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Evaluating the capacity to generate and preserve nitric oxide bioactivity in highly purified earthworm erythrocruorin: a giant polymeric hemoglobin with potential blood substitute properties.

Roche CJ, Talwar A, Palmer AF, Cabrales P, Gerfen G, Friedman JM - J. Biol. Chem. (2014)

Bottom Line: A potentially important additional property is the capacity of the HBOC either to generate nitric oxide (NO) or to preserve NO bioactivity to compensate for decreased levels of NO in the circulation.The results show that LtHb undergoes the same reactions as HbA and that the reduced efficacy for these reactions for LtHb relative to HbA is consistent with the much higher redox potential of LtHb.Evidence of functional heterogeneity in LtHb is explained in terms of the large difference in the redox potential of the isolated subunits.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461.

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Evolution of a metHbA sample (0.29 mm heme) at pH 7 as a function of initial nitrite concentration upon addition of an aliquot of a NONOate solution yielding a final ratio of NONOate to heme of 1:1.A, 1:1 nitrite initially added prior to addition of NO; B, 1 mm nitrite initially added; C, 10 mm nitrite initially added. Insets show a representative time sequence of spectra from which the populations were derived.
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Figure 9: Evolution of a metHbA sample (0.29 mm heme) at pH 7 as a function of initial nitrite concentration upon addition of an aliquot of a NONOate solution yielding a final ratio of NONOate to heme of 1:1.A, 1:1 nitrite initially added prior to addition of NO; B, 1 mm nitrite initially added; C, 10 mm nitrite initially added. Insets show a representative time sequence of spectra from which the populations were derived.

Mentions: Earlier work on HbA demonstrated the formation of a spectroscopically distinct species formed when both NO and nitrite are added to metHbA (14, 34, 41). That species was assigned in those earlier studies as the NA intermediate in which N2O3 is coordinated to a ferrous heme. It was also shown that the species associated with the NA intermediate can generate S-nitrosothiols such as GSNO (14). It was shown based on the formation of this spectroscopically distinct species that for HbA the NA reaction can effectively compete with the RN reaction (14, 34). Here we addressed whether the intermediate can be generated by LtHb and, if so, whether the NA reaction can compete with the RN reaction given the high redox potential and fast rate of RN for LtHb. As was done with HbA, we started with an excess of nitrite (30 mm), thus creating a substantial population of met nitrite for LtHb. The addition of a small amount of NO (via NONOates) resulted in the transformation of the met nitrite spectrum to one that resembles the HbA intermediate. The same transition occurred when low levels of NO were slowly generated by deoxy-LtHb produced by the addition of the reductant l-Cys to an LtHb sample with a large initial population of met nitrite. Fig. 5 compares for both LtHb and HbA the Q band spectra of the intermediate with the Q band spectra from ferric and ferrous NO derivatives and from the met nitrite derivative. Table 6 lists the spectral peaks for all of these NOx-related species. The spectrum of the NA intermediate was then used as a member of the basis set to fit the evolving populations during the course of reactions in which both NO and nitrite were added or generated. Fig. 6 compares the evolution of populations for metLtHb upon addition of a very low amount of NONOates in the presence (A) and absence (B) of an excess of nitrite. In the presence of the large excess of nitrite, the addition of the NONOates generates primarily the intermediate, whereas in the absence of nitrite, the addition of the identical low amount of NONOate triggers the RN reaction. It can be seen that at this level of added NO the RN reaction does not go to completion but instead plateaus after a rapid initial buildup of the ferrous NO derivative of LtHb. The addition of a second aliquot does drive the reaction to near completion but at a slower rate with a noticeable buildup of the met NO derivative prior to formation of the ferrous NO derivative. Fig. 7 shows the evolution of an initial population of the met nitrite derivative of LtHb upon addition of very small aliquots of NONOates to a metLtHb sample in the presence of excess nitrite. The pattern is very similar to that reported previously for HbA in that there is a progressive buildup of intermediate with addition of NO until the population of intermediate approaches 80% after which the further addition of NO results in the formation of the ferrous NO derivative. Fig. 8 compares the evolution of the populations for a sample of metLtHb upon addition of a stoichiometric amount of NO (NONOates) as a function of initial nitrite concentration. Fig. 9 shows a comparable series of traces for HbA. In both cases, the fraction of formed intermediate increases with nitrite concentration; however, the fraction of intermediate relative to ferrous NO is consistently much higher for HbA. Fig. 10 shows that under conditions where reduction of metHbA by l-Cys in the presence of 4 mm nitrite results in the progressive buildup of intermediate the comparable set of conditions for metLtHb produces a substantial amount of deoxyheme and ferrous NO heme populations with a much lower intermediate population that decays with time. Similar patterns are seen for lower and higher concentrations of nitrite.


Evaluating the capacity to generate and preserve nitric oxide bioactivity in highly purified earthworm erythrocruorin: a giant polymeric hemoglobin with potential blood substitute properties.

Roche CJ, Talwar A, Palmer AF, Cabrales P, Gerfen G, Friedman JM - J. Biol. Chem. (2014)

Evolution of a metHbA sample (0.29 mm heme) at pH 7 as a function of initial nitrite concentration upon addition of an aliquot of a NONOate solution yielding a final ratio of NONOate to heme of 1:1.A, 1:1 nitrite initially added prior to addition of NO; B, 1 mm nitrite initially added; C, 10 mm nitrite initially added. Insets show a representative time sequence of spectra from which the populations were derived.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4281771&req=5

Figure 9: Evolution of a metHbA sample (0.29 mm heme) at pH 7 as a function of initial nitrite concentration upon addition of an aliquot of a NONOate solution yielding a final ratio of NONOate to heme of 1:1.A, 1:1 nitrite initially added prior to addition of NO; B, 1 mm nitrite initially added; C, 10 mm nitrite initially added. Insets show a representative time sequence of spectra from which the populations were derived.
Mentions: Earlier work on HbA demonstrated the formation of a spectroscopically distinct species formed when both NO and nitrite are added to metHbA (14, 34, 41). That species was assigned in those earlier studies as the NA intermediate in which N2O3 is coordinated to a ferrous heme. It was also shown that the species associated with the NA intermediate can generate S-nitrosothiols such as GSNO (14). It was shown based on the formation of this spectroscopically distinct species that for HbA the NA reaction can effectively compete with the RN reaction (14, 34). Here we addressed whether the intermediate can be generated by LtHb and, if so, whether the NA reaction can compete with the RN reaction given the high redox potential and fast rate of RN for LtHb. As was done with HbA, we started with an excess of nitrite (30 mm), thus creating a substantial population of met nitrite for LtHb. The addition of a small amount of NO (via NONOates) resulted in the transformation of the met nitrite spectrum to one that resembles the HbA intermediate. The same transition occurred when low levels of NO were slowly generated by deoxy-LtHb produced by the addition of the reductant l-Cys to an LtHb sample with a large initial population of met nitrite. Fig. 5 compares for both LtHb and HbA the Q band spectra of the intermediate with the Q band spectra from ferric and ferrous NO derivatives and from the met nitrite derivative. Table 6 lists the spectral peaks for all of these NOx-related species. The spectrum of the NA intermediate was then used as a member of the basis set to fit the evolving populations during the course of reactions in which both NO and nitrite were added or generated. Fig. 6 compares the evolution of populations for metLtHb upon addition of a very low amount of NONOates in the presence (A) and absence (B) of an excess of nitrite. In the presence of the large excess of nitrite, the addition of the NONOates generates primarily the intermediate, whereas in the absence of nitrite, the addition of the identical low amount of NONOate triggers the RN reaction. It can be seen that at this level of added NO the RN reaction does not go to completion but instead plateaus after a rapid initial buildup of the ferrous NO derivative of LtHb. The addition of a second aliquot does drive the reaction to near completion but at a slower rate with a noticeable buildup of the met NO derivative prior to formation of the ferrous NO derivative. Fig. 7 shows the evolution of an initial population of the met nitrite derivative of LtHb upon addition of very small aliquots of NONOates to a metLtHb sample in the presence of excess nitrite. The pattern is very similar to that reported previously for HbA in that there is a progressive buildup of intermediate with addition of NO until the population of intermediate approaches 80% after which the further addition of NO results in the formation of the ferrous NO derivative. Fig. 8 compares the evolution of the populations for a sample of metLtHb upon addition of a stoichiometric amount of NO (NONOates) as a function of initial nitrite concentration. Fig. 9 shows a comparable series of traces for HbA. In both cases, the fraction of formed intermediate increases with nitrite concentration; however, the fraction of intermediate relative to ferrous NO is consistently much higher for HbA. Fig. 10 shows that under conditions where reduction of metHbA by l-Cys in the presence of 4 mm nitrite results in the progressive buildup of intermediate the comparable set of conditions for metLtHb produces a substantial amount of deoxyheme and ferrous NO heme populations with a much lower intermediate population that decays with time. Similar patterns are seen for lower and higher concentrations of nitrite.

Bottom Line: A potentially important additional property is the capacity of the HBOC either to generate nitric oxide (NO) or to preserve NO bioactivity to compensate for decreased levels of NO in the circulation.The results show that LtHb undergoes the same reactions as HbA and that the reduced efficacy for these reactions for LtHb relative to HbA is consistent with the much higher redox potential of LtHb.Evidence of functional heterogeneity in LtHb is explained in terms of the large difference in the redox potential of the isolated subunits.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461.

Show MeSH