A Doubly Cross-linked Human Hemoglobin

Abstract

Human deoxyhemoglobin cross-linked with trimesyl tris(3,5-dibromosalicylate) produces the previously reported cross-linked hemoglobin in which the ⑀ amino groups of the two ␤ chain 82 lysyl residues are joined by a trimesyl bridge. Further specific modification of this protein directed to the ␣ subunits with bis(3,5-dibromosalicyl)fumarate gives a doubly cross-linked material in which the ⑀-amino groups of the two ␣ chain 99 lysyl residues are now joined by a fumaryl bridge. The singly cross-linked ␤ chain species binds oxygen cooperatively with a high oxygen affinity (P 50 ‫؍‬ 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 ␣ 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.

Application of zero-length cross-linking to form lysozyme, horseradish peroxidase and lysozyme–peroxidase dimers: Activity and stability

“…Intermolecular covalent cross-linking of functional groups in proteins has proved to be another very useful approach [6][7][8]. specifically cross-linked hemoglobins are useful for the investigation of structure-function relationships [9][10][11], as well as in the development of potential substitutes for red blood cell transfusion [12][13][14]. Zero-length cross-linking is defined when the peptide chains are covalently linked through existing functional groups without incorporation of a spacer group [15].…”

Molecular engineering of a polymer of tetrameric hemoglobins

“…In order to prevent the dissociation into dimers and, thus, increase the retention time in circulation, several types of chemical modifications have been implemented that stabilize the tetrameric form by introducing an intramoleular crosslink between either the α or the β‐subunits 8–10. These derivatives have an increased retention time in circulation 11.…”

Allosteric intermediates indicate R2 is the liganded hemoglobin end state

“…The crosslinked hemoglobins, which display only slightly lower oxygen affinities than uncrosslinked hemoglobin and normal cooperativities (14,15), were prepared by reacting human deoxyhemoglobin with the bis(methylphosphate) derivatives (15,16) resolution (Table 1). Fig.…”