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Got Electrophoresis? Funded by the National Science Foundation EPSCoR (05-589) Sickle-Cell Anemia University of Southern Mississippi Sherry Herron, Bridgette Davis, and Parker Nelson May 7, 2008 Agenda A.M. 8-9 Orientation, Survey, and Activity Overview 9-12 Electrophoresis Chamber Construction, Gel and Buffer preparation P.M. 12:30 Electrophoresis and Explanations 2-3 Post Activity Discussion and Evaluation Problem: The ubiquitous, yet obsolete and oversimplified presentation of sickle cell disease in biology textbooks. From newest innovative NSFfunded high school biology textbook ~ 2 pages Rationale for inclusion of Sickle Cell Topics Research in cognition has demonstrated that learning is most effective when topics are of human interest, relate to one another (theme-oriented), and relate to previously learned concepts (scaffolding). A focus on sickle cell anemia satisfies these criteria and can facilitate the teaching of a variety of biological, chemical, social, medical, and ethical concepts. The initial unit could serve as the basis for the development of additional units designed for evolution, genetics, medical school, biochemistry, biotechnology, and bioethics college courses. Statistics from the CBCF Health Organisation, 2004 Most cases occur in those with African ancestors in America 2 million people carry the sickle cell genetic trait in America 1 in 12 African Americans carry the sickle cell trait in America About 91,000 people with sickle cell anemia ("Orphan Products: Hope for People With Rare Diseases", By Carol Rados, FDA Consumer magazine, November-December 2003 Issue) 1 in 500 African American births in America 1 in 1,000-1,400 Hispanic American births in America Deaths from Sickle Cell Anemia: 501 deaths (NHLBI 1999) Death rate extrapolations for USA for Sickle Cell Anemia: 500 per year, 41 per month, 9 per week, 1 per day, 0 per hour, 0 per minute, 0 per second. Statistics from the CDC Sickle cell disease affects millions of people throughout the world and is particularly common among those whose ancestors come from sub-Saharan Africa, Spanish-speaking regions in the Western Hemisphere (South America, the Caribbean, and Central America), Saudi Arabia, India, and Mediterranean countries such as Turkey, Greece, and Italy. More than 70,000 people have sickle cell disease in the U.S. Sickle cell disease occurs in 1 in every 500 African American births. 2 million people have sickle cell trait in the U.S. 1 in 12 African Americans has sickle cell trait in the U.S. Did you know? Sickle cell disease occurs more often in people from parts of the world where malaria is or was common. It is believed that people who carry the sickle cell trait are less likely to catch malaria. Sickle cell disease is a major public health concern. From 1989 through 1993, there were an average of 75,000 hospitalizations due to SCD in the U.S., costing ~ $475 million. Sickle cell anemia is the most common inherited blood disorder in the U.S., affecting about 72,000 Americans or 1 in 500 African Americans. (Source: Genes and Disease by the National Center for Biotechnology) Malaria kills over 3000 children each day in Africa or 1 every 30 seconds Malaria is the # 1 killer of children under 5 in subSaharan Africa Malaria kills more than 1 million people each year Young children and pregnant women are most likely to become severely ill and die from malaria Malaria was eradicated from the United States over 50 years ago, yet more than 40 percent of the world's population is at risk http://www.malarianomore.org/index.php YOUR electrophoresis chamber! What is electrophoresis? Electrophoresis refers to a separation technique in which an electrical field causes charged molecules to move through a matrix (usually a gel). Electrophoresis is routinely used to separate DNA, protein and other polymeric molecules. Applications include forensics and biotechnology research. Electrophoresis of DNA Electrophoresis Equipment The power supply, black and red cords leading from the power supply are attached to the tray in which the gel is run, located on the white benchtop. Molecular Biology Cyberlab: http://www.life.uiuc.edu/molbio/geldigest/equipment.html Gel Casting The tray is the actual mold which provides a shape for the gel as it polymerizes. A comb is placed into slots in the tray, with the "teeth" down, when the agarose is still hot. The agarose polymerizes (after about 15 minutes) with small "wells" in it into which samples are added. The comb is gently lifted up out of the gel after running buffer has been poured over the gel. Adding the samples 5-10 µl is all that is typically loaded into each well Power Supply + - Cathode – Anode + (Red) Electrophoresis Chamber (Black) 500bp DNA ladder Contains 16 bands in 500bp increments; the smallest one is 500 bp Used to estimate DNA fragment sizes using a 0.8% - 1.0% agarose gel. The concentration of an agarose gel allows for the separation of different sizes of DNA molecules 0.5% 1.0% 100Kb 20Kb 20Kb (50bp) .5Kb (500bp) .05Kb 2.0% 4Kb (4000bp) Voltage The higher the voltage, the faster the rate of migration. However, Accompanying heat may melt the gel. (Don’t use more than 125 volts). Imperfections in the gel distort the bands and produce ambiguous results (slants and smiles). Electrophoresis Enzyme will cut 160 base pair normal protein into two 80 bp fragments; will not cut mutated protein. Measure against a 100 bp ladder What are the genotypes of the father, mother, and their newborn child? Gel stained with ethidium bromide and visualized with UV light Electrophoresis Ladder Diagram Hemoglobin: A Molecule to Breathe By In lungs: In tissues: Hb + 4 O2 ----------------> Hb.O8 Hb.O8 -----------------> Hb + 4 O2 In the tissues, some Hb picks up CO2 (~25% of total) and transports it back to the lungs where it is released. Basic Hemoglobin Structure 2 alpha globin subunits 2 beta globin subunits Each globin subunit consists of 8 alpha helices folded together into an identical shape. Each globin subunit contains an identical heme group. Model of the beta globin chain The heme group (in red) is held in place by interactions with histidine side chains (shown in green). The iron atom is located in the middle of the heme molecule. Protoporphyrin and Heme Hemoglobin’s affinity for Oxygen Fe 2+ (in middle of the heme group) lies slightly below the plane of the ring. When bound to O2,, Fe lies in the plane of the heme group. Heme absorbs green and yellow wavelengths; reflects orange and red. The Genes that produce Adult Human Hemoglobin 2 α globin: gene on chromosome 16 2 β globin: gene on chromosome 11 Alpha Globin (HbA) Gene Locus Chromosome 16 2 ζ genes expressed only first few weeks of development 2 α genes expressed thereafter 5’----ζ2--ζ1--α1--α2--α1----3’ Beta Hemoglobin (HbB) Gene loci: 11p 15.5. 3 exons (coding regions) scattered over 1600 base pairs Yields a 626-bp mRNA transcript Translated into a 147 amino acid polypeptide Beta Globin (HbB) Locus: multiple genes arranged sequentially from 5’ to 3’ Epsilon ε – expressed during first trimester Gamma γ – “ during fetal development Delta δ - “ in small quantities Beta β – most abundant 5’--- ε---Gγ--Aγ---β1—δ---β---3’ Human hemoglobin Embryonic: 2ζ, 2ε; 2α, 2ε Fetal (HbF): 2α, 2γ Adult (HbA2): 2α, 2δ Adult (HbA): 2α, 2β HbF has a much higher affinity for oxygen than HbA. A significant amount of HbF persists for ~8 months after birth. Bioinformatics… The Evolution of Hemoglobin is a story of… Duplications Mutations Transpositions Over billions of years through plants and all animals (see color page) Evolution of Hemoglobin http://www.people.virginia.edu/~rjh9u/slidlist.html Anemia Any significant decrease in amount of functional Hb Due to a number of causes All forms have serious physiological effects Sickle Cell Anemia: A Series of Firsts 1st known molecular disease. Thousands of such diseases (most rare), including over 150 mutants of hemoglobin alone, are now known. 1st disease by which electrophoretic analysis was applied (by Linus Pauling and Harvey Itano). Microscopic observations showed that individuals with sickle cell trait had about half normal and half sickle cell hemoglobin 1st verified case of a genetic disease that could be localized to a defect in the structure of a specific protein molecule. HbA HbS Sickled Red Blood Cells contain Deoxy HbS HbS polymerizes when deoxygenated More rigid than HbA Stickier than HbA: adheres to walls of blood vessels Clogs the arterioles and capillaries preventing the blood from delivering oxygen and nutrients, and removing carbon dioxide and wastes from the tissues. Deoxy HbS DeoxyHbS DeoxyHbS molecules lock together, line up into long fibers inside the RBC and become rigid, precipitate out of solution and cause the RBC to collapse. End on view (Stetson, J. Exp. med. 123:341-346, 1966.) Longitudinal view of deoxyHbS [From G. Rykes, R.H. Crepeau, and S.J. Edelstein. Nature 72(1978):509.] Sickle Cell Anemia Genotype: HbS HbS Short-Term: Frequently out of breath and easily tired Crisis: acute musculo-skeletal pain caused by loss of oxygen in tissues Long-Term: Hand-foot syndrome: dactylitis damages small bones of hands and feet during first 2 years of life. Tissue death in top of femoral bone often crippling Other complications include stroke, organ damage or failure, leg ulcers. Shortened life span Sickle Cell Trait Heterozygous condition: HbA and HbS Individual is asymptomatic most of the time Crises occur when the oxygen saturation falls below 40% or when barometric pressure falls to 50 mm of mercury (1/3 normal level) Malaria: A 2-host disease The sexual stage develops in the mosquito. The asexual stage (rapid cell division) of the protozoan, Plasmodium vivax, occurs within the red blood cells of humans. The RBCs burst accompanied by a very high fever; followed by lower temperature and the sensation of "chills”. An Adaptive Response: Resistance to Malaria In HbA HbS individuals, half their hemoglobin will sickle when the oxygen tension becomes very low. These sickled cells are removed from the body by the spleen, along with the merozoites inside of them. Thus, heterozygotes remove the infected cells from their body before the protozoans can produce a large infectious population. Balanced polymorphism Balanced polymorphism: When the heterozygote in any population is selectively favored over either homozygote. In certain parts of Africa today, the frequency of HbS is very high (10-20%). HbS HbS may also have an advantage against malaria, but they have all the other problems associated with sickle cell disease, and hence are severely selected against and seldom reproduce. Malaria/Sickle Cell Geographic Correlation Other Hemoglobin Diseases that also offer immunity to malaria Thalessemia Ovalocytosis Favism (G6PD deficiency) Why is Electrophoresis a Tool? HbA and HbS have different charges! Glutamic acid (in HbA) is acidic Valine (in HbS) is neutral Therefore, HbS has 2 fewer negative charges that HbA – which changes the pH, pI, tertiary structure, quaternary structure, and oxygen affinity (function) of hemoglobin. Polymerizes when deoxygenated. Glycine, the smallest amino acid Main Chain Amine group: NH2 Alpha carbon Carboxylic acid group: COOH Side group: H –makes it aliphatic (linear) In others, makes it aromatic (ring), acidic, basic, hydroxylic, sulphur containing, or amidic (containing amide group) Primary Structure of Proteins: A Review Acidic amino acids Basic amino acids Aspartic Acid Glutamic Acid Arginine Lysine Histidine Neutral amino acids – all the rest! If # of + and – ions are equal: NH3+ = COODepending on the functional groups, the side chains are aliphatic, aromatic, acidic, basic, hydroxylic, sulphur containing, or amidic (containing amide group). Acids and Bases An acid is a proton donor A base is a proton acceptor COOH NH3 + COONH2 The ratio of acid to base is dependent upon the pH and pKa. pH – pKa = log[unprotonated] [protonated] At pH 3, pKa = 0, the ratio of COO- / COOH is 1/1 At pH 9, pKa = 0, the ratio of NH2 /NH3 + is 1/1 Isoelectric Point The pH at which an amino acid or protein bears no net charge and, therefore, does not migrate in an electric field. pI of neutral amino acids is around pH 6 pI of acidic amino acids is close to pH 3 Aspartic Acid Glutamic Acid pI of basic amino acids is close to pH 9 Arginine: 10.8 Lysine: 9.7 What happens to an amino acid in a strong acid solution? In an acid solution (excess H+) The amine groups become “protonated” (positively charged) NH2 → NH3 + The carboxyl groups are “unprotonated” (not ionized) Which way will they migrate during electrophoresis? What happens to an amino acid in a strong alkaline solution? In an alkaline (basic) solution (excess OH-) The carboxyls are negatively charged COOH → COO- + H + H+ + OH- → H2O The amino groups are not ionized. Which way will they migrate during electrophoresis? Importance of the Buffer The pH of the buffer we will use is 8.6. Most proteins are negatively charged at 8.6. Which way will they travel during electrophoresis? How fast will they travel? The rate of migration depends upon its net charge; the higher the charge the faster the protein will travel. Important Links http://globin.cse.psu.edu/ http://www.accessexcellence.org/AE/AEPC/WWC/1993/hemoglo bin.php National Heart, Lung and Blood Institute, National Institute of Health. Sickle cell anemia: Who is at risk? Available at www.nhlbi.nih.gov/health/dci/Diseases/Sca/SCA_WhoIsAtRisk.ht ml Accessed November 3, 2006. Ashley-Koch, A., et al. Centers for Disease Control and Prevention. Sickle Hemoglobin (HbS) Allele and Sickle Cell Disease. American Journal of Genetics. May 1, 2000. Accessed March 26, 2007.