Hemoglobin Disorders: Molecular Methods and ProtocolsRonald L. Nagel Hemoglobin and Hemoglobinologists This volume, Hemoglobin Disorders: Molecular Methods and Protocols, will be introduced with a review of the great milestones in the field, and the scientists responsible for those achievements. The history of hemoglobin can be divided into three periods: the Classical period, the Modern period, and the Post-Modern period. I am inclined to include as the four major members of the classical period Francis Roughton, Quentin Gibson, Jeffries Wyman, and Linus Pauling, not only because of their achievements, but also because of the superb scientists they trained and/or influenced. Francis John Worsely Roughton (1899–1972) (Fig. 1), in his laboratory at Trinity College in Cambridge, England, made the first measurements of the rapid reaction of oxygen with hemoglobin at the millisecond scale, at first by flow-mixing methods and later by flash photolysis. He not only opened an era of molecular research of hemoglobin, but also invented the methodology for fast reactions through the use of laser technology, which was later improved by others so that even faster reactions could be detected. Another contribution of Roughton was the education of Quentin H. Gibson (Fig. 2), his favorite s- dent, who, in his laboratory in Sheffield, continued to expand the horizon of ligand binding to hemoglobin, defining the oxygen binding constants for each of the hemes of hemoglobin. Though this did not, as expected, solve the und- lying mechanism of ligand cooperativity as discussed below, it was nonet- less an important milestone. |
Contents
Purification and Molecular Analysis of Hemoglobin | 31 |
Oxygen Equilibrium Measurements of Human Red Blood Cells | 49 |
Measurement and NO of Rate with Hemoglobin Constants for Reactions of O 2 | 65 |
Electrophoretic Methods for Study of Hemoglobins | 93 |
DNA Diagnosis of Hemoglobin Mutations | 101 |
Methods for Analysis of Prenatal Diagnosis | 117 |
Hemoglobin Fluorescence | 133 |
Seetharama A Acharya and Sonati Srinivasulu 12 βGlobinlike Gene Cluster Haplotypes in Hemoglobinopathies | 177 |
Transgenic Mice and Hemoglobinopathies | 213 |
Recombinant Single GlobinChain Expression and Purification | 243 |
Nuclear Magnetic Resonance of Hemoglobins | 251 |
Solubility Measurement of the Sickle Polymer | 271 |
Index | 289 |
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Common terms and phrases
a-globin Acad acetate acid allosteric analysis Biol blood Bohr effect centrifuge Chem chromatography column concentration containing crystals CSAT cuvet deletion deoxy deoxygenation deoxyHb detection dimers dissociation dithionite electrophoresis elution emission equilibrated expression Fabry fetal fluorescence front-face gene cluster genetic globin globin chains globin gene gradient haplotype Hb solution Hb variants HbCO HbO2 heme hemoglobinopathies homozygous HPLC human Hb human hemoglobin kinetics ligand measurements Methods and Protocols mg/mL mixture Molecular molecules mouse mutation Nagel Natl normal nucleation oxidation oxygen affinity Pharmacia phosphate buffer photolysis polymer polymerization polymorphisms prenatal diagnosis primers protein protons Purification R-state rate constants RBCs reaction recombinant red cells resonance RP-HPLC sample semisynthetic sequence sickle cell anemia sickle cell disease solubility structure studies Subheading subunit Superose technique temperature tetramer thalassemia tion transgenic transgenic mice vacutainer