Glutaraldehyde-Polymerized Hemerythrin: Evaluation of Performance as an Oxygen Carrier in Hemorrhage Models.

Hemoglobin-based oxygen carriers (HBOCs) have been proposed and tested for several decades for the treatment of hemorrhage. We have previously proposed replacing hemoglobin (Hb) in HBOC with the oxygen-carrying protein hemerythrin (Hr), from marine worms, showing that Hr-based derivatives can perform at least as well or even better than Hb-based HBOC in a range of in vitro assays involving oxidative and nitrosative stress as well as in top-up animal models, where small amounts of Hr- or Hb-HBOC were injected into rats. Here, these experiments are extended to a hemorrhage experiment, in which Hr polymerized with glutaraldehyde, alone or conjugated with human serum albumin, is administered after a loss of 20-30% blood volume. The performance of these preparations is compared with that of Hb-based HBOC measured under the same conditions. Polymerized Hr is found to decrease the survival rate and can hence cannot be used as an oxygen carrier in transfusions. On the other hand, an Hr-albumin copolymer restores survival rates to 100% and generally yields biochemical and histological parameters similar to those of glutaraldehyde-polymerized bovine hemoglobin, with the exception of an acid-base imbalance. The latter may be solved by employing an allogeneic albumin as opposed to the human albumin employed in the present study.

Glutaraldehyde-Polymerized Hemoglobin: In Search of Improved Performance as Oxygen Carrier in Hemorrhage Models.

Hemoglobin- (Hb-) based oxygen carriers (HBOC) have for several decades been explored for treatment of hemorrhage. In our previous top-up tests, HBOC with lower in vitro prooxidant reactivity (incorporating a peroxidase or serum albumin to this end) showed a measurable but small improvement of oxidative stress-related parameters. Here, such HBOCs are tested in a hemorrhage set-up; ovine hemoglobin is also tested for the first time in such a setting, based on in vitro data showing its improved performance versus bovine Hb against oxidative and nitrosative stress agents. Indeed, ovine Hb performs better than bovine Hb in terms of survival rates, arterial tension, immunology, and histology. On the other hand, unlike in the top-up models, where the nonheme peroxidase rubrerythrin as well as bovine serum albumin copolymerized with Hb were shown to improve the performance of HBOC, in the present hemorrhage models rubrerythrin fails dramatically as HBOC ingredient (with a distinct immunological reaction), whereas serum albumin appears not feasible if its source is a different species (i.e., bovine serum albumin fares distinctly worse than rat serum albumin, in HBOC transfusions in rats). An effect of the matrix in which the HBOCs are dissolved (PBS versus gelofusine versus plasma) is noted.

On the Stability of Glutaraldehyde in Biocide Compositions.

Glutaraldehyde (GA) is used as biocide in hospitals. Recent public investigations on the chemical composition of biocides used in Romania have in some cases found GA, as a key ingredient, to be apparently diluted. However, these data did not explicitly consider the complex chemical equilibria inherent to GA. An investigation of experimental and theoretical data is reported here, assessing the stability of GA solutions relevant for biocide compositions. GA solutions of various chemical composition and under varying circumstances were analyzed using spectroscopy (UV-VIS, Raman, NMR) coupled with density functional theory (DFT) calculations, as well as chemically, such as via the formation of imines in reaction/titration with glycine monitored at 270 nm; using LC-MS; or using SDS-PAGE analysis with GA as reagent in the polymerization of two test proteins- hemoglobin and myoglobin. The spectral properties of GA changed significantly over time, in a temperature-dependent manner; titration with glycine confirmed the spectral data. SDS-PAGE experiments demonstrated a non-linear and apparently unpredictable change in the reactivity of GA over time. The results may be relevant for the determination of GA concentration in various settings such as biocide analysis, hospital wastewaters, and others.

Excess Ascorbate is a Chemical Stress Agent against Proteins and Cells.

Excess ascorbate (as expected in intravenous treatment proposed for COVID-19 management, for example) oxidizes and/or degrades hemoglobin and albumin, as evidenced by UV-vis spectroscopy, gel electrophoresis, and mass spectrometry. It also degrades hemoglobin in intact blood or in isolated erythrocytes. The survival rates and metabolic activities of several leukocyte subsets implicated in the antiviral cellular immune response are also affected. Excess ascorbate is thus an unselective biological stress agent.

The high affinity of small-molecule antioxidants for hemoglobin.

Hemoglobin has previously been shown to display ascorbate peroxidase and urate peroxidase activity, with measurable Michaelis-Menten parameters that reveal a particularly low Km for ascorbate as well as for urate - lower than the respective in vivo concentrations of these antioxidants in blood. Also, direct detection of a hemoglobin-ascorbate interaction was possible by monitoring the 1H-NMR spectrum of ascorbate in the presence of hemoglobin. The relative difference in structures between ascorbate and urate may raise the question as to exactly what the defining structural features would be, for a substrate that binds to hemoglobin with high affinity. Reported here are Michaelis-Menten parameters for hemoglobin acting as peroxidase against a number of other substrates of varying structures - gallate, caffeate, rutin, 3-hydroxyflavone, 3,6-dihydroxyflavone, quercetin, epicatechin, luteolin - all with high affinities (some higher than those of physiologically-relevant redox partners of Hb - ascorbate and urate). Moreover, this high affinity appears general to animal hemoglobins. 1H-NMR and 13C-NMR spectra reveal a general pattern wherein small hydrophilic antioxidants appear to all have their signals affected, presumably due to binding to hemoglobin. Fluorescence and calorimetry measurements confirm these conclusions. Docking calculations confirm the existence of binding sites on hemoglobin and on myoglobin for ascorbate as well as for other antioxidants. Support is found for involvement of Tyr42 in binding of three out of the four substrates investigated in the case of hemoglobin (including ascorbate and urate, as blood-contained relevant substrates), but also for Tyr145 (with urate and caffeate) and Tyr35 (with gallate).

The Reaction of Oxy Hemoglobin with Nitrite: Mechanism, Antioxidant-Modulated Effect, and Implications for Blood Substitute Evaluation.

The autocatalytic reaction between nitrite and the oxy form of globins involves free radicals. For myoglobin (Mb), an initial binding of nitrite to the iron-coordinated oxygen molecule was proposed; the resulting ferrous-peroxynitrate species was not detected, but its decay product, the high-valent ferryl form, was demonstrated in stopped-flow experiments. Reported here are the stopped flow spectra recorded upon mixing oxy Hb (native, as well as chemically-derivatized in the form of several candidates of blood substitutes) with a supraphysiological concentration of nitrite. The data may be fitted to a simple kinetic model involving a transient met-aqua form, in contrast to the ferryl detected in the case of Mb in a similar reaction sequence. These data are in line with a previous observation of a transient accumulation of ferryl Hb under auto-catalytic conditions at much lower concentrations of nitrite (Grubina, R. et al. J. Biol. Chem. 2007, 282, 12916). The simple model for fitting the stopped-flow data leaves a small part of the absorbance changes unaccounted for, unless a fourth species is invoked displaying features similar to the oxy and tentatively assigned as ferrous-peroxynitrate. Density functional theory (DFT) calculations support this latter assignment. The reaction allows for differentiating between the reactivities of various chemically modified hemoglobins, including candidates for blood substitutes. Polymerization of hemoglobin slows the nitrite-induced oxidation, in sharp contrast to oxidative-stress type reactions which are generally accelerated, not inhibited. Sheep hemoglobin is found to be distinctly more resistant to reaction with nitrite compared to bovine Hb, at large nitrite concentrations (stopped-flow experiments directly observing the oxy + nitrite reaction) as well as under auto-catalytic conditions. Copolymerization of Hb with bovine serum albumin (BSA) using glutaraldehyde leads to a distinct increase of the lag time compared to native Hb as well as to any other form of derivatization examined in the present study. The Hb-BSA copolymer also displays a slower initial reaction with nitrite under stopped-flow conditions, compared to native Hb.

In vivo evaluation of hemerythrin-based oxygen carriers: Similarities with hemoglobin-based counterparts.

We have previously proposed the annelid-derived protein, hemerythrin, as a viable replacement for hemoglobin in the synthesis of semi-synthetic oxygen carriers ("blood substitutes"). Here, we report the first in vivo tests for potential hemerythrin-based oxygen carriers (HrBOC), using a battery of experiments involving Wistar rats and previously tested on a series of hemoglobin-based oxygen carrier candidates (HBOC). At the concentrations tested, hemerythrin appears to behave similarly to hemoglobin - including, importantly, immunological effects. The antioxidant strategies based on albumin as well as based on rubrerythrin appear to offer observable physiological advantages.

Copolymerization of recombinant Phascolopsis gouldii hemerythrin with human serum albumin for use in blood substitutes.

Hemerythrin is an oxygen-carrying protein found in marine invertebrates and may be a promising alternative to hemoglobin for use in blood substitutes, primarily due to its negligible peroxidative toxicity. Previous studies have shown that glutaraldehyde-induced copolymerization of hemoglobin with bovine serum albumin increases the half-life of the active oxy form of hemoglobin (i.e. decreases the auto-oxidation rate). Here, we describe a protocol for glutaraldehyde copolymerization of Hr with human serum albumin and the dioxygen-binding properties of the co-polymerized products. The copolymerization with HSA results in alteration of hemerythrin's dioxygen-binding properties in directions that may be favorable for use in blood substitutes.

Comparative In Vivo Effects of Hemoglobin-Based Oxygen Carriers (HBOC) with Varying Prooxidant and Physiological Reactivity.

A series of hemoglobin-based oxygen carrier candidates (HBOC), previously noted for their differences in prooxidative and physiological reactivity, were compared in terms of the negative effects displayed upon injection in Wistar rats. At the concentrations tested, antioxidant strategies based on albumin as well as based on rubrerythrin appear to offer observable physiological advantages.

Nitrite binding to globins: linkage isomerism, EPR silence and reductive chemistry.

The nitrite adducts of globins can potentially bind via O- or N- linkage to the heme iron. We have used EPR (electron paramagnetic resonance) and DFT (density functional theory) to explore these binding modes to myoglobin and hemoglobin. We demonstrate that the nitrite adducts of both globins have detectable EPR signals; we provide an explanation for the difficulty in detecting these EPR features, based on uniaxial state considerations. The EPR and DFT data show that both nitrite linkage isomers can be present at the same time and that the two isomers are readily interconvertible in solution. The millisecond-scale process of nitrite reduction by Hb is investigated in search of the elusive Fe(II)-nitrite adduct.

Involvement of ferryl in the reaction between nitrite and the oxy forms of globins.

The reaction between nitrite and the oxy forms of globins has complex autocatalytic kinetics with several branching steps and evolves through chain reactions mediated by reactive species (including radicals) such as hydrogen peroxide, ferryl and nitrogen dioxide, starting with a lag phase, after which it proceeds onto an autocatalytic phase. Reported here are UV-Vis spectra collected upon stopped-flow mixing of myoglobin with a supraphysiological excess of nitrite. The best fit to the experimental data follows an A â†’ B â†’ C reaction scheme involving the formation of a short-lived intermediate identified as ferryl. This is consistent with a mechanism where nitrite binds to oxy myoglobin to generate an undetectable ferrous-peroxynitrate intermediate, whose decay leads to nitrate and ferryl. The ferryl is then reduced to met by the excess nitrite. DFT calculations reveal an essentially barrierless reaction between nitrite and the oxy heme, with a notable outer-sphere component; the resulting metastable ferrous-peroxynitrate adduct is found to feature a very low barrier towards nitrate liberation, with ferryl as a final product-in good agreement with experiment.

Oxidative protection of hemoglobin and hemerythrin by cross-linking with a nonheme iron peroxidase: potentially improved oxygen carriers for use in blood substitutes.

The nonheme peroxidase, rubrerythrin, shows the ability to reduce hydrogen peroxide to water without involving strongly oxidizing and free-radical-creating powerful oxidants such as compounds I and II [formally Fe(IV)] formed in peroxidases and catalases. Rubrerythrin could, therefore, be a useful ingredient in protein-based artificial oxygen carriers. Here, we report that the oxygen-carrying proteins, hemoglobin (Hb) and hemerythrin (Hr), can each be copolymerized with rubrerythrin using glutaraldehyde yielding high molecular weight species. These copolymers show additional peroxidase activity compared to Hb-only and Hr-only polymers, respectively and also generate lower levels of free radicals in reactions that involve hydrogen peroxide. Tests on human umbilical vein endothelial cells (HUVEC) reveal slightly better performance of the Rbr copolymers compared to controls, as measured at 24 h, but not at later times.

Hemoglobin-albumin cross-linking with disuccinimidyl suberate (DSS) and/or glutaraldehyde for blood substitutes.

Hemoglobin (Hb) derivatization for blood substitute purposes often involves multi-step processes including redox reagents such as borohydride and periodate, with possible subsequent side effects. Disuccinimidyl suberate (DSS) allows protein cross-linking without toxic side-products, forming one-step peptide bonds with the lysine residues. Here, we report that Hb polymers were obtained using DSS, making this the first report of a single-step polymerization for blood substitutes. The increase in autooxidation rate incurred by this polymerization is completely reversed when BSA is copolymerized with Hb. Copolymerization of Hb with BSA appears to be beneficial for alleviating pro-oxidant effects, regardless of the polymerizing agent employed.