Hemoglobin tetramers stabilized by a single intramolecular cross-link.
A specific intramolecular cross-link was introduced into bovine and human hemoglobin by reaction of the deoxyhemoglobin with the dialdehyde, bispyridoxal tetraphosphate (bisPL)P4, followed by reduction with NaBH4. The yield of cross-linked hemoglobin is 80% in both cases, using 1 mol of (bisPL)P4 per mol of Hb. The crosslink is confined to the beta chains, where it connects the N-terminal residue (valine and methionine, respectively) to a lysine on the other beta chain across the central cavity. The stereochemical requirements for the reaction were probed by using a rigid analogous cross-linking reagent, as well as with a mutant Hb, which has a shorter distance between the residues to which the cross-link is attached. Introduction of the cross-link into human and bovine Hb results in a five-fold and four-fold reduction in the oxygen affinity and a decrease in the Bohr Effect by 1/3 and 1/2, respectively. Oxygenation remains cooperative, albeit with a decreased Hill coefficient. The cross-linked hemoglobins are oxidized more rapidly to the ferric form, but their resistance to heat denaturation is increased. The stability of the link between the beta chains and their hemes is 10 times greater in both cross-linked hemoglobins that in their native counterparts. The possible application of this chemical modification for the preparation of hemoglobin-based blood substitutes is discussed.
Benesch
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Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032.
Kwong
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S
D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents; D004355:Drug Stability; D006454:Hemoglobins / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011489:Protein Denaturation; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives
D003432:Cross-Linking Reagents; D006454:Hemoglobins; D046911:Macromolecular Substances; C073543:bispyridoxal tetraphosphate; D011732:Pyridoxal Phosphate; D010100:Oxygen