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BIOCHEMISTRY SECOND EXAM FALL 2020 Part 1 Chapter 5. Instrumental methods In your lab, you have the following instruments available: NMR spectrometer, mass spectrometer, high pressure liquid chromatograph, x-ray crystallography, SDS-PAGE electrophoresis, Western blotting, ELISA, (assume an ELISA assay is available for your protein of interest) ion-exchange chromatography, affinity chromatography (assume that an affinity column is available for the protein you are studying),, fluorescence microscopy. Tell which instrument or method you would use for each of these studies: 1. You are determining the sequence of a protein using the Edman method 2. The protein was isolated from kidney cells. You are trying to find in which part of the kidney cell the protein is most concentrated. 3. You wish to isolate more of this protein from kidney tissue 4. You wish to separate your protein which has an isoelectric point of 10.5 from another protein whose isoelectric point is 3.5. 5. An SDS-PAGE gel indicates that homogenized kidney tissue shows 27 protein bands. You wish to determine which of these bands represents your protein of interest. 6. You wish to determine the 3-dimensional structure of the kidney protein in aqueous solution 7. You wish to determine the 3-dimensional structure of the kidney protein in the solid state 8. You wish to determine the exact molar mass of this protein Part 2 Chapter 6. Enzyme basics Write a short essay describing the major features of enzymes and how they play a part in catalysis (the more the better!) Chapter 7 Enzyme Kinetics Using the data shown on the graph below, answer the following questions: The y-axis represents velocity in mole per liter per second units and the x-axis represents substrate concentration in millimole per liter units. 1. Find Km both with and without inhibitor. Show how you got it. 2. Find Km both with and without inhibitor. Show how you got it. 3. Which type of inhibitor is caffeic acid? Explain why 4. Is caffeic acid binding at the active site? Explain why or why not. 5. Arachidonate-5-lipoxygenase has a molar mass of 65 kilodaltons. In order to generate the results shown in the graph above, 0.50 gram of enzyme is dissolved in 100.0 mL of aqueous buffer. From this reaction, calculate Km both with and without caffeic acid. Chapter 8 Enzyme inhibition and regulation 1. In one or two sentences, describe how each of these types of enzyme inhibitors work. A. Competitive inhibition B. Noncompetitive inhibition C. Uncompetitive inhibition D. Irreversible inhibition E. Allosteric inhibition 2. In one or two paragraphs, describe what allosteric enzymes are, their major features and how they work. Chapter 9 Hemoglobin and Myoglobin 1. Describe the major structural and functional differences between hemoglobin and myoglobin. 2. For each of these molecules, describe how they affect the functioning of hemoglobin. A. Heme B. Proximal His C. Distal His D. BPG E. CO2 3. Explain why hemoglobin tends to absorb oxygen in lung tissue but release it in other tissues. Chapter 10 Carbohydrates Draw the chemical structures of two monosaccharides which fit each of these descriptions: 1. Two hexoses which are constitutional isomers 2. Two aldopentoses which are epimers 3. Two ketohexoses which are diastereomers but not anomers 4. Two hexoses which are anomers 5. Two aldopentoses which are not isomers of any type 6. Describe in as much detail as possible the structural features of amylose, amylopectin, glycogen and cellulose. Remember: Too much is better than too little! Arachidonate-5-lipoxygenase Kinetics 0123456789101112131415567891011121314151617181920012345678910111213141510111213141516171819202122232425 CHAPTER 5 QUESTIONS 1. Using a cationic, anion exchange column at pH 7.0, which amino acid would be the last to leave the column? A) Arg b) Lys c) Ala d) Asp 2. Which does SDS-PAGE not do? A) trap proteins in SDS micelles b) denature proteins c) make all proteins negatively charged d) break proteins into amino acids 3. The Edman method is used for what? A) locating proteins in cells b) determining sequence of proteins c) generating 3-D images of proteins d) separating proteins based on molar mass 4. The analytical instrument used in conjunction with the Edman method is: a) gel electrophoresis apparatus b) NMR spectrometer c) high pressure liquid chromatograph d) mass spectrometer 5. X-ray crystallography is used for what? A) locating proteins in cells b) determining sequence of proteins c) generating 3-D images of proteins d) separating proteins based on molar mass 6. Fluorescence microscopy is used for what? A) locating proteins in cells b) determining sequence of proteins c) generating 3-D images of proteins d) separating proteins based on molar mass 7. NOESY is an analytical technique which involves which instrument? A) gel electrophoresis apparatus b) NMR spectrometer c) high pressure liquid chromatograph d) mass spectrometer 8. MALDI is an analytical technique which involves which instrument? A) gel electrophoresis apparatus b) NMR spectrometer c) high pressure liquid chromatograph d) mass spectrometer 9. ELISA is an analytical technique which uses which instrument? A) microplate reader b) gel electrophoresis apparatus c) fluorescence microscope d) ultracentrifuge 10. Western blotting is an analytical technique which uses which instrument? A) microplate reader b) gel electrophoresis apparatus c) fluorescence microscope d) ultracentrifuge 11. If you wish to identify a specific protein from a mixture of proteins seen on an SDS-PAGE gel, the best method to use would be: a) NMR b) mass spectrometry c) ELISA d) Western blotting 12. If you wish to develop a quantitative assay for a specific protein, the best method to use is: a) NMR b) mass spectrometry c) ELISA d) Western blotting 13. If you wish to generate a 3-D image of a protein as it exists in aqueous solution, the best method is: a) NMR b) mass spectrometry c) ELISA d) Western blotting 14. If you wish to determine the exact molar mass of a protein, the best method is: a) NMR b) mass spectrometry c) ELISA d) Western blotting 15. If you wish to determine which cellular region contains the highest concentration of a specific protein, the best method to use is: a) differential centrifugation b) analytic microtomography c) microparticle analysis d) fluorescence microscopy CHAPTER 6 HOMEWORK 1. An enzyme which catalyzes the hydrolytic cleavage of peptide bonds is called a _______? A) oxidoreductase b) lyase c) protease d) ligase 2. Trypsin catalyzes the cleavage of: a) all peptide bonds b) peptide bonds after Lys or Arg c) peptide bonds before Lys or Arg d) peptide bonds after Met 3. Papain catalyzes the cleavage of: a) all peptide bonds b) peptide bonds after Lys or Arg c) peptide bonds before Lys or Arg d) peptide bonds after Met 4. The molecule which binds to an enzyme and undergoes chemical reaction is called a: a) substrate b) cofactor c) coenzyme d) prosthetic group 5. A metal ion which binds to an enzyme and assists it in catalysis is called: a) substrate b) cofactor c) coenzyme d) prosthetic group 6. Proteases are a subclass of which larger class? A) isomerases b) oxidoreductases c) lyases d) hydrolases 7. Transferases which catalyze the transfer of a phosphate group are called: a) lyases b) ligases c) kinases d) phosphatases 8. If a reaction is exergonic, which of these thermodynamic parameters is necessarily negative? A) ∆G b) ∆H c) ∆S d) ln K 9. Which statement is always true about exergonic reactions? A) they give off heat b) they proceed very rapidly c) they have the potential of proceeding without energy input d) they require enzyme catalysis 10. If the value of the equilibrium constant K is zero, this tells you: a) the reaction requires an enzyme b) the reaction is at equilibrium c) the reaction is endergonic d) the reaction is exergonic 11. Which of these thermodynamic parameter are changed by enzymes? A) Gibbs free energy b) entropy c) activation energy d) all of these 12. The place on the enzyme where the substrate binds is called the: a) coenzyme b) transition state c) active site d) enzyme-substrate complex 13. The enzyme-bound substrate is also called: a) coenzyme b) transition state c) active site d) enzyme-substrate complex 14. The enzyme-bound substrate facilitates the formation of the: a) coenzyme b) transition state c) active site d) enzyme-substrate complex 15. The binding of substrates to most enzymes may be best described as: a) lock and key b) induced fit c) backside attachment d) cofactor displacement