The quaternary structure of tetrameric hemoglobin regulates the oxygen affinity of polymerized hemoglobin

Abstract

This study focuses on the effect of the initial quaternary structure of bovine hemoglobin (Hb) on the physical properties of glutaraldehyde polymerized Hb (PolyHb) solutions. Tense (T) state PolyHb was synthesized by maintaining the pO(2) of Hb before and after polymerization at 0 mm Hg. In contrast, relaxed (R) state PolyHb was generated by maintaining the pO(2) of Hb before and after polymerization to >749 mm Hg. PolyHb solutions were characterized by measuring the pO(2), methemoglobin (metHb) level, molecular weight distribution, O(2) affinity and cooperativity coefficient. The metHb level of all PolyHb solutions was low (<2%). Analysis of the molecular weight distribution of PolyHb solutions indicates that in general, the molecular weight of PolyHb solutions increased with increasing cross-link density. T-state PolyHb solutions exhibited lower O(2) affinity compared to unmodified Hb, whereas R-state PolyHb solutions exhibited higher O(2) affinity compared to unmodified Hb. In addition, the polymerization reaction resulted in a significant decrease in cooperativity that was more pronounced at higher cross-link densities. All of these results were explained in terms of the quaternary structure of Hb. Taken together, our results yield more insight into the importance Hb's quaternary structure plays in defining the physical properties of glutaraldehyde PolyHb solutions. This information will be useful in designing optimized glutaraldehyde PolyHb oxygen carriers for various applications in transfusion medicine.

Tissue oxygenation after exchange transfusion with ultrahigh-molecular-weight tense- and relaxed-state polymerized bovine hemoglobins

“…Hence, T-state PolybHb was frozen in the low-O 2 affinity state via intra-and intermolecular glutaraldehyde cross-links within the bHb tetramer and between tetramers. The P 50 of high-MW T-state PolybHb (40 mmHg) was statistically different to that of unmodified bHb (26 mmHg) (30) and similar to the reported value of 38 mmHg for Hemopure (a glutaraldehyde cross-linked bHb manufactured by Biopure) (36). In the case of R-state PolybHb, bHb was maintained in the R state (high O 2 affinity) during the polymerization process and subsequently quenched with both NaCNBH 3 and NaBH 4 .…”

“…The P 50 of R-state PolybHb is 1 mmHg. The cooperativity of the two PolybHb solutions is much less compared with the reported value for unmodified bHb (2.5) (30). This behavior is explained by the glutaraldehyde cross-links present within and between the globin chains, which restrict the transmission of any quaternary changes in the Hb structure to other neighboring globin chains within the Hb tetramer.…”

Biophysical Properties and Oxygenation Potential of High-Molecular-Weight Glutaraldehyde-Polymerized Human Hemoglobins Maintained in the Tense and Relaxed Quaternary States

“…[18][19][20] PolyhHb synthesis T-state PolyhHb was synthesized as previously described in the literature. 9,[21][22][23] hHb was diluted with 20 mM phosphate buffer (PB; pH 8.0) to yield 1.5 L of 0.3 mM hHb that was stored in a 2 L glass bottle submerged in an ice bath. The contents of the glass bottle were then subjected to vacuum for 1 min, followed by argon purging for 1 min.…”

“…[21][22][23] About 1.5 L of 0.3 mM hHb in 20 mM PB was purged with pure O 2 for 2 h in an ice bath, and its pO 2 was continuously monitored with a RapidLab 248 Blood Gas Analyzer. After the pO 2 was out of range (>749 mm Hg), glutaraldehyde was added at the following glutaraldehyde to hHb molar ratios-30:1 and 20:1.…”

Synthesis, biophysical properties and pharmacokinetics of ultrahigh molecular weight tense and relaxed state polymerized bovine hemoglobins

“…In a recent study, we polymerized Hb exclusively in either the tense (T) or relaxed (R) quaternary states at different cross-link densities [46]. However, in this study no attempt was made to separate unpolymerized Hb from Hb polymers.…”