title: A doubly cross-linked human hemoglobin. Effects of cross-links between different subunits.
abstract: Human deoxyhemoglobin cross-linked with trimesyl tris(3,5-dibromosalicylate) produces the previously reported cross-linked hemoglobin in which the epsilon amino groups of the two beta chain 82 lysyl residues are joined by a trimesyl bridge. Further specific modification of this protein directed to the alpha subunits with bis(3,5-dibromosalicyl)fumarate gives a doubly cross-linked material in which the epsilon-amino groups of the two alpha chain 99 lysyl residues are now joined by a fumaryl bridge. The singly cross-linked beta chain species binds oxygen cooperatively with a high oxygen affinity (P50 = 4.8 torr at pH 7.4). The addition of the second cross-linking reduces the oxygen affinity to 15.9 torr, which compares with 13.0 torr for the singly cross-linked alpha chain species. The doubly cross-linked hemoglobin retains significant cooperativity with a Hill coefficient of 2.3 compared with 3.0 for unmodified hemoglobin. Because some of the groups responsible for the Bohr effect are acylated, this doubly cross-linked hemoglobin exhibits 25% of the normal Bohr effect and less than 20% of the normal chloride effect. The use of two distinct cross-links within the same tetramer provides a material for physical and structural analysis as well as for further modifications for specific applications. The results indicate that the cross-link introducing the lowest oxygen affinity in the two singly cross-linked species appears to control the overall affinity in this doubly cross-linked species.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, Oregon Health Sciences University, Portland 97201, USA.
authors.forename: R T
authors.initials: RT
authors.lastname: Jones
authors.forename: D T
authors.initials: DT
authors.lastname: Shih
authors.forename: T S
authors.initials: TS
authors.lastname: Fujita
authors.forename: Y
authors.initials: Y
authors.lastname: Song
authors.forename: H
authors.initials: H
authors.lastname: Xiao
authors.forename: C
authors.initials: C
authors.lastname: Head
authors.forename: R
authors.initials: R
authors.lastname: Kluger
mesh_terms: D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D006454:Hemoglobins; D010100:Oxygen