title: N-terminal acetylation and protonation of individual hemoglobin subunits: position-dependent effects on tetramer strength and cooperativity.
abstract: The presence of alanine (Ala) or acetyl serine (AcSer) instead of the normal Val residues at the N-terminals of either the alpha- or the beta-subunits of human adult hemoglobin confers some novel and unexpected features on the protein. Mass spectrometric analysis confirmed that these substitutions were correct and that they were the only ones. Circular dichroism studies indicated no global protein conformational changes, and isoelectric focusing showed the absence of impurities. The presence of Ala at the N-terminals of the alpha-subunits of liganded hemoglobin results in a significantly increased basicity (increased pK(a) values) and a reduction in the strength of subunit interactions at the allosteric tetramer-dimer interface. Cooperativity in O(2) binding is also decreased. Substitution of Ala at the N-terminals of the beta-subunits gives neither of these effects. The substitution of Ser at the N terminus of either subunit leads to its complete acetylation (during expression) and a large decrease in the strength of the tetramer-dimer allosteric interface. When either Ala or AcSer is present at the N terminus of the alpha-subunit, the slope of the plot of the tetramer-dimer association/dissociation constant as a function of pH is decreased by 60%. It is suggested that since the network of interactions involving the N and C termini of the alpha-subunits is less extensive than that of the beta-subunits in liganded human hemoglobin disruptions there are likely to have a profound effect on hemoglobin function such as the increased basicity, the effects on tetramer strength, and on cooperativity.
authors.affiliation: Department of Biology, Mugar Life Sciences Building, Room 134, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ashiuchi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yagami
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Willey
authors.forename: Julio C
authors.initials: JC
authors.lastname: Padovan
authors.forename: Brian T
authors.initials: BT
authors.lastname: Chait
authors.forename: Anthony
authors.initials: A
authors.lastname: Popowicz
authors.forename: Lois R
authors.initials: LR
authors.lastname: Manning
authors.forename: James M
authors.initials: JM
authors.lastname: Manning
mesh_terms: D000107:Acetylation; D000409:Alanine / Q000737:chemistry / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D006441:Hemoglobin A / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D010100:Oxygen / Q000737:chemistry; D020836:Protein Structure, Quaternary / Q000235:genetics; D011522:Protons; D012694:Serine / Q000737:chemistry / Q000235:genetics
chemical_list: D011522:Protons; D012694:Serine; D006441:Hemoglobin A; D000409:Alanine; D010100:Oxygen