1 Lab 2 Biochemistry Learning Objectives The lab has the following learning objectives. Investigate the role of double bonding in fatty acids, through models. Developing a calibration curve for a Benedict s colorimetric test for glucose Measure the concentration of glucose in an unknown of set concentration using the calibration curve you developed. Measuring the concentration of glucose in various food stuffs Perform a qualitative evaluation of protein levels in various food stuffs using the Biuret reaction. Introduction The chemistry of life is in turn the chemistry of the macromolecules. While we have discussed the properties and structure of the macromolecules in class we haven t gotten into how these chemicals are detected. In this lab we will be looking at how different classes of macromolecular classes can be chemically detected and using one of these techniques, the Benedict s Test for glucose, to investigate how we develop a standard calibration curve. The first part of the lab will be an informal investigation of lipid structure and its use in food. You will be looking at a few models of fatty acids and then determining the different types of lipids that are present in various foodstuffs. The second part of the lab will be using the Biuret reaction, a colorimetric assay (a test where the color changes in proportion to the activity/substance measured, to make a qualitative determination as to the presence of proteins in various foodstuffs. The final section will be using another colorimetric assay, the Benedict s Test, in a quantitative manner to determine the concentration of glucose in an unknown sample provided by the instructor (with stringent precision requirements) and in a selected foodstuff. Lipid Structure and Role in Food The lipids are the group of organic macromolecules that includes the fats, oils and waxes. While the four subgroups of lipids can be structurally quite different from each other, they are all insoluble in water. The biological version of the phrase "Oil and water don't mix" is "Lipids and water don't mix".
2 The four subgroups of the lipids are: 1. Triglycerides - "neutral fats" (most of your dietary fat is this type) 2. Phospholipids - the components of the plasma membrane 3. Waxes - as on leaves and in honeycombs 4. Steroids - including cholesterol, cortisone and the sex hormones Saturated and Unsaturated Fatty Acids Fatty acids are the long chains of primarily carbon and hydrogen that are found primarily in two groups of lipids, the triglycerides and the phospholipids. They are the "tails" of a phospholipid, and the three "chains" attached to the glycerol in a triglyceride. It is these fatty acids that make the triglycerides of our dietary fat saturated or unsaturated. Examine the models of the saturated, monounsaturated and polyunsaturated fatty acids. Answer the following questions for the Lab Report 1. Compare the shapes of the monounsaturated and saturated fatty acid models. In molecular terms, what causes the difference in the shape of the molecules? 2. Read the labels of the food products available to fill out the data table on the data sheet. 3. Are saturated fats more likely to be solids, or oils? Unsaturated fats? Explain why this difference exists. 4. Are saturated fats more likely to be found in foods that come from animal or plant sources? Unsaturated fats? Give examples. 5. In which way do peanut butter and margarine not seem to fit the pattern you have described in the previous question? 6. Note that these two foods are high in "hydrogenated oils". Does adding hydrogen to fatty acids make them more saturated, or less saturated? 7. Why do doctors advise minimizing the saturated fats in our diet
3 "Cis" and "Trans" Fats Cis and trans are terms that refer to the structure of unsaturated fatty acids. When the hydrogens on either side of a double bond are on the same side of the fatty acid molecule, we call the arrangement Cis. When they are on opposite sides we call the arrangement Trans. See the figures below. Trans fats are not normally found in naturally occurring foods, but are produced during hydrogenation of unsaturated fats. This is done by converting oils into solids, such as converting corn oil into margarine. O HO C C H C H Cis: (same) arrangement of H CH 3 O H C C C CH 3 HO H Trans: (opposite) arrangement of H Answer the following questions for the Lab Report 1. Go back to the table of information for the food items on page 3. Which foods would most likely have "trans" fats? 2. Health research is showing that trans fatty acids raise the level of LDL (bad) cholesterol and reduce the level of HDL (good) cholesterol. 3. Using what you have learned about the structure of fatty acids, explain why this may be. (Hint consider the shape of trans fatty acids)
4 Qualitative Analysis of Proteins A protein s structure is determined by its amino acid subunits. The amino acids are linked by covalent bonds called peptide bonds. Peptide bonds are detected by a combination of two chemicals in the biuret s test. In the presence of these peptide bonds, biuret s reagent changes from blue to a light violet or lavender. Procedure: 1. Thoroughly rinse the spot plate to remove all traces of solution from the previous procedure. Do not use soap. 2. Add 5 drops of each of the solutions indicated in the data table to separate wells of the spot plate. Record the color of the test solution. 3. Add 5 drops of sodium hydroxide (NaOH) to each of the solutions. Then add 5 drops of copper sulfate (CuSO4) to each well. Answer the following questions for the Lab Report 1. Record the final color for each solution in the table on the data sheet. 2. Which of the above tested positive for protein? 3. Explain what the R on an amino acid diagram indicates 4. You should have found that the amino acid solution did yield a positive result for proteins. How can this be, when amino acids are the subunits of proteins? (Hint: Review the description of the biuret's test on page 6).
5 Quantitative Analysis of Glucose Glucose is the main product of photosynthesis and primary energy source for most things on earth. It also serves at your blood sugar. Glucose is an example of a monosaccharide, a single sugar unit. There are three groups of carbohydrates, monosaccharides, disaccharides, and polysaccharides. A common polysaccharide in nature, and in foods, is starch. Starch is a long chain of glucose molecules. We will use chemical tests to identify the presence of glucose and starch. Calibration Curve for the Benedict s Test The Benedict s Test is a colorimetric reaction where the presence of reducing sugars, upon heating causes the solution to go from blue to green, then red and finally brown. We will be looking at this reaction in a quantitative manner, so we will be using a spectrophotometer to measure the absorbance of light by the solution at 735 nm. This absorbance will increase in proportion to the amount of glucose present in the sample. Colorimetric assays are very convenient and can be extremely accurate, but they do have a few significant drawbacks. First, they have a limited range over which the color response increases in a linear manner with the concentration of glucose. Second, since the assay is based on a specific wavelength of light any interference at this wavelength (random scattering due to food particles or colored food sample for examples) can lead to false readings. We will be addressing the first problem in this lab and will address how we might address the third. The Benedict s test detects all reducing sugars (including all monosaccharide and lactose), though most commonly it is used for the detection of glucose (specifically it was used to detect urine in patients with diabetes mellitus). If the sugars are bound in such a way that they cannot isomerize to an aldehyde form (i.e. sucrose and starches) very little reaction is seen. The reagent used is a copper solution and the reducing sugars cause a precipitation of red copper (I) oxide (Cu 2 O). The absorbance of this precipitate at 735 nm is what we are measuring. To use any colorimetric assay you first need to make a calibrated curve. The purpose of this curve is to determine the linear range of the assay. In other words, the analysis range over which the color response is in a linear relationship to the measured substance. You will determine the linear range for your assay through experimentation. Each group will be provided with tubes, pipettes, distilled water and a 20% (m/v) glucose solution. You will need to make a series of dilutions that can be tested to determine an
6 approximate linear range. I would suggest that you think about serial dilutions (a procedure you did in both BIO161 and Chemistry lab) and also consider replicates. We have a limited number of spectrophotometers so we will have to share, though measurements are quick. While you are not required to produce the calibration curve in class (you will use a linear regression analysis outside of class), it would help to do a rough plot in your lab notebooks to determine the ballpark you are looking for. After you have established you calibration curve ask your instructor for your unknown sample. These samples have a known concentration of glucose in them and as such serve as a test of your calibration curve. Your group must determine (using a linear regression model, see the appendix if you need to know how to do this by hand, or use a spreadsheet) the concentration of the sample. Your answer is to be recorded on the lab data sheet and will be evaluated as follows. Percentage off from value Points deducted from lab <2.0% 0 2.0 3.9% -2 4.0 6.0% -4 >6.0% -6 After you have measured your unknown sample you will also need to determine the approximate concentration for the food stuffs listed in the data table. On a side note, remember that any values you are going to use to determine concentration must be within the linear range of the assay (meaning you may have to dilute samples) and that any dilutions must be accounted for in your calculations. Procedure for Benedict s Test 1. Fill a 600 ml beaker ½ full with water, and heat to gently boiling on a hot plate. 2. Place 0.5 ml of each test solution in a separate test tube. 3. Add 2 ml of Benedict s reagent to each tube. 4. Heat for 2 minutes in boiling water. 5. Remove tubes from the water with a test tube holder, place in test tube rack and allow cooling.
7 6. Pipette the cooled solutions into spectrophotometer tubes for measurement. Be sure to use a blank, heated, control for your zero value and to wash the spectrophotometer tube between analyses. Answer the following questions for the Lab Report 1. Why cannot we not exactly determine the value of sugars in the foodstuff tested, but we can in the glucose unknown provided to you? (hint, think about the calibration curve) Lab Report Requirements The following materials should be submitted at the start of the next lab period. 1. Completed Lab Data Sheet 2. Copies of all Lab Notebook Pages (including any calculations) 3. Graph for linear regression either done by computer (with linear equation on graph) or done by hand with hand-done calculations of linear formula included. 3. Answers to questions in each section, organized by section. (must be typed) Please staple these together prior to class.
8 Lab 2 Data Sheet Lipids Structure and Their Role in Food Properties of Lipids Found in Food Items Food Serving Fat Types (g) Hydrogenated Stuff Size (g) Total Saturated Trans Monounsaturated Oil Present (Y/N) Qualitative Analysis of Proteins Biuret Reaction Results Well # Solution Final Color 1 Protein Standard 2 Honey 3 Amino Acid Solution 4 Distilled Water 5 Bean Extract 6 Milk 7 Sardines Quantitative Analysis of Glucose Concentration of Unknown # = % (m/v) Concentration of Sugars in Honey (approx.) = % (m/v)