Practical 2 : Protein Experiment

Practical no. : 2

Experiment Title : Protein Experiment

Objectives :

1.      Learn the principles of protein assays.

2.      Determine protein concentrations using the Biuret Protein Assay.

3.      Determine protein concentrations using the Lowry Assay.

a)      Biuret assay

Abstract :

This experiment is conducted to learn the principles of protein assays. This exercise introduces students to method of determining protein concentrations. The determination of protein concentration is an essential technique in all aspects of protein studies and proteomics. This lab activity is designed to teach students the principles behind a common protein estimation assay known as the Biuret Protein Assay (absorbance at 540nm).

            Standard Biuret reagent is already prepared. The 0.50mL of protein is mixed with the prepared Biuret Reagent. The solution is mixed well and allows standing for 10 minute. The absorbance for each tube is read against the blank at 540 nm. The standard curve is plotted using concentration of standard (mg/mL) against the absorbance at 540 nm. The standard is used to determine the concentration of the protein in the five egg samples from regular chicken, quail, duck, and omega. To analyze the data, a line or curve is fit through the standards. For the sample, read across the graph from the spot on the Y-axis that corresponds to absorbance of the sample until the standard curve is intersected. Read down the graph until intersect the X-axis. The concentration of protein in the sample is the value on the X-axis.

           

Introduction :

The Biuret reaction can be used for both qualitative and quantitative analysis of protein. The biuret method depends on the presence of peptides bonds in proteins. When a solution of proteins is treated with cupric ions (Cu²⁺) in a moderately alkaline medium, a purple colored Cu²⁺- peptide complex is formed which can be measured quantitatively by spectrophotometer in the visible region. So, biuret reagent is alkaline copper sulfate solution.

The intensity of the color produced is proportional to the number of peptide bonds that are reacting, and therefore to the number of protein molecules present in the reaction system. The reaction do not occur with amino acids because the absence of peptide bonds, and also that with di-peptide because presence of only one peptide bond, but do with tri-, oligo-, and poly-peptides. Biuret reaction needs presence of at least two peptide bonds in a molecule.The reaction occurs with any compound containing at least two bonds of:

The reaction takes its name "Biuret Reaction" from the fact that biuret itself, obtained by heating urea, gives a similar colored complex with cupric ions.

Apparatus and Materials :

Protein samples

Standard solutions

Distilled water

Biuret reagent

Test tubes

Label

Test tube rack

Pipettes

Methods :

Result:

Calculation:

Protein’s content:

Ayam biasa                                         : 4.55mg/ml x 50x dilution = 227.5 mg/ml

Ayam kampung                                   : 4.70mg/ml x 50x dilution = 235.0 mg/ml

Ayam omega                                       : 3.40mg/ml x 50x dilution = 170.0 mg/ml

Puyuh                                                  : 5.40mg/ml x 50x dilution = 270.0 mg/ml

Itik                                                       : 4.79mg/ml x 50x dilution = 239.5 mg/ml

Discussions:

In this experiment, we have to investigate about the protein contents (concentration) in the five egg’s samples which are telur ayam biasa, ayam kampung, ayam omega, puyuh and itik. We used Biuret reagent test to test the content of protein in all those egg samples. The Biuret test is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper(II) ion forms violet-colour coordination complexes in an alkaline solution. The Biuret reaction can be used to assess the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the colour and hence the absorption at 540 nm is directly proportional to the protein concentration according to the Beer-Lambert law.

Before we tested the sample, we have plot the standard curve. After that, we have to dilute the egg samples 50x because if we do not dilute those sample, we cannot get the amount of the protein content. This is because the amount of protein have exceed the standard scale. From our results, we found out that quail egg have highest content of protein followed by duck egg, ayam kampung, ayam biasa and lastly ayam omega. The amount of protein contents in those eggs were 270.0 mg/ml, 239.5 mg/ml, 235.0 mg/ml,  227.5 mg/ml and 170.0 mg/ml.

As we have known before, omega egg should have higher content of nutrition. But it actually have higher amount of fatty acid. This is because sources of omega-3 fatty acids have been mixed in with the chicken feed but their protein contents was the lowest compared to the other eggs.

The new information about the omega-3 egg, egg whites contain more protein than egg yolks and they also have less fat. The egg white from one large omega-3-enriched egg contains 3.6 g of protein, while the egg yolk contains only 2.7 g. Most of an egg's fat is in the yolk. The yolk from one large omega-3-enriched egg contains 4.51 g of fat, including 1.6 g of saturated fat. The unsaturated fat in these yolks provides a good source of omega-3 fatty acids. The egg white, on the other hand, contains only 0.06 g of fat and no saturated fat. The yolk contains 210 mg of cholesterol, while the white contains no cholesterol.

References:

Biuret test. Retrieved on April 1, 2013 from http://en.wikipedia.org/wiki/Biuret_test

Lisa Porter (2011). Egg protein levels. Retrieved on April 1, 2013 from http://www.livestrong.com/article/470871-egg-protein-levels/#ixzz2PH9KhV7I

Cavemangreg (2011). All about eggs. Retrieved on April 1, 2013 from http://www.paleodietandliving.com/paleo-diet/all-about-eggs/

Title: Lowry Assay

Abstract : 

The determination of protein concentration is an essential technique in all aspects of protein studies and proteomics. This lab activity is designed to teach students the principles behind a common protein estimation assay known as Lowry Protein Assay. Although there are a wide variety of protein assays available, none of the assays can be used without first considering their suitability for the application.  Each method has its own advantages and limitations and often it is necessary to obtain more than one type of protein assay for research applications. Protein assays based on these methods are divided into two categories, dye binding protein assays and protein assays based on alkaline copper. Under alkaline conditions cupric ions (Cu²⁺) chelate with the peptide bonds resulting in reduction of cupric ions (Cu²⁺) to cuprous ions (Cu). The Cuprous ions can also be detected with Folin Ciocalteu Reagent (phosphomolybdic/phosphotungstic acid) and this method is commonly referred to as the Lowry method.  Cuprous ions (Cu⁺) reduction of Folin Ciocalteu Reagent produces a blue color that can be read at 750nm.  The amount of color produced is proportional to the amount of peptide bonds, i.e. size as well as the amount of protein/peptide. An experiment on Lowry Assay is conducted to learn the principles of protein assays and to determine the protein concentration using Lowry Protein Assay.

Introduction:

The “Lowry Assay: Protein by Folin Reaction” (Lowry et al., 1951) has been most widely used method to estimate the amount of proteins (which is already in solution or easily-soluble in dilute alkali) in biological samples. First the proteins are pre-treated with copper ion in alkali solution, and then the aromatic amino acids in the treated sample reduce the phosphomolybdatephosphotungstic acid present in the Folin Reagent. The end product of this reaction produced a blue color solution. The amount of proteins in the sample can be estimated via reading the absorbance at 750nm of the end product of the Folin reaction against a standard curve of a selected standard protein solution and sample protein solution. Lowry's assay is not without problems. In particular, it is sensitive to "interference" by many other compounds. Interference, the production of color by substances other that the analyte of interest, is a common problem with indirect colorimetric assays. In an attempt to overcome some of the problems of Lowry's method, many other assays for protein have been proposed. Two other colorimetric methods commonly used for the assay of protein are the "Bradford" assay and the "BCA assay". The Bradford" is based on a shift in the spectrum of a dye upon binding to proteins. The "BCA assay" is based upon the detection of Cu(I) (produced when proteins react with alkaline Cu(II)) using bicinchoninic acid (BCA).

Materials and apparatus:

Solutions of standard protein

Solutions of sample protein (telur ayam biasa, telur puyuh, telur itik, telur ayam kampung, telur ayam omega)

Lowry reagents 1 and 2

Distilled water

Test tubes

Test tube rack
Absorbance machine

Methods

Discussion

Lowry assay

The Lowry method is one of the most sensitive and widely used. The Lowry procedure can detect protein levels as low as 5 µg. There are several limitations to this method. Several contaminants interfere with the assay. With most protein assays, sample protein concentrations are determined by comparing their assay responses to that of a dilution-series of standards whose concentrations are known. Protein samples and standards are processed in the same manner by mixing them with assay reagent and using a spectrophotometer to measure the absorbance. The responses of the standards are used to plot or calculate a standard curve. Absorbance values of unknown samples are then interpolated onto the plot or formula for the standard curve to determine their concentrations.

Standard Curve

This comparative method for determining the concentration of an "unknown" is conceptually simple and straightforward. However, its implementation in an assay protocol is complicated by pipetting and dilution steps, evaluation of replicates, blank-corrections and other factors. These steps frequently cause confusion with regard to the calculations that are necessary to obtain a final determination.

The Lowry method relies on two different reactions. The first is the formation of a copper ion complex with amide bonds, forming reduced copper in alkaline solutions. This is called a "Biuret" chromophore. The second is the reduction of Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate) by tyrosine and tryptophan residues. The reduced Folin-Ciocalteu reagent is blue and thus detectable with a spectrophotometer in the range of 500-750 nm. The Biuret reaction itself is not all that sensitive. Using the Folin-Ciocalteu reagent to detect reduced copper makes the assay nearly 100 times more sensitive than the Biuret reaction alone.

The assay is relatively sensitive, but takes more time than other assays and is susceptible to many interfering compounds. The following substances are known to interfere with the Lowry assay: detergents, carbohydrates, glycerol, Tricine, EDTA, Tris, potassium compounds, sulfhydryl compounds, disulfide compounds, magnesium and calcium. Most of these interfering substances are commonly used in buffers for preparing proteins. This is one of the major limitations of the assay. The Lowry assay is sensitive to variations in the content of tyrosine and tryptophan residues. If the protein we are assaying has an unusual content of these residues, an appropriate substitute standard is required. The standard curve is linear in the 1 to 100 ug protein region. The absorbance can be read in the region of 500 to 750 nm. Most researchers use 660 nm, but other wavelengths also work and may reduce the effects of contamination (e.g. chlorophyll in plant samples interferes at 660 nm, but not at 750 nm).

The Lowry protein assay offered a significant improvement over previous protein assays. The Modified Lowry Protein Assay uses a stable reagent that replaces two unstable reagents described by Lowry. Essentially, the assay is an enhanced biuret assay involving copper chelation chemistry. Although the mechanism of color formation for the Lowry assay is similar to that of the BCA protein assay, there are several significant differences between the two. The exact mechanism of color formation in the Lowry assay remains poorly understood. The assay is performed in two distinct steps.

First, protein is reacted with alkaline cupric sulfate in the presence of tartrate for 10 minutes at room temperature. During this incubation, a tetradentate copper complex forms from four peptide bonds and one atom of copper (this is the "biuret reaction"). Second, a phosphomolybdic-phosphotungstic acid solution is added. This compound (called Folin-phenol reagent) becomes reduced, producing an intense blue color. It is believed that the color enhancement occurs when the tetradentate copper complex transfers electrons to the phosphomolybdic-phosphotungstic acid complex. The blue color continues to intensify during a 30 minute room temperature incubation. It has been suggested that during the 30 minute incubation, a rearrangement of the initial unstable blue complex leads to the stable final blue colored complex which has higher absorbance. The final blue color is optimally measured at 750nm, but it can be measured at any wavelength between 650nm and 750nm with little loss of color intensity. It is best to measure the color at 750nm since few other substances absorb light at that wavelength.

For small peptides, the amount of color increases with the size of the peptide. The presence of any of five amino acid residues (tyrosine, tryptophan, cysteine, histidine and asparagine) in the peptide or protein backbone further enhances the amount of color produced because they contribute additional reducing equivalents to further reduce the phosphomolybdic/phosphotungstic acid complex. With the exception of tyrosine and tryptophan, free amino acids will not produce a colored product with the Lowry reagent, however, most dipeptides can be detected. In the absence of any of the five amino acids listed above in the peptide backbone, proteins containing proline residues have a lower color response with the Lowry reagent due to the amino acid interfering with complex formation.

There are several ways to measure protein concentration, and each of them has its own advantages and disadvantages. The three different methods for measuring protein concentration are absorbance at 280 nm, the Bradford assay, and the BCA assay.

Absorbance at 280 nm

How it works:

Aromatic residues, like tyrosine and tryptophan, absorb UV light at 280 nm.  So, if we have an extinction coefficient for the  protein , we can measure the absorbance in a UV/Vis spectrometer and calculate the concentration of your protein using Beer’s law (A = elc, where l is the path length of the spectrometer).  We can estimate the extinction coefficient of the protein based on the sequence using Expasy’s ProtParam tool.  Because ProtParam only considers the linear sequence of the protein and doesn’t take into account the structure, which can affect the extinction coefficient, we’ll want to denature the protein before we measure the absorbance.

Advantages:

This technique is quick and doesn’t require any special reagents, except for the guanidinium, which you may have on hand anyway.

Disadvantages:

This method relies on having an accurate extinction coefficient for the protein, which depends on the number of aromatic residues.  If there aren’t a decent number of aromatic residues, our extinction coefficient will be quite low, and we will need a fairly concentrated sample to get a reasonable absorbance (generally an absorbance between 0.1 and 1.0 is considered within the “linear range”).  Also, ProtParam warns that there may be at least a 10% error in the extinction coefficient if there are no tryptophans in your protein.  Therefore, if the extinction coefficient is low, which is likely the case if there are no tryptophans in the sequence, a 10% error could significantly throw off the assessment of the final protein concentration.

Bradford Assay

How it works:

The Bradford assay is a colorimetric assay based on the interaction between Coomassie brilliant blue and the arginine and aromatic residues in the protein.  When the dye binds to these residues, its maximum absorption shifts from 470 nm to 595 nm.  In general, we measure the absorbance of a series of known concentrations of a standard protein, generally BSA, and create a standard curve.  We then use that standard curve to calculate the concentration of your protein sample based on its absorbance.

Advantages:

 This assay is quick, and the reagent is not affected by the presence of reducing agents.

Disadvantages: 

Basic conditions and detergents, such as SDS, can interfere with the dye’s ability to bind to the protein; however, there are detergent-compatible Bradford reagents. Also, like the absorbance at 280 nm technique, the Bradford assay depends on the sequence of the protein.  If the protein doesn’t contain a decent number of arginine and/or aromatic residues, then the dye will not bind to the protein as efficiently, resulting in an underestimation of the protein concentration.

BCA Assay

How it works:

The BCA assay is another colorimetric assay like the Bradford assay.  It makes use of the biuret reaction, in which the protein backbone chelates Cu2+ ions and reduces them to Cu1+ ions.  The Cu1+ ions then react with bicinchoninic acid (BCA) to form a purple-colored product that absorbs at 562 nm.  The procedure is similar to that of the Bradford assay, in which we create a standard curve based on a series of known protein standards.

Advantages:

Because the peptide backbone is involved in the reaction, the BCA assay is less sensitive to the types of amino acids in the protein.  However, the reaction is influenced by cysteine, tyrosine, and tryptophan residues. The reagent is not sensitive to detergents and denaturants, so it’s okay to have those in buffer.

Disadvantages: 

The presence of reducing agents in your buffer can interfere with the dye, but there are reducing agent-compatible dyes available. The reaction takes some time to proceed.  Usually, the samples are incubated at 37°C for 15-30 min.  Also, as in the Bradford assay, we determine the protein concentration by creating a standard curve from a known, standard protein.  So again, if the protein doesn’t interact with the dye in a similar way as the standard protein, the concentration could be off.

Spectrophotometer calibration is a process in which a scientific instrument known as a spectrophotometer is calibrated to confirm that it is working properly. This is important, as it ensures that the measurements obtained with the instrument are accurate. A spectrophotometer is capable of both transmitting and receiving light. The device is used to analyze samples of test material by passing light through the sample and reading the intensity of the wavelengths. Different samples impact the light in different ways. Spectrophotometer calibration is necessary to confirm that the results are accurate. In spectrophotometer calibration, a reference solution is used to zero out the equipment. Blank solution provides a base or zero reading. The device is calibrated by placing the reference solution inside the spectrophotometer, zeroing out the settings, and running the instrument. Then, samples of an actual test material can be subjected to spectrophotometry in confidence that the machine has been calibrated and is working properly.

References