5 Chapter 5 – Analysis of Proteins

Analysis of Proteins


Proteins are of . There are twenty different naturally occurring amino acids in . The type, number and sequence of amino acids making up the backbone differs between proteins. This variation accounts for the different molecular structures, nutritional attributes and physiochemical properties of proteins.

In the body, proteins can be a or a . Structural proteins provide scaffolding that gives stiffness and rigidity to otherwise fluid biological components (collagen, keratin, actin, etc.). Functional proteins are the building blocks that do all the work in the body (enzymes, hormones, neurotransmitters, etc.). Many proteins are not confined to being either structural or functional, but can serve as both.

Proteins are important constituents of foods for a number of reasons. They are a major source of energy, and they contain essential amino-acids such as lysine, tryptophan, methionine, leucine, isoleucine and valine. s are those required for human health but which the body cannot synthesize. Proteins are also the major structural components of many natural foods, often determining their overall texture such as tenderness of meat or fish. Isolated proteins are often used as ingredients in food because of their unique functional properties, for example the ability to provide desirable appearance, texture, or stability. Typically, proteins are used as gelling agents, emulsifiers, foaming agents and thickeners. Many food proteins are enzymes which are capable of enhancing the rate of certain biochemical reactions. These reactions can have either a favorable or detrimental effect on the overall properties of food. Food analysts are interested in knowing the total concentration, type, molecular structure and functional properties of the proteins in foods.

Enzymes are crucial contributors to protein digestion. Protein-digesting enzymes are referred to as . These enzymes must break down the bonds binding the complex molecule together. This digestive process begins in the stomach with the secretion of the stomach’s gastric acid. in the gastric juices attacks the protein molecules, separating them and breaking them down into amino acids. The gastric enzyme then begins to digest the amino acids.

Digestion of proteins continues in the duodenum, the first segment of the small intestine. The pancreas assists in the process by secreting the pancreatic protease enzymes and chymoffypsin. Like pepsin, trypsin breaks down the proteins into single amino acid molecules through a process called . During hydrolysis, a water molecule is inserted between the two amino acids which are bonded together. This breaks the bond between them.

Absorption of the small amino acids is accomplished by the finger-like projections on the wall of the small intestine called . Once in the bloodstream, the amino acids are distributed by both red blood cells and the blood plasma to tissues throughout the body. These tissues then use the amino acids for creation and repair of cell structures.

The presence of proteins in a solution can be determined using a few simple tests. The uses a reagent made of potassium or sodium hydroxide (KOH/NaOH) and hydrated copper (Il) sulfate mixed with potassium sodium tartrate. It is mixed with a solution and allowed to stand for a few minutes before qualitative analysis of proteins can be done. When in the presence of proteins, the reagent turns from blue to violet. The reagent will turn pink when combined with short-chain polypeptides. A semi-quantitative analysis may be concluded on the variation of the darkness of the solution color.

The method was devised to measure protein concentration. This method uses either the natural ability of proteins to absorb or scatter light in the UV-visible region of the electromagnetic spectrum, or the proteins are chemically or physically modified to absorb or scatter light in this region. To use the spectroscopy method, a calibration curve of absorbance versus protein concentration is first prepared using a series of protein solutions with known concentration. The absorbance of the solution being analyzed is then measured at the same wavelength, and its protein concentration determined from the calibration curve.

For a more accurate quantitative analysis with , let the solution stand for 25 to 30 minutes before reading the absorbance at 540nm. The major advantage of this technique is the lack of interference from materials that absorb at lower wavelengths. The technique is also less sensitive to protein type because it utilizes absorption involving peptide bonds that are common to all proteins, rather than specific side groups.


Key Terms

  • Biuret method
  • Essential amino acid
  • Functional protein
  • Hydrochloric acid
  • Hydrolysis
  • Pepsin
  • Protease
  • Proteinase
  • Structural protein
  • Trypsin
  • UV-visible spectroscopy
  • Villi



•   Test for the presence of proteins in foods.

•   Determine the effect of HCI and pepsin on protein digestion.

•   Understand the function of proteins and their digestion.




  • 100x12mm test tubes (15)
  • Egg albumin (2%)
  • Vegetable Oil
  • Starch solution (2%)
  • 1M HCl
  • Graduated cylinder (10ml)
  • Pepsin
  • Amylase
  • Biuret Reagent*
  • Food Samples (5, A-E)
  • Test tube rack
  • Pipettes

*Biuret Reagent: 4ml of 10M sodium hydroxide + 0.5ml 1 % copper sulfate solution.

  1. The primary structure of a protein is held together by what type of bonds?
  2. Proteins are made from long, folded chain molecules. What are these chains called?
  3. Amino acids contain carbon, hydrogen, oxygen, and what other main element?
  4. How many different types of amino acids are used to make proteins?
  5. What is the difference between structural proteins and functional proteins?
  6. What are essential amino acids, and why are they important?
  7. What is the role of enzymes in protein digestion?
  8. How does hydrochloric acid in the stomach aid in protein digestion?
  9. Which enzymes are secreted by the pancreas to further break down proteins in the small intestine?
  10. What are the principles behind the Biuret method and UV-visible spectroscopy for protein analysis?

Exercise 1: Determination of Presence Proteins


  1. Place 2ml of a sample in a clean test tube.
  2. Add 2ml of Biuret reagent.
  3. Repeat for all other samples (see Data Table 1).
  4. Wait for 10 minutes.
  5. Record the data for the presence or absence of protein in Table 1.


Exercise 2: Effect of HCL and Pepsin Protein Digestion


  1. Place 2ml of a protein positive sample in each of 5 clean test tubes. Label them 1-5.
  2. Add the following solutions to the specified tube:
  • Test Tube 1: 2ml water
  • Test Tube 2: 2ml Pepsin
  • Test Tube 3: 2ml HCl
  • Test Tube 4: 2ml HCI and 2ml Pepsin
  • Test Tube 5: 2ml Amylase
  1. Wait for 15 minutes, then add 2ml of Biuret reagent to each tube.
  2. Wait for 10 minutes, then record your observations in data table 2.
Table 1: Presence of Proteins
Test Tube # Material Tested Biuret Test Result (Protein +/-)
1 Vegetable Oil
2 Starch Solution
3 Egg Albumin
4 Unknown A
5 Unknown B
6 Unknown C
7 Unknown D
8 Unknown E


Table 2: Effect of HCl and Pepsin on protein digestion
Test Tube # Experimental Sample Observation
1 Sample + H20 + Biuret
2 Sample + Pepsin + Biuret
3 Sample + HCI + Biuret
4 Sample + HCI + Pepsin+ Biuret
5 Sample + Amylase + Biuret



  1. Based on your results, what can you conclude about the effect of HCI, pepsin, and amylase on proteins?
  2. What results would you expect if you ran the second experiment on the other samples?
  3. Disulfide bridges help to maintain which aspect of protein structure?
  4. Which of these is a protein: catalase, chlorophyll, cholesterol, or cytosine?
  5. What type of reaction is catalyzed by protease enzymes?
  6. Enzymes are catalysts. What does this mean?
  7. Name two protein-digesting enzymes made by the pancreas.
  8. What are the functions of HCI and pepsin in protein digestion?

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Biology I Cellular Processes Laboratory Manual by The authors & Hillsborough Community College is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, except where otherwise noted.

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