Oxidation rates and stability in solution of mono- and bivalently cross-linked human hemoglobin

V. W. Macdonald, K. D. Vandcgriff, R. M. Winslow, D. Currell, C. Fronticelli, J. C. Hsia, J. C. Bakker

Research output: Contribution to journalConference articlepeer-review

6 Scopus citations


The purposes of this study were to determine: 1) how changes in oxygen affinity induced by different chemical modifications of hemoglobin affect rates of autooxidation and 2) the degree to which mono- or bivalent crosslinkers prevent precipitation of oxidation products. We studied human stroma-free Hb bivalently crosslinked between alpha globin chains (ααHb) or between beta chains (ββHb) with bis(3,5-dibromosalicyl)fumarate and between beta chains with 2-nor-2-formylpyridoxal 5′-phosphate (HbNFPLP). We also studied the 64KD fractions of Hb reacted with a crosslinker derived from peroxidate oxidized raffinose under oxy- (Hb[R]) or deoxy- (Hb[T]) conditions. Monovalent derivatives included Hb with mono(3,5-dibromosalicyl)fumarate bound to the beta chains (HbFMDA) and Hb in which alpha and beta chains were reacted with 2-isothiocyanato-benzene-1,4-disulfonic acid (HbICB) or 4-isothiocyanatobenzene sulfonic acid (HbICBS). Anion-induced autooxidations of Hbs were carried out with azide in PO4 buffer in a rapid scanning spectrophotometer. Pseudo-first-order rate constants (K′1) were calculated for each Hb and compared with those of unmodified HbAo. P50s (torr) were measured in BisTris (pH 7.4, 25°C) and in PO4 buffer (pH 7.2, 27°C). Decreased Hb oxygen affinities were associated with increased rates of autooxidation. Menadione-induced oxidation in the presence of 2-propanol caused precipitation of HbAo within 35-45 minutes, whereas precipitation of the bivalently crosslinked Hbs occurred much later (> 24 hrs). Precipitation of the monovalent Hb derivatives occurred within 1-2 hrs. This work provides further evidence that modifying Hb to enhance oxygen off-loading in tissues will increase intrinsic rates of methemoglobin formation. In addition, greater stabilization of the oxidized tetramers in solution is achieved with bivalent as opposed to monovalent crosslinkers.

Original languageEnglish (US)
Number of pages1
JournalBiomaterials, Artificial Cells, and Immobilization Biotechnology
Issue number2
StatePublished - Dec 1 1991
Externally publishedYes
Event8th World Congress of the International Society for Artificial Organs in conjunction with the 4th International Symposium on Blood Substitutes - Montreal, Que, Can
Duration: Aug 19 1991Aug 23 1991

ASJC Scopus subject areas

  • Medicine(all)


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