AMINO ACID SEQUENCE FOR SICKLE CELL Sickle cell anemia is a genetic disorder that affects the protein

hemoglobin, of which red blood cells are primarily composed. Individuals with sickle cell anemia have misshapen red blood cells that tend to accumulate in vessels and create small clots, leading to organ damage and pain. The disease arises from a miscoded portion of the genes, or DNA, for hemoglobin, which results in a faulty amino acid sequence. FEATURES Sickle cell anemia, explains the National Heart Lung and Blood Institute, is the result of a genetic mutation that can be spread from parent to offspring and produces misshapen red blood cells. While normal red blood cells have a biconcave shape, making them similar in appearance to a doughnut with an incomplete hole in the center, sickled cells look like quarter moons. They stick to one another, instead of slipping past each other in blood vessels, and have short life spans compared to healthy cells. CHEMICAL BASIS The reason sickled red blood cells have such an odd shape is that they're made up of misshapen hemoglobin. Hemoglobin is a protein, a member of the same class of molecules that make up muscles and connective tissue like collagen. Instead of acting as structural material like collagen, however, the hemoglobin protein carries oxygen. Like all proteins, explain Drs. Mary Campbell and Shawn Farrell, hemoglobin is made up of a long string of amino acids. MUTATIONS The body makes proteins by stringing amino acids together in a specific sequence, the instructions for which are coded into the genetic material, or DNA. In their text "Biochemistry," Drs. Reginald Garrett and Charles Grisham explain that mistakes in DNA result in mistakes in amino acid sequences. There are 20 types of naturally occurring amino acids, which can be put together in any sequence or combination to make proteins that vary in length from tens to thousands of amino acid units. Even a single incorrect amino acid significantly affects the function of a protein. HEMOGLOBIN MUTATION The hemoglobin protein normally consists of four strands of around 140 amino acids each, the seventh of which in two of the strands is the compound glutamic acid, explain Drs. Campbell and Farrell. In sickle cell patients, the seventh amino acid in two of the strands is not glutamic acid but is instead a very different amino acid, called valine. Valine and glutamic acid have significant differences in their chemical structures and properties, meaning that the mutated strands act and are shaped differently, leading to mutant hemoglobin.

Definition Thalassemia is an inherited disorder that leads to the decreased production and increased destruction of red blood cells. Hemoglobin, found in red blood cells, is responsible for carrying oxygen through the body to all of the organ systems. Low hemoglobin, due to loss of red blood cells, leads to anemia and the inability of the body to deliver oxygen and maintain normal functions.

Thalassemias are named for the amino acid chain in the hemoglobin molecule that is affected. Amino acids are the building blocks of protein. Hemoglobin has two separate amino acid chains, alpha and beta. Thalassemias are also categorized by the number of genes that are absent or defective:

Alpha thalassemiathe alpha chain is affected Silent carrierone gene affected Thalassemia traittwo genes affected Hemoglobin H diseasethree genes affected Alpha hydrops fetalisfour genes affected Beta thalassemiathe beta chain is affected Thalassemia minorone abnormal gene Thalassemia major (Cooley's anemia)two abnormal genes

Spherocytosis Nondominant hereditary spherocytosis (ndHS) is a disorder characterized in some patients by severe hemolytic anemia and marked deficiency of erythrocyte spectrin. This report describes the identification of a variant spectrin chain, alpha-spectrin Bughill or alpha(BH), that is associated with this disorder in a number of patients. Tryptic maps of spectrin from affected individuals revealed an acidic shift in isoelectric point of the alphaII domain peptides at 46 kD and 35 kD. A point mutation at codon 970 of the alpha-spectrin gene (GCT-->GAT), that changes the encoded amino acid from an alanine to an aspartic acid, was identified in genomic DNA of affected patients. The alpha(BH) variant was present in 8 patients with ndHS from five different kindreds but was absent in 4 patients from two other kindreds. The 8 ndHS patients with the alpha(BH) variant appeared to be homozygous for the alpha(BH) variant by analysis of peptide maps of limited tryptic digests of erythrocyte spectrin. However, following genomic DNA analysis, only 2 of these patients were true homozygotes, whereas 6 were found to be doubly heterozygous for the alpha(BH) allele and a second, presumably abnormal, alpha-spectrin gene. These results suggest that, in these 6 patients, the second alpha-spectrin allele is in fact associated with one or more genetic defect(s), causing decreased accumulation of alpha-spectrin. The pattern of transmission of the alpha(BH) allele in certain families suggests that the alpha(BH) amino-acid substitution is not itself responsible for ndHS but is more likely a polymorphic variant that, in some but not all cases, is in linkage disequilibrium with another uncharacterized alpha-spectrin gene defect that itself is a cause of ndHS.

RBC LYSIS Red blood cell (RBC) lysis and iron release contribute to intracerebral hemorrhage (ICH)-induced brain injury. Tissuetype transglutaminase (tTG), which has a role in neurodegeneration, is upregulated after ICH. The current study investigated the effect of RBC lysis and iron release on brain tTG levels and neuronal death in a rat model of ICH. This study had three parts: (1) Male Sprague-Dawley rats received an intrahippocampal injection of 10 L of either packed RBCs or lysed RBCs; (2) rats had a 10 L injection of either saline, hemoglobin or FeCl2; (3) rats received a 10 L injection of hemoglobin and were treated with an iron chelator, deferoxamine or vehicle. All rats were killed 24 h later, and the brains were sectioned for tTG and Fluoro-Jade C staining. Lysed but not packed RBCs caused marked tTG upregulation and neuronal death in the ipsilateral hippocampus CA-1 region. Both hemoglobin and iron mimicked the effects of lysed RBCs, resulting in tTG expression and neuronal death. Hemoglobin-induced tTG upreglution and neuronal death were reduced by deferoxamine. These results indicate that RBC lysis and iron toxicity contribute to neurodegeneration after ICH.

The hemoglobin molecule is made up of four polypeptide chains: two alpha chains < >of 141 amino acid residues each and two beta chains < > of 146 amino acid residues each. The alpha and beta chains have different sequences of amino acids, but fold up to form similar three-dimensional structures. The four chains are held together by noncovalent interactions. There are four binding sites for oxygen on the hemoglobin molecule, because each chain contains one heme group < >. In the alpha chain, the 87th residue is histidine F8 < >and in the beta chain the 92nd residue is histidine F8 >. A heme group is attached to each of the four histidines. The heme consists of an organic part and an iron atom < >. The iron atom in heme binds to the four nitrogens in the center of the protoporphyrin ring. The hemoglobin molecule is nearly spherical, with a diameter of 55 angstroms . The four chains are packed together to form a tetramer. The heme groups are located in crevices near the exterior of the molecule, one in each subunit. Each alpha chain is in contact with both beta chains< >. However, there are few interactions between the two alpha chains or between the two beta chains >. Each polypeptide chain is made up of eight or nine alpha-helical segments < >and an equal number of nonhelical ones placed at the corners between them and at the ends of the chain. The helices are named A-H, starting from the amino acid terminus, and the nonhelical segments that lie between the helices are named AB, BC, CD, etc. The nonhelical segments at the ends of the chain are called NA at the amino terminus and HC at the carboxyl terminus. To form the tetramer < >, each of the subunits is joined to its partner around a twofold symmetry axis, so that a rotation of 180 degrees brings one subunit into congruence with its partner. One pair of chains is then inverted and placed on top of the other pair so that the four chains lie at the corners of a tetrahedron. The four subunits are held together mainly by nonpolar interactions and hydrogen bonds. There are no covalent bonds between subunits. The twofold symmetry axis that relates the pairs of alpha and beta chains runs through a water-filled cavity >at the center of the molecule. This cavity widens upon transition form the R structure to the T structure to form a receptor site for the allosteric effector DPG (2,3 diphosphoglycerate) between the two beta chains. The heme group is wedged into a pocket of the globin with its hydrocarbon side chains interior and its polar propionate side chains exterior. There are nine positions in the amino acid sequence that contain the same amino acid in all or nearly all species studied thus far. These conserved positions are especially important for the function of the hemoglobin molecule. Several of them, such as histidines F8 (His87)< > and E7 (His63)< >, are directly involved in the oxygen-binding site< > . Phenylalanine CD1 (Phe43) < > and leucine F4 (Leu83) < > are also in direct contact with the heme group< >. Tyrosine HC2 (Tyr140) < >stabilizes the molecule by forming a hydrogen bond between the H< > and F helices< >. Glycine B6 (Gly25)< >is conserved because of its small size: a side chain larger than a hydrogen atom would not allow theB< > and E helices< > to approach each other as closely as they do. Proline C2 (Pro37)< > is important because it terminates the C helix. Threonine C4 (Thr39) and lysine H10 (Lys127) are also conserved residues, but their roles are uncertain.