Polyethylene Glycol Conjugation Enhances the Nitrite Reductase Activity of Native and Cross-Linked Hemoglobin

Abstract

Although stabilized hemoglobins have been evaluated as oxygen-carrying replacements for red cells in transfusions, in vivo evaluations have noted that these materials are associated with vasoactivity, a serious complication. Scavenging of endogenous nitric oxide by the deoxyheme sites of the stabilized proteins is one likely source of vasoactivity. Recent reports indicate that modification of cell-free hemoglobin derivatives with multiple chains of polyethylene glycol (PEG) suppresses vasoactivity. Gladwin and co-workers observed that the nitrite reductase activity of hemoglobin serves as a major endogenous source of nitric oxide. If PEG conjugation leads to enhanced nitrite reductase activity, this could compensate for scavenged endogenous nitric oxide. To test this possibility, the rates of conversion of nitrite ion to nitric oxide by altered hemoglobins with and without PEG were measured at 25 degrees C. Fumaryl (alpha99-alpha99) cross-linked hemoglobin reacts with nitrite with a bimolecular rate constant of 0.52 M (-1) s (-1), which is comparable to that associated with native hemoglobin (0.25 M (-1) s (-1)). Addition of PEG chains to the cross-linked hemoglobin at beta-Cys93 (alphaalpha-Hb-PEG5K 2) results in a material that produces nitric oxide much more rapidly ( k = 1.41 M (-1) s (-1)). R-State-stabilized hemoglobins with multiple PEG chains (Hb-PEG5K 2 and Hb-PEG5K 6) react 10 times faster with nitrite to produce nitric oxide than does native hemoglobin ( k = 2.5 and 2.4 M (-1) s (-1), respectively). These results, showing enhanced production of nitric oxide resulting from an increased proportion of the protein residing in the R-state, are consistent with the decrease in vasoactivity associated with PEG conjugation.

Enhancing Nitrite Reductase Activity of Modified Hemoglobin: Bis-tetramers and Their PEGylated Derivatives

“…In this work we demonstrate the effects of various methods of hemoglobin crosslinking and conjugation on the protein's nitrite reductase activity (60). We show that PEG-hemoglobins react at enhanced rates to produce NO, a reaction that is related to the protein's affinity and cooperativity for oxygen binding and release.…”

“…follows that an HBOC with an increased molecular size, high affinity and cooperativity for oxygen and enhanced nitrite reductase activity will have some of the necessary properties for a safe blood substitute. We have now shown that the addition of PEG to hemoglobin at β-Cys93 significantly increases the rate of reduction of nitrite to NO compared to native and cross-linked hemoglobin (60). The increased rate of NO production may therefore also contribute to the decreased hypertensive effect observed with larger PEGylated hemoglobins (39,55,58,67,68).…”

“…We have also previously examined the nitrite reductase activity of BT-Hb and BT-PEG in comparison to other existing HBOCs and found that the correlation between nitrite reductase activity and the oxygen binding properties (P 50 and n 50 ) may play a significant role in making these materials non-vasoactive (60). The site at which the rate of NO production is fastest depends directly on the HBOC's P 50 (64,90).…”

Structural and Functional Studies Indicating Altered Redox Properties of Hemoglobin E

“…Other reactions and mechanisms are still under investigation (30, 36, 89 -91). Nonetheless, there are clear indications of the role of this reaction as seen in studies that show that the efficacy of potential Hb-based oxygen carriers are correlated with nitrite reductase activity (92)(93)(94).…”

Caracterização e aplicação analítica de eletrodos modificados com sistemas porfirínicos supramoleculares

“…Entretanto, esses procedimentos são laboriosos e requerem, em geral, 1 hora para obter curva de processo de reação [154,157] A Figura 63 mostra a relação entre a intensidade de corrente amostrada em -1,1 V e a concentração de NaNO 2 para cada um dos seis filmes estudados. Entretanto, a falta de reticulação em estrutura da Hb-PEG5K 2 desfavorece a sua aplicabilidade clínica pelo seu potencial risco de dissociação em espécies diméricas in vivo.…”