Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Cut Parafilm into 2-cm squares.
2. Prepare 250 mL of a 10 mg/mL albumin stock solution.
   1. Using a graduated cylinder, add 250.0 mL of distilled water to the albumin bottle.
   2. Cap and shake gently to dissolve. More extreme shaking or agitation of the albumin solution may cause the proteins to denature and precipitate out of solution.
   3. The albumin solution may be prepared up to one week before use if it is stored in a refrigerator.
   4. For easier dispensing during the student laboratory, premeasure 20 mL of the albumin solution for each student group.
3. Prepare two unknown samples for each student group using the table below. Each group will need 5 mL of unknown prepared for them.
   {10977_Preparation_Table_2}

Safety Precautions

Biuret quantitative assay solution contains copper sulfate in a sodium hydroxide solution. It is corrosive to all body tissue, especially eyes. It is also moderately toxic by ingestion. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Excess Biuret quantitative assay solution and the reacted protein solution may be neutralized using dilute hydrochloric acid solution and flushed down the drain with excess water according to Flinn Suggested Disposal Method #10. Excess albumin may be disposed of according to Flinn Suggested Disposal Method #26b.

Lab Hints

• Enough materials are provided in this kit for 30 students working in groups of three or for 10 groups of students. This laboratory activity can reasonably be completed in one 50-minute class period if the prelaboratory assignment is completed before coming to lab, and the data compilation and calculations are completed the day after the lab.
• Biuret solution can also be used as a simple qualitative test for proteins. A pinkish or purplish-violet color indicates the presence of proteins with at least two peptide linkages. Proteases and peptones give a pink color; gelatin turns a bluish color.
• The name of the biuret test is frequently confusing to students and teachers alike. The test solution consists of copper sulfate in basic solution. The name of the test actually derives from the name of the reagent chemical, called biuret that gives a characteristic positive control test with the test solution. Biuret is a derivative of urea that contains two amide (–CONH₂) groups in its structure and thus forms a purple coordination complex with copper ions. The biuret test solution itself does not contain any biuret!
• Extend the activity by having students determine the optimal wavelength for analysis using the spectrophotometer.
• If no spectrophotometer is available, create a series of samples with known concentrations against which students can do a visual color comparison. Have students create mid-point knowns as necessary to determine the concentration of the unknown.

Answers to Prelab Questions

1. Why is it important that a best fit trend line be drawn through 0,0 on the graph?

   The spectrophotometer is blanked using all of the same solutions except the protein. Therefore the absorbance reading for zero protein is zero absorbance. The trend line should take this data point into account when it is drawn.

2. Why is it important that the reaction between the proteins and the test solution produce a colored product and not a colorless product?

   The spectrophotometer measures the amount of light absorbed by a solution at a specific wavelength. A colorless product does not absorb light within the visible spectrum.

Sample Data

Answers to Questions

1. Plot the absorbance versus concentration and draw a best fit line graph that includes 0,0.
   {10977_Answers_Figure_3}
2. What is the protein concentration of the unknown solution in cuvet 6?

   In our example the calculated concentration is 2.5 mg/mL.

3. What is the protein concentration of the unknown solution in cuvet 7?

   In our example the calculated concentration is 4.5 mg/mL.

4. Kidney damage leads to protein in the urine. The amount of protein in the urine is directly related to the severity of kidney damage. Using the information and procedure above, design a lab procedure for determining the amount of protein in a urine sample. Keep in mind that the maximum absorbance may not be 540 nm due to interference created by the other components in a sample of urine.

   The blank must include a non-protein-containing urine component to ensure the urine itself does not add to the absorbance. The procedure must also include a scan of all possible wavelengths to find the maximum absorbance before a calibration curve and analysis can be executed. All standards must be made using a non-protein-containing urine sample. In all other ways the actual procedure should be similar to the one outlined in this lab.

References

Morholt, E.; Brandwein, P. F. A Sourcebook for the Biological Sciences, 3rd ed.; Harcourt Brace Jovanovich: Fort Worth, TX, 1986; p 179.

Introduction

The amount of protein present in a solution can be determined using a colorimetric indicator and a spectrophotometer.

Concepts

• Proteins
• Peptide bonds
• Spectrophotometric analysis

Background

Proteins represent the most diverse class of biological compounds within cells. It is estimated that a single bacteria cell contains more than 3,000 different types of proteins. Proteins are of primary importance in terms of both their occurrence within cells and their function in cell activities. The functions of proteins are at the very center of life itself—proteins catalyze all of our metabolic reactions, carry oxygen to our body tissues, protect the body from infection, and maintain cell and tissue structure.

Proteins are composed of amino acid molecules joined together in a chain-like fashion via peptide linkages. Amino acids are thus often referred to as the “building blocks” of protein structure. The size of the amino acid chain in a single protein molecule can vary from around 50 amino acid residues in insulin to more than 500 in hemoglobin and more than 5,000 in some viruses.

All amino acids have two structural features in common—they contain a carboxylic acid group (–COOH) on one end and an amine group (–NH₂) on the other end. Peptide linkages are created when the carboxyl group of one amino acid reacts with the amino group of the next amino acid in the sequence. As each amino acid is added to the growing polypeptide chain, a molecule of water is formed as a byproduct, as shown in Figure 1.

All proteins are derived from about 20 different, naturally occurring amino acids, which can be arranged in an almost infinite number of ways, giving rise to the thousands of unique proteins found in nature. The primary structure of a protein is determined by the number and identity of amino acids within the protein and the order in which they are joined together via peptide linkages. Higher levels of protein structure (called secondary, tertiary, and quaternary structure) result as the polypeptide chains form ribbons, sheets and coils that ultimately fold in on themselves to form more compact and more stable three-dimensional arrangements.

Proteins can be identified using a simple color test based on the reaction of their polypeptide backbones with copper ions in basic solution. When molecules containing two or more peptide linkages react with copper sulfate in the presence of a strong base, a purple complex is formed. This is called the biuret test. The colored product is the result of coordination of peptide nitrogen atoms with copper ions. The amount of product that is formed and thus the intensity of the purple color depend on the nature of the protein and on how much protein is present.

The light-absorbing ability of a material is called the absorbance. Since more protein equates to a deeper color, the more protein present in the sample, the greater the amount of light absorbed by the sample. The absorbance of a colored solution is directly proportional to the concentration of the substance responsible for the color (see Equation 1). Spectrophotometers and colorimeters are common laboratory instruments that are used to measure the absorbance of light by a solution. For a given substance at a specific concentration, the amount of light absorbed varies according to the wavelength of light shone through the solution. In general, the wavelength or color of light at which maximum light absorbance occurs is complementary to the color of light transmitted by the solution (the color we see). The wavelength of maximum absorbance for a positive biuret test for proteins is 540 nm, within the green range of visible light. The maximum absorbance can be determined for any solution by blanking the spectrophotometer at a specific wavelength and then inserting a single sample with a known concentration and reading the absorbance. This process is repeated for each wavelength.

In order to calculate the amount of protein in an unknown sample using a biuret test, a series of “known” protein standards must be prepared. Standards must be made with extreme care since any mistakes in calculating the concentration of a standard will lead to an incorrect calculated concentration in the unknown samples. In order to make a standard, a known amount of protein (usually in milligrams) is dissolved into a known amount of water (usually in milliliters). A set amount of biuret test solution is added to a known volume of the prepared standard protein solution. The process is repeated using three to five different known concentrations of protein. These three (or five) standards are analyzed using the spectrophotometer.

The absorbance readings are recorded and a standard curve or graph is constructed by plotting concentration of protein on the x-axis and absorbance on the y-axis. Since zero protein should equal zero absorption, a best-fit trend line is drawn through 0,0 as well as through the three (or five) data points (see Figure 2). Finally, the unknown protein sample is analyzed using the spectrophotometer. Its absorbance is plotted onto the standard curve and the concentration is read directly from the graph.

Experiment Overview

In this experiment the concentration of protein in a solution will be determined using a color change reaction with a copper solution and its absorbance at a wavelength of 540 nm.

Materials

Prelab Questions

1. Why is it important that a best fit trend line be drawn through 0,0 on the graph?
2. Why is it important that the reaction between the proteins and the test solution produce a colored product and not a colorless product?

Safety Precautions

Biuret quantitative assay solution contains copper sulfate in a sodium hydroxide solution. It is corrosive to all body tissue, especially eyes. It is also moderately toxic by ingestion. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

1. Turn on and warm up the spectrophotometer.
2. Obtain 20 mL of a 10.0 mg/mL albumin stock solution, a 10-mL serological pipet and five large test tubes.
3. Label the test tubes 1 to 5 using a marker or wax pencil.
4. Prepare five standard solutions using the following chart Fill all of the test tubes with albumin solution (step a) before proceeding to steps b–d.
   1. Use a 10-mL serological pipet to transfer the required volume of albumin stock solution into each test tube 1–5. Note: Always use a pipet bulb. Never pipet by mouth.
   2. Rinse the pipet thoroughly with DI water.
   3. Use the pipet bulb to evacuate as much water as possible from the pipet.
   4. Pipet the required volume of DI water into each test tube 1–5.
      {10977_Procedure_Table_1}
5. Seal the top of each test tube with Parafilm and invert 10–15 times to thoroughly mix the contents and create a homogeneous solution.
6. Place eight clean and dry cuvets into the test tube rack. On a sheet of white paper label the first seven cuvets with the numbers 1 to 7. The last cuvet should be labeled “blank.” Do not write on the cuvets themselves.
7. Using the same pipet as in step 4d, add 5.0 mL deionized water to the blank cuvet. This blank cuvet will be used to zero the spectrophotometer.
8. Use the pipet bulb to evacuate the remaining water from the pipet into an empty beaker.
9. Add 5.0 mL of the 1.0 mg/mL albumin solution from test tube 1 to cuvet 1.
10. Rinse the pipet with deionized water. Dry it both inside and outside as much as possible.
11. Repeat steps 9 and 10 to add 5.0 mL of each standard to the corresponding cuvet 2–5. Always work from the lowest concentration to the highest.
12. After pipetting the last standard solution, rinse the pipet and dry it as much as possible.
13. Add 5.0 mL of the first unknown sample into cuvet 6.
14. Rinse the pipet and dry it as much as possible.
15. Add 5.0 mL of the second unknown sample into cuvet 7.
16. Rinse the pipet and dry it as much as possible.
17. Use the pipet to add 2.5 mL of Biuret Quantitative Assay solution to each cuvet 1–7 and the blank.
18. Mix the solutions in the cuvets using the Parafilm inversion method described in step 5.
19. Wait 10 minutes for any color to fully develop. The fully-developed color is stable for at least an hour.
20. During this time, set a wavelength of 540 nm on the spectrophotometer, or choose the closest wavelength available on the colorimeter (565 nm).
21. Close the cover on the cuvet holder and follow the instructions for your instrument to adjust the spectrophotometer for 0% T, if needed.
22. Handle the cuvets by their top rims to avoid getting fingerprints on the surface. Wipe the exterior of each cuvet with either a clean Kimwipe or with lens paper just prior to inserting it into the spectrophotometer.
23. Insert the “blank” cuvet into the holder. Be sure it is clean, free of fingerprints, and the line marker is aligned with the mark on the holder. Close the cover on the cuvet holder and once again adjust the instrument to read zero absorbance. Note: The zero cuvet is called a “blank.” Before each subsequent reading check that the instrument is stable by re-inserting the blank and checking that the blank continues to read zero absorbance.
24. Begin measuring absorbance readings for the seven cuvets that contain albumin.
25. Insert the first cuvet into the cuvet holder and close the cover.
26. Record the absorbance reading in the data table on the Worksheet.
27. Repeat steps 25 and 26 for the remaining albumin solutions.
28. Plot the absorbance of the five standard solutions versus the concentration of albumin in each standard on graph paper.
29. Draw a linear best fit line from 0,0 through the five data points plotted on the graph.
30. Using the graph, determine the theoretical concentrations of the unknowns in cuvets 6 and 7.
31. Pour the contents of the seven cuvets into the excess biuret test solution bottle. The excess albumin and water solutions may be flushed down the drain with plenty of tap water.

Student Worksheet PDF

10977_Student1.pdf