Enhanced Nitrite Reductase Activity and Its Correlation with Oxygen

Jul 25, 2016 - In addition, hemoglobin bis-tetramers can produce nitric oxide in circulation through their intrinsic nitrite reductase activity. Exami...
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Enhanced Nitrite Reductase Activity and Its Correlation with Oxygen Affinity in Hemoglobin Bis-Tetramers Aizhou Wang and Ronald Kluger* Davenport Chemical Laboratories, Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 3H6 ABSTRACT: The vasoactivity of circulating cross-linked hemoglobin is consistent with the acellular protein penetrating the endothelial lining of blood vessels where hemoglobin can bind nitric oxide, the signal for relaxation of the muscles that surround blood vessels. In an important contrast, derivatives of bis-tetramers that are produced from hemoglobin by chemical coupling do not cause vasoconstriction in animal models. Presumably, they are unable to enter the endothelia where hemoglobin tetramers bind to nitric oxide. In addition, hemoglobin bis-tetramers can produce nitric oxide in circulation through their intrinsic nitrite reductase activity. Examination of this activity for hemoglobin-derived bis-tetramers that are acetylated at lysyl amino groups in their α subunits reveals enhanced activity (k = 2.21 M−1 s−1) compared to that of nonacetylated bis-tetramers (k = 0.70 M−1 s−1). Plots of nitrite reductase activities as a function of the corresponding oxygen affinities of certain allosteric-state-stabilized derivatives reveal a significant correlation, providing a basis for interpretation of the correlated functions.

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added PEG adds nonfunctional mass, and the resultant derivative has a very high oxygen affinity. We developed an alternative size-increase strategy combining hemoglobin tetramers, thereby producing combined sets of functional oxygen-binding sites in a chemically connected double protein, a bis-tetramer.16 We initially produced the bistetramers by connecting two cross-linked Hb tetramers using multifunctional reagents.17,18 The most effective reagent in terms of function of the resulting bis-tetramer combines the features of two conventional cross-linking agents and a diphenyl sulfone group in the connector (Figure 1A). Animal tests of the resulting materials reveal that both the native and PEGylated bis-tetramers (BT-Hb and BT-Hb-PEG5K4) do not produce increases in blood pressure in normal and NOsensitive diabetic mice, consistent with the hypothesis that it does not extravasate.19 In addition, the bis-tetramer possesses enhanced nitrite reductase activity (NiR).20,21 Gladwin and coworkers showed that nitrite serves as a stable circulating precursor of NO that lowers blood pressure.22 The hememediated conversion of nitrite to NO was characterized by Brooks23 in 1937 (eq 1) and Doyle et al.41 in 1981. The formed NO is then captured by vicinal deoxy hemoglobin (eq 2) to complete the nitrite reductase reactions:

nterest in the potential for acellular oxygen carriers as replacements for red cells in transfusions has led to the design and implementation of chemically stabilized hemoglobin (Hb).1 Outside the cell, circulation of the native tetrameric (α2β2) protein results in its dissociation into nonfunctional αβ dimers, with large amounts causing renal damage.2 An evaluation of clinical outcomes of potential hemoglobin-based oxygen carriers (HBOCs) revealed the presence of complications that were likely to arise from the vasoactivity from the acellular circulation of those proteins.3−6 Thus, we were aware that alterations to hemoglobin must maintain effective oxygen transport, prevent dissociation into dimers, and also minimize vasoactivity.1 This increases the challenge of producing chemical alterations to Hb beyond those that stabilize the tetrameric protein.7 It appears likely that the vasoactivity of the potential HBOCs can arise from extravasation of the circulating tetrameric protein into the endothelial lining of blood vessels.1 Nitric oxide (NO) is the endothelium-derived relaxing factor for the smooth muscle surrounding blood vessels.8,9 There, scavenging of NO prevents vasodilation that would otherwise have been signaled by its presence as result of conversion of arginine by the highly regulated NO synthase.10−12 Because hemoglobin’s ligand-binding site makes little distinction between the similar diatomics, O2 and NO,13 the endothelial coexistence of heme and NO leads to irreversible NO scavenging.14 The depletion of endothelial NO would cause the smooth muscles surrounding the blood vessels to remain in a contracted state, retarding blood flow and thereby leading to hypertension and its consequent effects. Vandegriff and co-workers found that upon administration of Hb conjugated to large chains of polyethylene glycol (PEG), there is minimal hypertension induced.15 This is consistent with the large assembly not undergoing extravasation while in circulation. However, the © XXXX American Chemical Society

HbFe 2 + + NO2− + H+ → HbFe3 + + NO + OH−

(1)

HbFe 2 + + NO → HbFe 2 +NO

(2)

The nitrite reductase activity of hemoglobin is considered an important source of NO under low-oxygen conditions where NO from arginine is less available.24 We previously reported an analysis of the rates of production of NO from the heme nitrite Received: May 27, 2016 Revised: July 14, 2016

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DOI: 10.1021/acs.biochem.6b00542 Biochemistry XXXX, XXX, XXX−XXX

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Figure 1. Structures of hemoglobin derivatives. (A) Bis-tetramer [BT-Hb (tetra-ester)] formed from aminolysis of β-Lys82 residues by a tetrafunctional cross-linker.20 (B) Bis-tetramer [BT-Hb (triazole)] formed from copper-catalyzed alkyne azide cycloaddition (CuAAC).26,27 (C) Bistetramer (BT-acHb) formed from acetylation and CuAAC.28 (D) Acetylated hemoglobin (acHb). (E) Cross-linked acetylated hemoglobin (acHb>N3).28 All cross-linking processes mentioned above target β-Lys82 residues, leading to the formation of homogeneous cross-linked tetramers and bis-tetramers.

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MATERIALS AND METHODS Reagents and chemicals were obtained from commercial suppliers. Highly purified human hemoglobin A was a gift from Oxygenix, Inc. Methods for purifying, analyzing, and storing hemoglobin were performed according to the procedures described by Jones.29 Concentrations of hemoglobin solutions were determined using the cyanomethemoglobin assay.30 The azide-functionalized cross-linker 2,2′-[(5-{[4(azidomethyl)-2-bromophenyl]carbamoyl}isophthaloyl)bis(oxy)]bis(3,5-dibromobenzoic acid) was synthesized according to the method of Singh et al.31 The bis-alkyne N,N′[sulfonylbis(4,1-phenylene)]dipropiolamide was prepared according to the method of Yang et al.27 Acetylated hemoglobin derivatives (acHb, acHb>N3, and BT-acHb) were prepared as reported previously.28 Chromatographic Analysis of Modified Hemoglobins. The molecular size distributions of modified hemoglobins were analyzed using size-exclusion chromatography on a Superdex G-200 HR column (300 mm × 10 mm). Protein samples were eluted under partially dissociating conditions in which unmodified hemoglobin tetramers dissociate into dimers [37.5 mM Tris-HCl (pH 7.4) and 0.5 M magnesium chloride]. The effluent was monitored at 280 nm. Isolation of Hemoglobin Bis-Tetramers. The hemoglobin bis-tetramers were purified by gel-filtration chromatography

reaction of a bis-tetramer and its PEGylated derivatives. In those systems, we observed significantly elevated nitrite reductase activity.20,25 These are the same modified proteins that exhibit low vasoactivity in the murine models.19 Solubility-directed coupling increases the efficiency of formation and homogeneity, providing a new class of bistetramers. The coupling process forms triazoles using coppercatalyzed azide−alkyne cycloaddition [SD-CuAAC (Figure 1B)].26,27 The formation of a cross-linked azido derivative of hemoglobin with ε-amino-acetylated lysines in its α-subunits forms bis-tetramers (Figure 1C) in SD-CuAAC with an especially high degree of selectivity.28 Although the overall structure of the resulting materials (Figure 1B,C) is similar to those of previously reported bis-tetramers (Figure 1A), the chemical constituents of the interprotein links and the presence of acetylated residues may affect dynamics and reactivity. Thus, we examined the nitrite reductase activities of a triazole-linked bis-tetramer (Figure 1C) as well as the activities of modified native and cross-linked hemoglobins, each of which was acetylated at ε-amino groups of lysines in the α-subunits (Figure 1D,E). Our quantitative examination of catalytic activity and oxygen affinity reveals a significant correlation, indicating that there is a common basis that can be used for further analysis. B

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Biochemistry on a Sephadex G-100 column (1000 mm × 35 mm) under partially dissociating conditions [37.5 mM Tris-HCl (pH 7.4) and 0.5 M magnesium chloride]. Collected fractions were concentrated and analyzed using Superdex G-200 size-exclusion chromatography, reverse-phase C4 analytical high-performance liquid chromatography (HPLC), and sodium dodecyl sulfate− polyacrylamide gel electrophoresis. Oxygen-Binding Properties. Oxygen affinity was measured (28 °C, pH 7.4) with a Hemox analyzer that measures hemoglobin’s fractional saturation of oxygen (Y) and oxygen partial pressure (PO2). Hemoglobin samples (∼1 g/L), in sodium phosphate buffer (0.01 M, pH 7.4), were converted to the oxygen-bound form before being analyzed. Oxygenated hemoglobin was then deoxygenated by bubbling nitrogen through the sample. The deoxygenation process was monitored with the Hemox analyzer until PO2 reached a minimal value. The half-saturation oxygen partial pressure (P50) and Hill coefficient at half-saturation (n50) were derived by fitting data to Adair’s equation.32 Nitrite−Heme Kinetics. Kinetic studies were conducted in the same manner as described by Lui et al. at 28 °C.25 Briefly, hemoglobin samples (1 mL, 20−40 μM) in Bis-Tris buffer (0.05 M, pH 7.2) were thoroughly flushed with nitrogen, and the resulting solutions were transferred anaerobically into a sealed cuvette using a gas-tight syringe. Oxygen-free solutions of nitrite were then added to the cuvette to give a final nitrite concentration of 0.2−1.0 mM. The formation of methemoglobin (metHb) and iron-nitrosylhemoglobin (NOHb) was followed by recording spectra from 500 to 650 nm. The spectral data were deconvoluted by multiple-linear regression analysis using separately acquired spectra of deoxyHb, oxyHb, metHb, and NOHb as a basis set.25,33 The initial rate of metHb formation was obtained via linear regression analysis over the first 100 s of reaction. Modified hemoglobin-based entities were isolated and then characterized prior to kinetic measurements. For cases in which complete conversion to cross-linked hemoglobin derivatives was not achieved, heat treatment28 was applied to give pure cross-linked species (acHb>N3), free of unmodified species. Separation of bis-tetramers (BT-acHb) from cross-linked tetramers was achieved via size-exclusion chromatography under partially dissociating conditions.28 The purity of these materials was assessed by Superdex G-200 size-exclusion HPLC (Figure 2) as each of the derivatives elutes with a unique retention time.

Figure 2. Size-exclusion G-200 chromatography indicates formation of acetylated bis-tetramers. The elution order is as follows: BT-acHb (128 kDa), acHb>N3 (64 kDa), and dimers of acHb (32 kDa).

Table 1. Nitrite Reductase Activities and Oxygen Affinities oxygen binding propertiesc nitrite reductase activity k (M−1 s−1)b

P50 (torr)

HbA

0.25 ± 0.02

5

acHb Hb-PEG5K2a Hb-PEG5K6a αα-Hba αα-HbPEG5K2a acHb>N3 BT-Hba BT-HbPEG5K4a BT-acHb

0.43 2.50 2.40 0.52 1.40

± ± ± ± ±

0.04 0.03 0.05 0.03 0.03

10.1 3.6 3.7 13.9 7.9

2.9 1.8 1.8 2.6 2.4

0.50 ± 0.04 0.70 ± 0.05 1.80 ± 0.05

7.7 9.3 4.1

2.5 2.7 2.4

2.21 ± 0.21

4.7

2

Hb variant non-crosslinked

cross-linked

bis-tetramer

n50 3

Reference data.34 bExperiments conducted at 28 °C in Bis-Tris buffer (0.05 M, pH 7.2). cExperiments conducted at 28 °C in phosphate buffer (0.01 M, pH 7.4). a

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RESULTS AND DISCUSSION Oxygenation properties of bis-tetramers are listed in Table 1 along with those of native and modified tetrameric hemoglobin. The oxygen dissociation curves of Hb derivatives (Figure 3) are from least-squares fitting of data to Adair’s equation.32 Oxygen affinities (P50) are the values of oxygenation curves at halfsaturation (i.e., Y = 0.5). Cooperativities (n50) are from fitting data to the Hill equation.32 Oxygen-binding properties of HbA, acHb, and BT-acHb are similar to those reported previously.28 acHb>N3 has an oxygen affinity (P50 = 7.7 Torr) slightly lower than that of native hemoglobin (P50 = 5 Torr; n50 = 3) while producing cooperativity similar to that of the native protein (n50 = 2.5). The oxygen-binding properties of cross-linked acHb>N3 provide further insights into the relationship between function and structural alteration. The oxygen affinity of acHb>N3 (P50

Figure 3. Oxygen dissociation curves for hemoglobin and derivatives. The fractional saturation of oxygenated hemoglobin (Y) was plotted against the partial pressure of oxygen (PO2).

C

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(Figure 4A). The resulting spectra were deconvoluted by multiple linear regressions using a basis set of spectra of pure deoxyHb, oxyHb, NOHb, and metHb (Figure 4B). The regression analysis reveals changes in the concentration of each reaction component over time (Figure 4C). The results show that deoxyHb is converted to metHb and NOHb to approximately the same extent. The observed first-order rate constant for the reaction was derived from the initial rate plot of metHb formation versus nitrite concentration (Figure 4D) under pseudo-first-order conditions. The data were used to obtain the values of second-order rate constants by dividing the observed first-order rate constant by the concentration of the catalytic protein. Kinetic data for the hemoglobin derivatives are summarized in Table 1 and compared with previous results.34 In general, the rates of reactions promoted by acHb and acHb>N3 are greater than those of their analogues that lack acetyl groups. We observe the greatest activity for the acetylated bis-tetramer BT-acHb. The rate constant of BTacHb is almost one order of magnitude larger than that provided by the native protein. Figure 5 summarizes the effects of chemical modifications of hemoglobin derivatives on NiR. Acetylation of amino groups in α subunits results in small increases in the second-order rate constant (acHb, 0.43 M−1 s−1) compared to that of HbA (0.25 M−1 s−1). Cross-linking acHb enhances the rate (acHb>N3, 0.50 M−1 s−1) marginally above that of its non-cross-linked counterpart, acHb. Moreover, acHb>N3, which is a ββ crosslinked derivative, produces a rate similar to that of the αα crosslinked derivative (αα-Hb, 0.52 M−1 s−1). The common structural feature observed in all of the derivatives, i.e., acHb (non-cross-linked and with amino groups in α subunits acetylated), acHb>N3 (ββ cross-linked and amino groups in α subunits acetylated), and αα-Hb (αα cross-linked), is that they all have alterations on the α subunits, while only some have cross-linked β subunits. Considering the comparable nitrite reductase activities of these derivatives with their common structural feature, it is likely that the rate-enhancement is a consequence of chemical alterations within the α subunits. Examining the extent of the effect of PEGylation on rate34 (Figure 5, clusters 1−3), we note that the magnitude of the rate enhancement varies for various Hb species: the enhancement is 10-fold for Hb-PEG5K2 and Hb-PEG5K6 and 3-fold for αα-HbPEG5K2 and BT-Hb-PEG5K4. The differential effects of PEG conjugation on rate enhancement imply that while PEGylation on Hb is a key factor in increasing nitrite reductase activity, the enhancement also depends on the conformation of the protein. The results indicate that the rate enhancement induced by PEGylation is most pronounced where no additional modification is applied to hemoglobin. Where PEGylation is combined with other chemical modifications, the rate enhancement is smaller. We also assessed the nitrite reductase activities of bistetramers. The α-N-acetylated bis-tetramer (BT-acHb) exhibits a 3-fold rate enhancement compared to that of the nonacetylated bis-tetramer BT-Hb (2.21 M−1 s−1 vs 0.70 M−1 s−1). While the conformations of the two bis-tetramers are similar (panels A and C of Figure 1), the increase in rate indicates that the chemical constituents of the interprotein linkers (tetra-ester for BT-Hb and triazole-containing linker for BT-acHb) affect nitrite reductase activity. The acetylated amino groups are far from the heme, so they are unlikely to have an effect. Whether the differing capabilities for NiR of the two bis-tetramers arise

= 7.7 Torr) is significantly greater than that of its non-crosslinked counterpart, acHb (P50 = 10.1 Torr). The apparent decrease in P50 is a consequence of cross-linking the two βsubunits of hemoglobin. Because the azide-functionalized crosslinker targets the β-Lys82 residues in the cationic DPG-binding site of hemoglobin,28,31 the results suggest that cross-linking from this site increases the modified hemoglobin’s oxygen affinity. Alterations on the α subunits, such as acetylation (acHb; P50 = 10.1 Torr) or cross-linking by acylation (ααHb; P50 = 13.9 Torr),34 decrease the oxygen affinity of the hemes. In the development of safe and effective HBOCs or in the formulation of protein design in general, a primary goal is to predict protein functions. Those properties of hemoglobin are closely related to its conformation, with cooperativity being the result of factors inducing the transitions between T- and R-state conformations (Figure 6).32 Therefore, chemical modifications that produce changes in oxygen-binding shift allosteric equilibria of modified proteins relative to that of the native state. R-State stabilization produces reduced cooperativity and higher oxygen affinity. T-State stabilization reduces cooperativity and oxygen affinity. The effects of chemical modifications on oxygen-binding and protein conformation are summarized in Table 2. We note that Table 2. Effects of Chemical Modification on Hemoglobin’s Oxygen-Binding Properties and the Allosteric Statea Hb variant acHb acHb>N3 BT-acHb αα-Hb αα-HbPEG5K2 BT-Hb BT-HbPEG5K4 HbPEG5K2 HbPEG5K6

modifications

n50

b

P50

c

favored allosteric stated

α acetylation α acetylation and ββ α acetylation, ββ, and bistetramer (triazole) αα αα and PEG2

− − −−

++ + −

T′ R′ R

− −−

++ +

T′ R

bis-tetramer (tetra-ester) and ββ bis-tetramer (tetra-ester), ββ, and PEG2 PEG2

−

+

R′

−−

−

R

−−−

−

R

PEG6

−−−

−

R

a

With respect to the protein’s allosteric state, the modified Hbs are assigned T if T-state-stabilized and Hbs are assigned T′ if the oxygen affinity is decreased while cooperativity is maintained, and vice versa for R and R′. bExtent of change in n50: −, 2.5−3.0; −−, 2.0−2.49; −−−, 1.5−1.99. cExtent of change in P50: −, 1−4.9 Torr; +, 5−9.9 Torr; ++, 10−14.9 Torr. dFavored allosteric state: T, ++ for P50 and −− and −−− for n50; R, − and + for P50 and −− and −−− for n50; T′, ++ for P50 and − for n50; R′, − and + for P50 and − for n50.

modifications of amino groups in α-subunits generally decrease the affinity for ligands without affecting cooperativity. Conjugation of PEG to the thiol at β-Cys93 increases oxygen affinity while decreasing cooperativity. Where PEG conjugation occurs in combination with other modifications, the effect of PEG on ligand-binding is most significant. Moreover, the formation of bis-tetramers induces conformations in which the R state is stabilized. We examined the rates of nitrite−heme reactions of native Hb, acHb, acHb>N3, and BT-acHb. We incubated deoxyHb derivatives with excess nitrite solutions and recorded spectra at specific time intervals throughout the course of the reaction D

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Figure 4. Rates of reaction between deoxyhemoglobin and excess nitrite solution. (A) Spectra taken at 30 s intervals for the reaction of deoxygenated BT-acHb (0.02 mM) with nitrite (0.43 mM). (B) Basis set of standard spectra of deoxyHb, oxyHb, NOHb, and metHb used in the regression analysis. (C) Time course of the reaction between 0.02 mM BT-acHb and 0.43 mM nitrite. (D) Initial rate plot showing the effect of nitrite concentration for NiR of acHb, acHb>N3, and BT-acHb. Rates are normalized for 0.04 mM hemoglobin so the plots can be comparable for all samples.

more conducive to nitrite-binding and subsequent reaction. This is consistent with the observation that R-state Hb has a redox potential lower than that of T-state Hb.36 Therefore, when nitrite binds to an R-state Hb, it becomes more likely that the rates of electron transfer and reduction of nitrite will increase. Furthermore, deoxymyoglobin reduces nitrite 36 times faster than deoxyhemoglobin does, which is also consistent with its lower heme redox potential.37 Studies of chemically modified hemoglobin20,25 and of haptoglobin-bound Hb dimers38 indicate that deoxyhemes produce greater initial rates for nitrite reduction where Hb derivatives are stabilized in the R state. Chemical modifications that shift the conformational equilibrium to the R state will act as allosteric activators, increasing the proportion of the heme protein that is in the R state and/or lowering the protein’s heme redox potential.

from the constituents or from the size of the linkers will be the subject of further investigation. Our study focused on producing functional and nonvasoactive derivatives of hemoglobin, using the properties of known nonvasoactive derivatives (BT-Hb and BT-HbPEG5K2)19 as comparators. On the basis of our results, we conclude that the acetylated bis-tetramer, with enhanced nitrite reductase activity and appropriate oxygenation properties, is sufficiently similar to the nonvasoactive derivatives for us to predict that they will have desirable properties in circulation. Hemoglobin’s Allosteric State and NiR. The rates of heme−nitrite reaction correlate with the allosteric state of hemoglobin (Figure 6).22 R-State Hb reduces nitrite faster than T-state Hb does.33,35 The enhanced rate that occurs when the protein is in the R state may arise from the lower redox potential of the heme iron or a heme pocket geometry that is E

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Figure 5. Effects of protein modifications on the rate of heme−nitrite reactions. PEG-conjugated derivatives are plotted with their non-PEGylated precursors (clusters 1−3) using literature values.34 Results for acetylated derivatives are presented in cluster 4.

Figure 6. Nitrite reductase activity is allosterically controlled. Maximal heme−nitrite reactivity (6 M−1 s−1) occurs in the R state, whereas the minimum (0.12 M−1 s−1) occurs in the T state. Maximal NO forming efficacy exists in the half-oxygenated Hb as a compromise between the higher reactivity of the R-state subunit and the availability of reactive nonligated heme sites.22

nitrite reduction rate. Inverse relationships between rate and P50 are also the case for BT-acHb, BT-Hb-PEG5K4, and ααHb-PEG5K2. On the basis of protein conformation, derivatives with the same extent of reduced cooperativity and different ligand-binding affinity will manifest the inverse P50−k correlation (i.e., a correlation along the P50−k plane in addition to the diagonal line). This can be seen from results for the two derivatives with the same degree of ligand-binding cooperativity, BT-Hb-PEG5K4 (P50 = 4.1 Torr, n50 = 2.4, and k = 1.8 M−1 s−1) and αα-Hb-PEG5K2 (P50 = 7.9 Torr, n50 = 2.4, and k = 1.4 M−1 s−1). The former derivative, with more R character (lower P50), displays a higher nitrite reductase activity. In the case of the clusters in the lower right section of Figure 7, for which n50 ≥ 2.5, the correlation between kinetics and oxygenbinding is weaker, where species that are neither R-state- nor Tstate-stabilized, P50 values are less predictive of nitrite reductase activity. To determine whether the k−P50 relationship is conformation-dependent, we plotted the negative log of the NiR rate constant against P50 for all derivatives (Figure 7B) and for those with n50 < 2.5 (Figure 7C). The better linear fit observed in the later case indicates that the correlation between

Previous studies report separate correlations between NiR and P50 and between NiR and n50.20 After examining the nitrite reductase activities of bis-tetramers with and without acetylated amino groups or cross-links, we compared the results to previous reports to discover correlations between allosteric state and nitrite reductase activity. To visualize the correlation, we plot the rate constant for nitrite reduction against P50 and n50 for the modified Hbs in our study (Figure 7A). It is important to note that only species with lowered ligand-binding cooperativity should be compared in assessing correlations between kinetics of nitrite reduction and the allosteric state of the protein. Only under those conditions will one of the conformational states be favored. This relationship can then be implied from values of P50. Derivatives that are assigned T or R in Table 2 were considered. There is good correlation along a diagonal in the P50−n50 plane. Modified hemoglobin with Rstate stabilization clusters on the top left corner of the plot. The two PEG-conjugated species, Hb-PEG5K2 (P50 = 3.6 Torr, and n50 = 1.8) and Hb-PEG5K6 (P50 = 3.7 Torr, and n50 = 1.8), which possess high oxygen affinity, are the most R-statestabilized derivatives of the present series and show the highest F

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Figure 7. (A) Second-order rate constants plotted vs oxygen affinity and cooperativity of hemoglobin derivatives. A combination of low P50 and n50 values reflects an R-state-stabilized allosteric protein conformation (R-stab.), whereas that of high P50 and low n50 values reflects a T-state-stabilized protein conformation (T-stab.). (B) The negative log of second-order rate constants plotted vs the oxygen affinity of all derivatives in this study reveals a poor correlation. (C) Better correlation is observed among the derivatives that are conformationally stabilized in one of the allosteric states (i.e., derivatives with n < 2.5).

derivatives led us to at least one derivative (BT-acHb) that provides the desired features: the acetylated hemoglobin bistetramer is sufficiently large with useful oxygen-binding parameters and enhanced nitrite reductase activity. We also note that oxygen affinity in conjunction with cooperativity correlates with heme−nitrite reduction rates, providing a set of chemical modifications with predictable functional effects.

NiR and allosterically stabilized hemoglobin derivatives is significant. Because we have shown that altered conformational equilibria can be characterized qualitatively by a protein’s oxygen affinity and Hill coefficient (Table 2), this can be used in combination with the allosteric state−NiR correlation (Figure 7) to consider sources of the altered states of reactivity.

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CONCLUSIONS A safe and effective HBOC must produce efficient tissue oxygenation and not induce hypertension.39 We have suggested that vasoactivity arises from extravasation that is defeated with Hb derivatives that are sufficiently large. It is also desirable to produce derivatives with an enhanced capacity to produce NO from nitrite.40 Functional characterization of a series of novel

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

This work was supported by an operating grant from the Canadian Blood Service through the Canadian Institutes of G

DOI: 10.1021/acs.biochem.6b00542 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

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Health Research (CIHR2013-N-3177905). A.W. is the recipient of an NSERC Canada postgraduate scholarship. Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.biochem.6b00542 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry (41) Doyle, M. P., Pickering, R. A., DeWeert, T. M., Hoekstra, J. W., and Pater, D. (1981) Kinetics and mechanism of the oxidation of human deoxyhemoglobin by nitrites. J. Biol. Chem. 256, 12393−12398.

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DOI: 10.1021/acs.biochem.6b00542 Biochemistry XXXX, XXX, XXX−XXX