment to assess whether these amino acids interfere with either of the two methods for the determination of protein.
• Write down your hypothesis:

• When designing your experiment to test your hypothesis, remember to think about suitable controls.
• Ensure that you test it using both methods of protein determination

• Which method is more specific? Why?
……………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………..

At the end of the lab class
When you have completed all the sections tidy your bench and leave the area in a safe condition.
• Non-hazardous used liquids should be washed down the sink.
• Used glassware should be emptied, rinsed and placed in the red trolley for washing.
• Used plastic ware (e.g. tips, graduated pipettes etc.) should be placed in the waste bin on your bench or in the yellow incineration bin.
• Have you completed your lab skills passport?
• Have you recorded all your results in your lab book?

Complete the data and question sheet associated with this lab.

Protein determination using spectroscopy
RESULTS

a) Preparation of protein standards
Volume (mL)
Stock albumin solution (20 g/L 5.0 4.0 3.0 2.0 1.0 0
Distilled water 0 1.0 2.0 3.0 4.0 5.0
Final protein concentration (g/L) 20 16 12 8 4 Black
0

a) Protein determination by visible spectroscopy- The Biuret Reaction
Using the data generated in the table’s pages, plot a calibration graph using the graph paper provided and this to work out the concentration for each of your repeat analyses of the unknown protein and QC samples. Record the concentrations, mean, standard deviation and coefficient of variation in following tables. Remember to add the appropriate units to table headings.

Record the wavelength of analysis
Biuret analysis of standards
Standard number Protein concentration
(g/L) absorbance
1 2 0.568
2 1.6 0.450
3 1.2 0.354
4 0.8 0.239
5 0.9 0.148
6 0 0

Biuret analysis of unknown protein sample
Repeat absorbance concentration
1 0.284
2 0.172
3 0.374
4 0.15
5 0.224

Mean concetration
Standard deviation
Coefficient of avariation

Biuret analysis of QC sample (4g/L)
Repeat absorbance Concentration ( )
1 0.167
2 0.171
3 0.161
4 0.172
5 0.169
Mean concentration
Standard deviation
Coefficient of variation

Biuret analysis of low QC protein sample (0.5 g/L)
Repeat Absorbance Concentration ( )
1 0.085
2 0.112
3 0.088
4 0.135
5 0.143
Man concentration
Standard deviation
Coefficient of variation

Biuret analysis of high QC protein sample (15 g/L)
Repeat Absorbance Concentration ( )
1 0.473
2 0.579
3 0.905
4 0.680
5 0.616
Man concentration
Standard deviation
Coefficient of variation

Why did you do 5 replicates for each sample?
……………………………………………
>>>>>>>>>>>>>>>>>>>
What conclusions can you draw about the accuracy and precision of the data you have collected from biuret experiment?
……………………………………
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,

b) UV absorbance method of protein determination
Plot a calibration graph using paper provided and use this to work out the concentration for each of your repeat analyses of the unknown protein and QC samples. Record the concentrations, mean, standard deviation and confident of variation in the table above. Remember to add the appropriate units to the table headings.
Record the wavelength of analysis:

UV analysis of standards
Standard number Protein concentration
(g/L) absorbance
1 2 0.609
2 1.6 0.512
3 1.2 0.358
4 0.8 0.402
5 0.4 0.438
6 0 0

UV analysis of unknown protein sample

Standard number Absorbance concentration
1 0.040 0
2 0.070
3 0.048
4 0.068
5 0.077
Mean concentration
Standard deviation
Coefficient of variation

UV analysis of QC protein sample (4g/L)

Standard number Absorbance concentration
1 0.046
2 0.075
3 0.106
4 0.084
5 0.060
Mean concentration
Standard deviation
Coefficient of variation

UV analysis of low QC protein sample (0.5 g/L)

Standard number Absorbance concentration
1 0.029
2 0.029
3 0.018
4 0.020
5 0.014
Mean concentration
Standard deviation
Coefficient of variation

What conclusions can you draw about the accuracy and precision of the data you have collected from the UV absorbance method?
……………………………………..
<<<<<<<<<<<<<<<<<<<<<<<,,
Use the data from the low QC sample and comment on the sensitivity of the two methods:
…………………………………………..
<<<<<<<<<<<<<<<<<<<<<<<<,

c) Investigation the specificity of each method
Briefly describe the experiment you have devised to test the specificity of the two methods:
…………………………………………..
<<<<<<<<<<<<<<<<<<<<<<<<<<<<,,,,,

Record the data you have collected from your investigation belo:

Biuret assay
water alanine tyrocine
1 0.116 0.105 0.129
2 0.102 0.093 0.130
3 0.124 0.096 0.124

UV assay
water alanine tyrocine
1 0.100 0.122 2.8600
2 0.121 0.135 2.659
3 0.1 0.4 2.541

Briefly explain the findings of your experiments
………………………………………………………..
/////////////////////////
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,…………..

Question sheet

a) Preparation of Protein Standards
Volume (mL)
Stock albumin solution (20 g/ L) 5.0 4.0 3.0 2.0 1.0 0
Distilled water 0 1.0 2.0 3.0 4.0 5.0
Final protein concentration (g/L) 20 BLANK
0

b) Protein determination by visible spectroscopy – The Biuret Reaction
Using the data generated in the tables on the following pages, plot a calibration graph using the graph paper provided and use this to work out the concentration for each of your repeat analyses of the unknown protein and QC samples. Record the concentrations, mean, standard deviation and coefficient of variation in the following tables. Remember to add the appropriate units to the table headings.

Record the wavelength of analysis:

Biuret Analysis of Standards
Standard number Protein concentration
( ) Absorbance
1
2
3
4
5
6

Biuret Analysis of Unknown Protein Sample

Repeat Absorbance Concentration
( )
1
2
3
4
5
Mean Concentration
Std dev
Coefficient of Variation

Biuret Analysis of QC Protein Sample (4g/L)

Repeat Absorbance Concentration
( )
1
2
3
4
5
Mean Concentration
Std dev
Coefficient of Variation

Biuret Analysis of low QC Protein Sample (0.5 g/L)
Repeat Absorbance Concentration
( )
1
2
3
4
5
Mean Concentration
Std dev
Coefficient of Variation

Biuret Analysis of high QC Protein Sample (30 g/L)

Repeat Absorbance Concentration
( )
1
2
3
4
5
Mean Concentration
Std dev
Coefficient of Variation

Why did you do 5 replicates for each sample?
……………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………
What conclusions can you draw about the accuracy and precision of the data you have collected from the Biuret experiment?
………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………
c) UV Absorbance method of protein determination
Plot a calibration graph using the graph paper provided and use this to work out the concentration for each of your repeat analyses of the unknown protein and QC samples. Record the concentrations, mean, standard deviation and coefficient of variation in the table above. Remember to add the appropriate units to the table headings.
Record the wavelength of analysis:

UV Analysis of Standards
Standard number Protein concentration
( ) Absorbance
1
2
3
4
5
6

UV Analysis of Unknown Protein Sample

Repeat Absorbance Concentration
( )
1
2
3
4
5
Mean Concentration
Std dev
Coefficient of Variation

UV Analysis of QC Protein Sample (4g/L)

Repeat Absorbance Concentration
( )
1
2
3
4
5
Mean Concentration
Std dev
Coefficient of Variation

UV Analysis of low QC Protein Sample (0.5 g/L)
Repeat Absorbance Concentration
( )
1
2
3
4
5
Mean Concentration
Std dev
Coefficient of Variation

What conclusions can you draw about the accuracy and precision of the data you have collected from the UV absorbance method?
……………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………
Using the data for the low QC sample comment on the sensitivity of the two methods:
……………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………

d) Investigation of the specificity of each method
Briefly describe the experiment you have devised to test the specificity of the two methods.

Record the data you collected from your investigation below:

Briefly explain the findings of your experiments.
…………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………

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