Q. 1. Explain how gel filtration chromatography (size exclusion chromatography) separates molecules on the basis of their mass/size.
A. In gel filtration, porous beads are packed into a column. The pores are of different size. The column is then packed with a buffer solution. This buffer solution is present inside the beads as well as around it. When molecules of different size pass through the columns, the smaller molecules pass through the pores and take a longer path. The larger molecules, however, travel around the beads. Thus, larger molecules travel faster than the smaller molecules, resulting in separation (Twyman 41).
Q. 2. For gel electrophoresis, what physical properties of proteins are used for their separation? Explain how SDS polyacrylamide gel electrophoresis is able to separate protein only on the basis of mass. Give the name of the type of gel electrophoresis you would run if you wanted to separate proteins only on the basis of their charge.
A. Size and charge can be used for separation of proteins in gel electrophoresis. In SDS polyacrylamide gel electrophoresis (SDS-PAGE), we reduce the effect of charge by breaking down the tertiary structure of proteins and forming linear molecules using SDS. Also, the SDS coats the proteins with a negative charge. These molecules can then be separated on the basis of weight alone. While all the molecules move towards the cathode they now move differently due their size with smaller molecules moving faster than the larger molecules (Twyman 28). Isoelectric focusing would be used to separate proteins based on charge alone (Twyman 25).
Q. 3. a. Discuss the steps for glycolysis
A. There are ten steps in glycolysis. In the first step, enzyme hexokinase phosphorylates glucose to form glucose-6-phosphate using an ATP molecule. After this, phosphoglucoisomerase converts glucose-6-phosphate to fructose 6-phosphate. The third step involves further phosphorylation by phosphofructokinase to form fructose-1, 6-biphosphate using another ATP molecule. The enzyme aldolase then acts on fructose-1, 6-biphosphate two form two sugars - dihydroxyacetone phosphate and glyceraldehyde phosphate. Enzyme triose phosphate isomerase then acts on dihydroxyacetone phosphate to form another molecule of glyceraldehyde phosphate. These molecules are then acted upon by triose phosphate dehydrogenase to form two molecules of 1, 3-bisphosphoglycerate. In this step we also see the formation of 2 NADH molecules. Phosphoglycerokinase then acts on the two molecules of 1, 3-bisphosphoglycerate to form 2 molecules of 3-phosphoglycerate and 2 ATP molecules. In the eighth step, Phosphoglyceromutase converts the molecules of 3-phosphoglycerate to 2-phosphoglycerate. Enolase then converts these two molecules to 2 molecules of phosphoenolpyruvic acid (PEP). In the final step, pyruvate kinase acts on the PEP molecules to form 2 molecules of pyruvic acid and two ATP molecules. In the whole process 2 ATP molecules are used and 4 ATP molecules are produced (Bailey).
Q. 3. b. Which steps of glycolysis are not reversible for gluconeogenesis? How are these steps reversed during gluconeogenesis?
A. The steps catalyzed by hexokinase, phosphofructokinase and pyruvate kinase are irreversible.
The pyruvate kinase reaction is reversed in two steps. The first step involves carboxylation of pyruvate into oxaoacetate by pyruvate carboxylase. This is an ATP-dependent reaction. In the next step, the oxaoacetate is acted upon by PEP-carboxykinase to form PEP. This step uses a molecule of GTP to provide the phosphate group. The remaining two steps are reversed by hydrolytic removal of phosphate. Fructose-1,6-biphosphatase and glucose-6-phosphatase act on fructose-1,6-biphosphate and glucose-6-phosphate, respectively. They form fructose-6-phosphate and glucose.
Q. 3. c. During fasting, explain what happens for carbohydrate metabolism (glycogen synthesis/breakdown; glucose synthesis/breakdown/insulin & glucagon levels)
A. During fasting the insulin levels fall and glucagon levels rise. Glucagon promotes cAMP-directed phosphorylation. Glycogen is broken down and gluconeogenesis takes place. Glycolysis activities are decreased.
Q. 4. Explain the major function of the pentose phosphate pathway (pentose shunt)?
A. The primary functions of the pentose phosphate pathway are the formation of NADPH molecules for reductive biosynthesis and the formation of ribose-5-phosphate for the synthesis of nucleotides and nucleic acids (Meisenberg).
Q. 5. a. Discuss the steps for lipid biosynthesis.
A. Lipid biosysnthesis consists of 3 steps – Initiation, elongation and release. Initiation starts with acetyl-CoA which transferred to a pantothenate group of acyl carrier protein on FAS and later to cysteine sulfhydral (–SH) group on FAS. This prepares the pantothenate of –SH group to accept a malonyl group from malonyl-CoA. Elongation starts with acceptance of the malonyl group by the pantothenate. Now, there are two activated substrates on FAS protein, acetyl on cysteine –SH and malonyl on pantothenate –SH. The 2 carbon acetyl unit is then transferred to the malonyl group. This forms a 4 carbon protein unit. This unit is then reduced, dehydrated and reduced again to form a 4 carbon saturated fatty acid. This carbon chain is now ready to accept another 2 carbon unit and the process of elongation continues till palmitoyl-ACP is formed. In the release step, thioesterase acts on FAS complex to release 16-carbon palmitate.
Q. 5. b. How much energy, in the form of ATP is released for each molecule of a 16 carbon long fatty acid? Explain why breakdown of fatty acids produces more energy that the break carbohydrate.
A. Each molecule of a 16 carbon fatty acid releases 131 ATP molecules but 2 ATP molecules are used for the initiation of the reaction. Breakdown of fatty acids produce greater energy as compared to carbohydrates because there are greater number of carbons to oxidize. Carbohydrates already have CO molecules. Thus, only one more oxygen molecule can be attached. Fatty acids on the other hand have carbon atoms with no oxygen molecules. It is the oxidation of carbon molecules which produces energy (Meisenberg).
Q. 6. Discuss the four “structures” for proteins. What are allosteric changes in protein structure? Which of these four structures would be affected by allosteric changes?
A. The four “structures” for proteins are the primary, secondary, tertiary and quaternary levels of protein structures. The primary structure is the linear sequence of amino acids making the protein. The secondary structure is due to the H-bond interactions in the chain leading to α-helix or a β-sheet form. In tertiary structure, the α-helix or β-sheet structures fold upon themselves through hydrophobic interactions, H-bonds and ionic bonds leading to a 3-dimensional structure. When two or more tertiary sub-units come together through similar interactions as above they lead to the formation of a quaternary structure (Meisenberg).
Allosteric changes in proteins are the changes in its structure due to binding of an effector molecule leading activation or de-activation of the protein. Usually, the allosteric changes require the protein to have a complex structure with their active sites. Hence, allosteric changes are seen only in quaternary or tertiary structures (e.g. Hemoglobin which has a quaternary structure) (Meisenberg).
Q. 7. Discuss the terms Vmax and Km. Discuss how metabolic control is exerted for a pathway (glycolysis for example). This may help Sephadex Column Preparation.
A. Vmax is the maximum reaction rate achieved by a system. Km is the concentration of substrate required to achieve half of Vmax. Metabolic control is exerted on a pathway through changes in concentrations of substrate and enzymes, through the allosteric regulation of enzymes and through covalent modification of enzymes. In glycolysis, for example, allosteric regulation of hexokinase, phosphofructokinase and pyruvate kinase is used for metabolic regulation (Meisenberg).
Twyman, Richard M. "Strategies for Protein Separation." Principles of Proteomics. New York: BIOS Scientific, 2004. 25. Print.
Bailey, Regina. "The 10 Steps of Glycolysis." About. Web. 8 Nov. 2014.
Meisenberg, Gerhard, and William H. Simmons. "Carbohydrate Metabolism." Principles of Medical Biochemistry. 3rd ed. Philadelphia: Elsevier/Mosby, 2012. Print.