Philip E. Hallaway

Hemoglobin potentiates central nervous system damage.

Iron and iron compounds--including mammalian hemoglobins--catalyze hydroxyl radical production and lipid peroxidation. To determine whether hemoglobin-mediated lipid peroxidation might be important in hemorrhagic injuries to the central nervous system (CNS), we studied the effects of purified hemoglobin on CNS homogenates and injected hemoglobin into the spinal cords of anesthetized cats. Hemoglobin markedly inhibits Na/K ATPase activity in CNS homogenates and spinal cords of living cats. Hemoglobin also catalyzes substantial peroxidation of CNS lipids. Importantly, the potent iron chelator, desferrioxamine, blocks these adverse effects of hemoglobin, both in vitro and in vivo. Because desferrioxamine is not known to interact with heme iron, these results indicate that free iron, derived from hemoglobin, is the proximate toxic species. Overall, our data suggest that hemoglobin, released from red cells after trauma, can promote tissue injury through iron-dependent mechanisms. Suppression of this damage by desferrioxamine suggests a rational therapeutic approach to management of trauma-induced CNS injury.

Erythrocyte catalase. A somatic oxidant defense

Mammalian erythrocytes have large amounts of catalase, an enzyme which catabolizes hydrogen peroxide (H2O2). Because catalase has a low affinity for H2O2, others have suggested that glutathione peroxidase clears most H2O2 within the erythrocyte and that catalase is of little import. We hypothesized that erythrocyte catalase might function to protect heterologous somatic cells against challenge by high levels of exogenous H2O2 (e.g., in areas of inflammation). We find that, whereas nucleated cells (L1210 murine leukemia) are readily killed by an enzymatically generated flux of superoxide (and, therefore, H2O2), the addition of human and murine erythrocytes blocks lethal damage to the target cells. Inhibition of erythrocyte superoxide dismutase, depletion of glutathione, and lysis of the erythrocytes do not diminish this protection. However, inhibition of erythrocyte catalase abrogates the protective effect and the addition of purified catalase (but not superoxide dismutase) restores it. Furthermore, erythrocytes derived from congenitally hypocatalasemic mice (in which other antioxidant systems are intact) do not protect L1210 cells. Our results raise the possibility that the erythrocyte may serve as protection against by-products of its own cargo, oxygen.

Acute iron poisoning. Rescue with macromolecular chelators.

Acute iron intoxication is a frequent, sometimes life-threatening, form of poisoning. Present therapy, in severe cases, includes oral and intravenous administration of the potent iron chelator, deferoxamine. Unfortunately, high dose intravenous deferoxamine causes acute hypotension additive with that engendered by the iron poisoning itself. To obviate this problem, we have covalently attached deferoxamine to high molecular weight carbohydrates such as dextran and hydroxyethyl starch. These macromolecular forms of deferoxamine do not cause detectable decreases in blood pressure of experimental animals, even when administered intravenously in very large doses, and persist in circulation much longer than the free drug. These novel iron-chelating substances, but not deferoxamine itself, will prevent mortality from otherwise lethal doses of iron administered to mice either orally or intraperitoneally. Further reflecting this enhanced therapeutic efficacy, the high molecular weight iron chelators also abrogate iron-mediated hepatotoxicity, suppressing the release of alanine aminotransferase. We conclude that high molecular weight derivatives of deferoxamine hold promise for the effective therapy of acute iron intoxication and may also be useful in other clinical circumstances in which control of free, reactive iron is therapeutically desirable.

Changes in conformation and function of hemoglobin and myoglobin induced by adsorption to silica

Abstract Adsorption of myoglobin (Mb) or hemoglobin (Hb) to silica (Cab-O-Sil) causes marked alterations in protein hydrogen exchange kinetics. The exchange is slower for cyanometMb and faster for both cyanometHb and oxyHb in adsorbed state than for the corresponding species in the free state. For Hb, adsorption increases oxygen affinity (P 50 = 12.9 mmHg vs. 16.7 for free) and decreases cooperativity (n = 2.05 vs. 2.87 for free). Myoglobin has the same oxygen affinity in both the free and adsorbed states.

Comparison of the state of deoxyhemoglobin S molecules in solution and in fibers by hydrogen exchange kinetics

Hydrogen exchange kinetics of deoxyhemoglobin S gel and deoxyhemoglobin A solution were compared at 4.8 mM tetramer concentration, 25 degrees C, and in sodium phosphate buffer at pH 7.0 with gamma/2 = 0.2 by means of microdialysis using tritium as a trace label. Cyanomethemoglobin A in solution and as crosslinked crystals were compared under the same conditions. The exchange values from 15 to 10(4) min were fitted to a power law, and the distribution function of exchange rates was calculated. There was no significant difference in the distribution for deoxyhemoglobin S gel and deoxyhemoglobin A. Exchange from crosslinked cyanomethemoglobin crystals was less in the early time region than for the solution state, but after 600 min the exchange curves were the same. This resulted in a larger area for the distribution function, although the predominate rates were nearly the same. The effect of polymerization on conformational fluctuations was very small, smaller than the effect of crosslinking hemoglobin crystals.