Lab 3 Testing for Biomolecules

Testing for Biomolecules Learning Outcomes: Upon successful completion of this lab, students should be able to: 1. Describe the distinguishing chemical structures of carbohydrates, lipids and proteins and use this information to identify images of chemical and/or structural formulas. 2. Perform and interpret test results for Benedict’s, Biuret, and Iodine indicator tests. 3. Explain the chemistry for why/how the Benedict’s, Biuret, and Iodine indicator tests work to detect specific categories of biomolecules. This lab focuses on the chemical structure of biomolecules. Because each biomolecule has a unique chemical structure, they can be identified in reactions designed to detect these unique structural differences. Chemical and Structural Formulas for Biomolecules There are four essential types of biomolecules found in living organisms: carbohydrates, proteins, lipids, and nucleic acids. Each has a unique molecular structure that can be easily recognized both by its chemical formula and structural formula. Carbohydrates Carbohydrates are among the most abundant types of biomolecule and are made solely of carbon, hydrogen, and oxygen atoms. Monomers (basic units) of carbohydrates (called monosaccharides) have these three elements in a characteristic ratio of 1:2:1 (1 carbon: 2 hydrogens: 1 oxygen). This ratio is often represented as (CH₂O)ₙ where “n” represents the number of times the ratio is raised depending on the size of the molecule. Many of you are already familiar with the chemical formula for glucose C₆H₁₂O₆. When monosaccharides combine to form more complex carbohydrates (such as polysaccharides), the ratio is slightly altered (slightly lower O and H), but is still relatively close to 1:2:1. This is due to the process of “dehydration synthesis”, whereby two monomers bond via the loss of one water (H₂O) molecule Lipids Though we often think of lipids as being our dietary “fats”, this biomolecule category also includes sterol molecules (like cholesterol and steroids) and waxes. Like carbohydrates, lipids are made of carbon, hydrogen, and oxygen atoms, but they are not in a 1:2:1 ratio. Instead, lipids have a disproportionate number of carbons and hydrogens compared to the number of oxygens. Structurally, this is due to the fact that many lipids (such as phospholipids and triglycerides) are composed of long chains of carbon and hydrogen (called hydrocarbons) to which a carboxyl functional group (-COOH) is attached. These hydrocarbon + COOH chains are called fatty acids and have a generic chemical formula of CH₃(CH₂)ₙCOOH where “n” represents the number of times CH₂ is repeated in the fatty acid tail. Proteins The monomer (basic unit) of a protein is the amino acid. All amino acids have the same basic chemical “backbone” comprised of carbon, hydrogen, oxygen, and nitrogen atoms: NH₂CH(R)COOH. Amino acids are so named due to the amino (NH₂) and carboxylic acid or carboxyl (COOH) functional groups. Each amino acid is distinguished, though, by the chemical make-up of their “R-group”. There are 20 amino acids, each with a unique chemistry to their R- group. The R-groups play an important role in the folding and unfolding properties of the protein. Nucleic Acids Nucleic acids are composed of monomer units called nucleotides. Each nucleotide has 3 key components: a 5 carbon sugar (deoxyribose or ribose), a phosphate group (PO₄), and a nitrogen containing base (adenine, thymine, cytosine, guanine or uracil). You are probably most familiar with the nucleic acids DNA and RNA, which are composed of numerous nucleotides bonded together. The molecule ATP, which is used by cells for energy, is another important biomolecule whose structure is composed of a nucleotide. Though we will not test for nucleic acids in this lab, we will spend a significant amount of time studying their structure in lecture. Use the information from above to help you determine if the following chemical and structural formulas are monomers of carbohydrates, lipids, or proteins. We are not identifying nucleic acids. Write your answer on the lines provided next to each molecule. proteins a. NHCH(H)COOH __________________________________ b. CH₂OHCHOHCHO __________________________________ c. CH₃CH₂COOH __________________________________ d. C₃H₆O₃ __________________________________ carbohydrates lipids carbohydrates e. proteins __________________________________ lipids f. C₈₈H₁₇₈O₂ __________________________________ g. (CH₂O)₁₂ __________________________________ carbohydrates h. i. carbohydrates lipids __________________________ ______________________________ Chemical Indicator Tests Since each biomolecule has a unique chemical make-up (based on the types and arrangements of atoms), it is possible to identify them in solution by conducting chemical tests, called indicator tests. The unique chemical make-up of the molecule being tested interacts in a specific way with a unique indicator solution, often resulting in a color change and/or the formation of a precipitate (a solid that no longer stays in solution). Benedict’s Reagent/Solution: Tests for presence of reducing sugars All monosaccharides have the ability to give electrons to (to reduce) other molecules. Reducing sugars have a free aldehyde or ketone group (a carbon with double bonded oxygen at the end or middle of a compound) located on an anomeric carbon (a carbon bonded to two different oxygen atoms). Frequently, when monosaccharides join via dehydration synthesis to form disaccharides and polysaccharides, the aldehyde group on the anomeric carbon gets tied up into the sugar-sugar bond so it is not available to release electrons, and thus, cannot act as a reducing sugar. Thus, Benedict’s reagent only detects the presence of single monosaccharides, such as fructose (the monomer sugar in fruit) and galactose (the monomer sugar in milk) or a small number of disaccharides that can give up electrons (such as lactose, but not sucrose). The blue color of the Benedict’s reagent is due to the presence of copper ions (Cu2+). When Benedict’s reagent is heated in the presence of reducing sugars, the copper ions are reduced to cuprous oxide (Cu2O) via a chemical reaction, forming a precipitate (a substance which settles out of solution) that varies in color from green to orange to brick red depending on the concentration of reducing sugars in the sample and thus the amount of cuprous oxide formed. GLUCOSE- reducing b/c the anomeric carbon has a free oxygen this oxygen is tied up this oxygen on the anomeric carbon is free LACTOSE- reducing b/c one of the two anomeric carbons (shown with arrows) has a free oxygen (the O below the green arrow) SUCROSE- non reducing b/c neither anomeric carbon (shown with arrows) has a free oxygen Iodine Solution: Tests for presence of the polysaccharide starch The iodine indicator test does not involve a chemical reaction. Instead, it relies on the physical interactions between the starch molecules and the iodine reagent. Amylose, more commonly known as starch, is a large, coiled polysaccharide (many glucose monosaccharides bonded together via glycosidic bonds). When combined with iodine, the molecules of iodine (I₂) can become trapped in the starch coils. This impacts the way that light passes through the solution. The less light that can pass through the filled starch coils, the darker the solution appears to your eye. A starch solution by itself is clear or cloudy white. The iodine solution by itself is yellow-orange-brown or “amber” (depending on the concentration). A starch plus iodine solution, though, has a dark purple or deep blue-black color. Note the change of the iodine to black on the potato (right) but not the apple (left). These results indicate that potatoes contain starch, but apples do not. Before learning about the Protein Indicator, take a moment to explain in your own words the differences in how the two carbohydrate indicators (learned above) work and why they are specific to certain types of carbohydrates. a. How does the Benedict’s test detect monosaccharides? (be specific about the molecule/indicator interaction and what causes a color change). b. A starch molecule is a polysaccharide, made of monosaccharide sugar units joined together, but Benedict’s can’t the monomer sugar units. Why not? *(answer a and b in this space) BenedictÕs!test!used!to!indicate!the!presence!of!monosaccharides!and!reducing!sugars .!Copper!sulphate!in!the!benedictÕs!reagent!will!react!with!monosaccharides!and!reduci ng!sugars!form!brick!red!precipitate.!Monosaccharides!and!reducing!sugars!can!react! with!a!benedictÕs!reagent!because!contain!free!aldehyde!or!ketone.!The!positive!result! is!indicated!by!the!color!of!the!solution!changes!to!green,!yellow,!orange,!or!brick!red!a nd!appears!green,!yellow,!orange!or!brick!red!sediment." c. How does the Iodine test detect starch (a polysaccharide)? (be specific about the molecule/indicator interaction and what causes a color change). d. Monosaccharide solutions (like glucose or fructose) are not detected with Iodine? Why not? * (answer c and d in this space) Starch!iodine!test!is!a!speciÞc!test!for!polysaccharides!(starch!positive).!Iodine!test!indi cates!the!presence!of!polysaccharides!in!any!solution.!When!iodine!solution!reacts!wit h!starch,!a!bluish!colour!is!obtained!indicating!the!presence!of!starch. Biuret Reagent: Tests for presence of peptide chemical bonds The Biuret reagent, like the Benedict’s reagent, is blue in color due to the presence of copper ions (Cu2+). The chemical reaction, unlike Benedict’s, does not require heating. The indicator detects the presence of proteins, which are chains of amino acids linked by special bonds called peptide bonds. The blue-colored copper II ion can form a complex with the peptide bonds since the peptide has unshared electron pairs in nitrogen and oxygen of water. Once this complex has been formed, the solution turns from blue to purple. The deeper the purple color, the higher is the number of peptidecopper complexes. N-C-C represents a single amino acid (the center C has the R-group attached). Shown here, nitrogen atoms involved in peptide bonds are forming attractions (- - -) to copper ions. Peptide bonds are shown in red. Apply/ Build your own explanation: A solution containing hundreds of individual amino acids will test negative with Biuret while a solution with only a dozen or so protein molecules will test positive. A protein is made of amino acids, so why does the Biuret detect (turn positive) the protein solution but not the amino acid solution? * Chemical Indicator Tests Analysis Questions 1. Would you say that indicator tests are qualitative or quantitative? Explain your answer. * It's!qualitive,!because!we!were!looking!for!the!change!of!the!colors,!not!only!speciÞc!a mount!of!numbers.! What do “positive” and “negative” test results mean? When you interpret results for an indicator test, you state the test was positive or negative. This means the indicator did (positive test. +) or did not (negative test, -) detect the presence of the biomolecule of interest in your sample. Using this information, answer the following questions. 2. Does a positive test result absolutely mean that biomolecule is present? Why or why not? * A!positive!test!result!means!that!the!desired!biomolecule!is!present!(if!the!indicator!wa s!supposed!to!react!with!the!desired!biomolecule).!And!that!is!why!the!indicator!is!able !to!react!with!the!Biomolecule!and!give!a!positive!result. 3. Does a negative test result absolutely mean that biomolecule is not present? Why or why not? * A!negative!test!result!means!that!the!desired!biomolecule!is!not!present!(if!the!indicato r!was!supposed!to!react!with!the!desired!biomolecule).!And!that!is!why!the!indicator!is! unable!to!react!with!the!Biomolecule!and!give!a!negative!result. 4. You sometimes hear about “false positive” and “false negative” results. If you look carefully, for example, on the Cologuard commercial it notes the chance for false positive/negative results in find print. What are “false positive” and “false negative” tests? Why might they occur? * !"False!positive"!occurs!when!the!desired!biomolecule!is!not!present!to!react!with!the!i ndicator!but!due!to!the!presence!of!any!similar!molecule!in!the!mixture!or!due!to!any!ot her!reason,!the!test!gives!a!positive!result.!Similarly,!"False!Negative"!occurs!whe!the! desired!biomolecule!reacts!with!the!indicator!added!to!react!with!but!gives!a!negative!r esult.!It!occurs!because!the!biomolecule!might!was!present!in!very!low!concentration!o r!any!other!molecule!present!suppressed!the!reaction!or!expression!of!the!biomolecule and indicator in the mixture What are controls and why are they important? In all of the tests conducted in the following interactive lab activity to detect biomolecules, water will be used as a negative control. Using this information, answer the following questions. 5. Why do we expect water to test negative (“because it is the control” is not the answer)? Think about molecule chemistry. * water!is!used!to!test!the!presence!of!biomolecules!like!simple!sugar.!water!is!used!as! a!negative!control!as!it!doesn't!contain!any!simple!sugar!so,!the!test!group!will!differ!in! terms!of!absorbance!value!during!a!spectrophotometric!analysis. 6. Why are we testing water if we already know it should test negative? How can you use the negative test results to your advantage? * to!conÞrme!it!if!it's!negative.! 7. What could it mean if your water tests positive? What could cause this? Give at least 2 specific examples. * It!could!mean!that!there!is!contamination.!1)!presence!of!insecticides!in!the!water.!2)!pr esence!of!metals!in!the!water.! 8. If water is a negative control, what could we use for a positive control? In other words, what is a “positive control”? * A!positive!control!is!a!gathering!in!a!test!that!gets!a!treatment!with!a!known!result!and! ought!to!along!these!lines!show!a!particular!move!during!the!investigation.!It!is!utilized! during!the!examination!to!manage!obscure!factors!and!to!offer!something!to!the!resear cher!to!contrast!and!the!test!gathering." " REAL WORLD CONNECTION #1: Forensic Pathology Directions: Read through the hypothetical scenario and analyze the data below to answer the following questions. You are a forensic pathologist who uses scientific methods and testing to solve crimes. As a member of Las Vegas’ CSI team, you have been enlisted to help police solve a possible triple murder case involving three top-level executives of a biotechnology company that clones pets. Upon arrival, police tell you that they received an anonymous phone call stating that one of CEOs was intentionally poisoned earlier that day while eating at a local “specialty” restaurant. The source of the poison was scorpion venom, which is protein-based. The three local restaurants include: • “Viva Las Steakas” which only serves a three-course meal of sirloin steak and fruit • “Cash In Your Chips” which only serves baked potatoes, French fries and homemade potato chips • “That Salad of Green” which only serves leafy green and fruit salads. Below is a data table (Table 1) of the test results from your analysis of each victim’s stomach contents. Use the information and your understanding of the colors and chemical reactions of the indicator tests learned in this lab to complete Table 2 and the two subsequent questions. Table 1. Results from Testing Victims’ Stomach Contents for Biomolecules. Indicator 1 Indicator 2 Indicator 3 Sample from victim 1 black blue blue Sample from victim 2 amber purple orange Sample from victim 3 black purple orange Table 2. Analysis of Table 1 Results Indicator 1 is Iodine! Victim 2 Victim 3 Biuret!reagen Indicator 3 is Benedjcts!rea _____________ _____________ which tests for which tests for which tests for polysaccharid Victim 1 Indicator 2 is _____________ proteins! _____________ _____________ Pos. or neg. result? Pos. or neg. result? Pos. or neg. result? ________ Pos. or neg. result? negative ________ Pos. or neg. result? positive! ________ negative ________ Pos. or neg. result? positive! ________ Pos. or neg. result? positive ________ Was the victim poisoned by snake venom? (yes, no, maybe) carbohydrate _____________ positive! Name of restaurant at which victim last ate? * * That!Salad!of!G no! negative reen ________ Pos. or neg. result? positive ________ Pos. or neg. result? positive! ________ * * Cash!In!Your!C hips no! * * Viva!Las!Steaka no! s Explain how you know which victim(s) was /were NOT poisoned. * victim!3!was!not!poisoned!because!the!results!from!Testing!VictimsÕ!Stomach!Contents !for!Biomolecules.! Were you able to make an exact determination about which CEO was poisoned? Why or why not? * Yes,!victim!1!was!poisoned!because!all!the!test!result!was!showed!are!Stomach!Conte nts!for!Biomolecules.! REAL WORLD CONNECTION #2: Detecting the presence of sugar, starch and/or protein in bodily fluids Detecting the presence of sugar, starch and/or protein in blood, sweat or urine is a perfect way to check for illnesses and diseases. • Benedict’s solution can be used to test for the presence of glucose in urine, which could indicate diabetes. Labs also use glucose strips to detect to sugars in urine (look like strips of paper that turn color in the presence of sugars). • The iodine-starch test is frequently used as a diagnostic tool to evaluate underactive (hypohidrosis) and overactive (hyperhidrosis) sweating, a common problem for amputees. • Protein in human urine, especially more than the normal limit may indicate kidney-related problems. It may also indicate other health conditions such as diabetes mellitus, leukemia, high blood pressure, lupus and pregnancy. Indicator tests are not just used to detect biomolecules. They can be used to detect the presence of certain hormones, such as with a home pregnancy test (which detects the hCG hormone), to monitor water quality in your home aquarium or garden soil (such as testing the pH, ammonium, etc.), or antibody indicator tests (a topic we hear about lately related to COVID-19 antibody testing). Assignment: Using the website Science Daily, conduct a search for a research article related to indicator tests. The article does not need to relate to biomolecule tests specifically but must link to the larger topic of indicator tests (not every article will be appropriate to the topic so don’t necessarily go with the first article listed) On the next page, provide the article title, website link, and write a brief paragraph overview of what you learned. Do not cut/paste direct words from the article. This is plagiarism and will result in a zero. Take the time to understand the article and provide me with your written description of what was learned and what you found interesting. Macroscopic!quantum!interference!in!an!ultra-pure!metal Article Title: _______________________________________________________________________ Paste the Website Link: https://www.sciencedaily.com/releases/2020/06/200626114740.htm _________________________________________________________________________________ Write a brief summary of what you learned and found interesting (do not plagiarize direct text): * As!highschool!college!see!in!experiments!with!water!waves,!and!we!word!and!use!with!gentl e!waves!in!lots!of!optical!devices,!interference!is!a!primary!property!related!to!wavelike!behavior.!Indeed,!Davisson!and!Germer\'s!popular!statement!of!interference!in!experime nts!with!dilute!beams!of!electrons,!just!about!a!period!of!one!hundred!years!ago,!gave!key!ex perimental!beef!up!to!the!correctness!of!the!then-new!quantum!theory.