Site-Specific Semisynthetic Variant of Human Hemoglobin

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 85; no. 3; pp. 709 - 713
• Main Authors: Hefta, Stanley A., Lyle, Stephen B., Busch, Mark R., Harris, David E., Matthew, James B., Frank R. N. Gurd
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01-02-1988; National Acad Sciences
• Subjects: Amino acids; Analytical, structural and metabolic biochemistry; Anhydrides; Biochemistry; Biological and medical sciences; Dialysis; Flow velocity; Fundamental and applied biological sciences. Psychology; heme; hemoglobin; Hemoglobins; Hemoglobins - chemical synthesis; Hemoglobins - metabolism; Hemoproteins; Humans; Ligands; Magnetic Resonance Spectroscopy; man; Metalloproteins; Molecules; Oxygen; Oxygen - metabolism; Oxyhemoglobins - biosynthesis; Protein Conformation; Protein Multimerization; Proteins; Salts; Hemoprotein; Oxygen; Substitution; Valine; Molecular association; Hemoglobin; Affinity; Chemical synthesis; N terminal aminoacid
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.85.3.709

A single round of Edman degradation was employed to remove the NH2-terminal valine from isolated α chains of human hemoglobin. Reconstitution of normal β chains with truncated or substituted α chains was used to form truncated (des-Val1-α 1) and substituted ([[1-13C]Gly1]α 1) tetrameric hemoglobin analogs. Structural homology of the analogs with untreated native hemoglobin was established by using several spectroscopic and physical methods. Functional studies indicate that the reconstituted tetrameric protein containing des-Val1-α chains has a higher affinity for oxygen, is less influenced by chloride ions or 2,3-bisphosphoglycerate, and shows lower cooperativity than native hemoglobin. These results confirm the key functional role of the α -chain NH2 terminus in mediating cooperative oxygen binding across the dimer interface. The NH2-terminal pK1/2 value was determined for the [13C]glycine-substituted analog to be 7.46 ± 0.09 at 15 degrees C in the carbon monoxide-liganded form. This value, measured directly by 13C NMR, agrees with the determination made by the less-direct 13CO2 method and confirms the role of this residue as a contributor to the alkaline Bohr effect; however, it is inconsistent with the presence of an NH2-terminal salt bridge to the carboxylate of Arg-141 of the α chain in the liganded form.