©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Heme Degradation in the Presence of Glutathione
A PROPOSED MECHANISM TO ACCOUNT FOR THE HIGH LEVELS OF NON-HEME IRON FOUND IN THE MEMBRANES OF HEMOGLOBINOPATHIC RED BLOOD CELLS (*)

(Received for publication, May 8, 1995)

Hani Atamna Hagai Ginsburg (§)

From the Department of Biological Chemistry, Institute of Life Sciences, Hebrew University, Jerusalem 91904, Israel

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Unstable hemoglobins and oxidative conditions tend to produce hemichromes which demonstrably release their heme to the erythrocyte membrane, with consequent lipid peroxidation and cell lysis. High levels of non-heme iron are also found in such circumstances, but the origin of this iron is uncertain. In the present work, we show that reduced glutathione (GSH) is able to degrade heme in solution with a pH optimum of 7. Degradation depended on the presence of oxygen and on heme and GSH concentrations. It was inhibited by catalase and superoxide dismutase, implicating the involvement of perferryl reactive species in the process of heme degradation. Heme degradation at pH 7 and 37 °C is rapid (t = 70 s) and results in the release of iron from heme. Heme that was dissolved in red blood cell ghosts is also degraded by GSH with a concomitant increase in non-heme iron, most of which (75%) remains associated with the cell membrane. Loading of intact erythrocytes with heme was followed by time-dependent decrease of membrane-associated heme and caused an acceleration of the hexose monophosphate shunt due to the production of H(2)O(2) and the oxidation of intracellular GSH. Most of the activation of the hexose monophosphate pathway was due to redox cycling of iron, since iron chelators inhibited it considerably. These results explain the origin of non-heme iron found in the membrane of sickle cells and the oxidative stress that is observed in these and other abnormal erythrocytes.


INTRODUCTION

The most sizable and concentrated store of heme in the body resides in erythrocytes hemoglobin (Hb). As long as heme is bound tightly to its hydrophobic pocket in globin, it mediates its normal physiological role in oxygen transport. However, in some pathological conditions or under oxidative stress, heme may be released and exert various noxious actions. The pathobiology of heme has been reviewed extensively(1, 2, 3, 4) , and the main points relevant to the present work will be mentioned briefly. The physiological oxidation of deoxyhemoglobin involves the sharing of an electron of hemoglobin's Fe with oxygen, leading oxyHb to form superoxiferriHb. Whereas this reaction is usually reversible upon deoxygenation, occasionally the oxygen dissociates as superoxide that creates oxidative stress and metHb. The normal red cell is endowed with efficient mechanisms to remedy these physiological deviations, but variant erythrocytes such as sickle and thalassemic cells are unable some times to do so, either because their Hb has a higher tendency to autooxidize or is less stable. Unstable metHb readily forms hemichromes which have a tendency to bind to the cell membrane and sometimes may release their heme. Normal HbA and HbS were also shown to release their heme to cell membranes and to liposomes made of aminophospholipids, although at a reduced extent compared with metHb or hemichromes. It seems that the red cell's membrane is the major ``sponge'' for free heme: some of it interacts nonspecifically with the membrane proteins, and some dissolves in the membrane's lipid bilayer. Heme has been shown to rapidly destabilize the bilayer structure, thus increasing its permeability to ions and leading to hemolysis(5) , and to induce the peroxidation of the membrane lipids(6) . Its binding to membrane proteins diminishes their reduced thiol content and leads to cross-linking. Both latter processes are apparently due to the chronic effect of increased membrane heme. Hence, efficient mechanisms must exist to prevent the build-up of heme in membranes. Since heme is able to translocate across membranes(7, 8) , it's fate and membrane concentration are determined by the presence of various ligands, such as serum's albumin and hemopexin, and their relative affinities to heme vis à vis that of the membrane(9) . Reduced glutathione (GSH) has also been shown to remove heme from membranes (10) , but it is not known if this scavenging is effective in vivo in protecting membranes from the deleterious effects of heme. Altogether, it is presumed that the concentration of free heme in the membrane is physiologically kept at low (micromolar) levels(11) . In conditions that favor higher membrane heme association, the concentrations of non-heme iron also increase in the membrane(12, 13) . The origin of this iron is not well established, but it has been suggested that it could result from the destruction of heme by organic and lipid peroxides and by H(2)O(2); peroxidation of sickle cell ghosts causes an increase in free iron and a parallel decrease in membrane-bound heme.

In the present work we have investigated the interactions between heme and GSH. We have shown that co-incubation of these compounds lead to the destruction of heme and the generation of oxidative radicals. The heme iron and oxygen are essential for these reactions. Addition of heme to intact red blood cells increases their hexose monophosphate shunt activity, and, in the presence of GSH, heme in ghost membrane is decomposed releasing its iron. These results indicate that intracellular GSH interacts with membrane heme to produce an oxidative stress and increase the membrane iron content.


MATERIALS AND METHODS

Materials were obtained from the following sources: GSH, diethylenetriaminepentaacetic acid (DTPA), (^1)catalase, superoxide dismutase, fatty acid-free bovine serum albumin (BSA), EDTA, deferoxamine (DFO), 3-amino-1,2,4-triazole (3-AT), Ferrozine (3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine), neocuproine (2,9-dimethyl-1,10-phenanthroline), and ascorbic acid from Sigma. [1-^14C]Glucose was from Amersham, United Kingdom, Chelex 100 from Bio-Rad, FeCl(3) and thiourea from B.D.H. Heme, Zn-protoporphyrin and hematoporphyrin IX were from Porphyrin Products Inc. (Logan, UT), 5,5`-dithiobis(2-nitrobenzoic acid) from Pierce, and Hepes from Research Organics Inc. (Cleveland, OH). Pyridoxal isonicotinoyl hydrazone (PIH) and salicylaldehyde isonicotinoyl hydrazone (SIH) were kindly donated by Dr. P. Ponka. Outdated human blood was a generous gift from the Blood Bank of the Shaarei Zedek Medical Center, Jerusalem. All other chemicals were of the best available grade. All solutions were prepared in deionized, charcoal-filtered water.

Investigation of the Destruction of the Heme Molecule by GSH: Spectral Changes in the Heme Molecule

Heme was prepared fresh at the beginning of each experiment as a stock solution of 1 mM in 0.2 N NaOH and was kept in the dark on ice. DTPA was prepared in Hepes buffer as a stock solution of 20 mM. Fresh GSH solution was prepared as a stock of 0.1 M in 0.2 M Hepes buffer, pH 7.0, or as detailed under ``Results.'' First, DTPA was added (1 mM final concentration) to the Hepes buffer prewarmed to 37 °C. DTPA is needed in order to prevent nonspecific oxidation of GSH(14) . Then GSH was added to a final concentration of 2 mM, and finally heme was added to the desired final concentration. The spectral changes between 300 and 800 nm were measured immediately after gentle mixing in a Milton Roy Spectronic 3000 Array spectrophotometer and thereafter at 100-s intervals. The rate of heme degradation was measured by calculating the decrease at 365 nm, a wavelength characteristic of the hemebulletGSH complex(10) . The pH dependence of heme degradation by GSH was measured as described above, in Hepes buffer or PBS adjusted to the desired pH. The pH was determined at the beginning and at the end of the reaction and was found to be constant. It was ascertained that heme did not precipitate out of solution during the measurement. For calculation of the amounts of heme that were degraded, we used a millimolar extinction coefficient which was determined in our laboratory to be 64.1 ± 1.3 at 365 nm. The effect of the hypoxic conditions were measured by bubbling nitrogen through the buffer and the stock solutions, and the degradation of heme was followed as described above under continuous exposure to nitrogen.

Iron Release from the Heme Molecule during the Degradation Reaction

A reaction mixture of the heme decomposing system was prepared in a final volume of 5 ml at 37 °C. The heme concentration was 10 µM, and the GSH concentration was 2 mM. DTPA was not added to this reaction because it interferes with iron determination. Free iron was measured by the Ferrozine method(15) . Briefly, a 0.45-ml sample taken from the reaction mixture was mixed with 50 µl of 100% (w/v) trichloroacetic acid. Then, 0.5 ml of 0.02% ascorbic acid in 0.1 N HCl was added, the system was incubated for 5 min at room temperature, and 0.4 ml of ammonium acetate (10%) and 0.1 ml of Ferrozine solution (75 mg of Ferrozine and 75 mg of neocuproine in 25 ml water) were added. After an additional incubation for 5 min at room temperature, the color developed was measured at 562 nm. The iron concentration was calculated using a millimolar extinction coefficient of 27.9.

Fluorometric Determination of GSH-dependent Heme Degradation

GSH (2 mM) and heme (10 µM) were mixed in Hepes buffer and incubated at 37 °C for 2 h to allow for complete degradation of heme. The excitation and emission spectra of the final product were determined in a SPEX Fluorolog II (all slits were set at 5 mm), and the maxima were found to be 473 and 550 nm, respectively. The evolution of the final product with time was followed in various incubation conditions as described under ``Results.''

GSH Oxidation during Heme Degradation

The oxidation of GSH in the presence of heme was measured by the method of Beutler (16) with a minor modification: DTPA was added at 1 mM to the assay medium to chelate heavy metals which may participate in redox reactions and lead to a further GSH oxidation. Hence, the measured decrease in GSH concentration was due to the interaction of heme with GSH.

Degradation of RBC Ghosts-associated Heme by GSH, Detection and Compartmentalization of the Iron Released

Ghosts from RBC were prepared by 1:10 dilution of a known number of RBC into an ice-cold solution of 5 mM NaHPO(4), pH 8.0 (5P8). Ghosts were washed with cold 5P8 until they became white (27,000 times g, 10 min at 4 °C; usually 5 wash cycles were needed with a 5-min incubation on ice between washes). White ghosts were suspended in 0.2 M Hepes, pH 7, to a concentration of 50-80 times 10^8 ghosts/ml. A known amount of the ghosts (usually 7.4-16 times 10^8) were suspended in 3 ml of Hepes buffer containing 30 µM heme and incubated for 60 min at 37 °C. Ghosts were then washed twice in 10 ml of ice-cold 5P8 and finally resuspended in Hepes buffer. In order to determine the heme inside the membranes, those were dissolved by adding SDS (1% w/v final concentration) and the absorption spectrum was measured between 300 and 800 nm. For the calculations of heme concentration, we used a millimolar extinction coefficient (in the presence of 1% SDS) of 83.5 ± 1.8 at 399 nm which was determined in our laboratory. The same amount of ghosts was taken for the measurement of total non-heme iron (free and membrane-bound). Iron was assayed as described before (13) with a minor modification. Briefly, 200 µl of the ghost suspension was dissolved with 500 µl of 0.6% SDS in 0.2 M sodium acetate, pH 4.5. A 400-µl aliquot of the SDS-dissolved ghosts were taken into 500 µl of reductants solution (0.2% ascorbic acid and 0.2% sodium dithionite dissolved in 0.2 M sodium acetate, pH 4.5) and incubated for 5 min at room temperature. Then, 100 µl of the color developing solution were added (200 mg of Ferrozine and 1.25 g of thiourea or 200 mg of neocuproine dissolved in 50 ml of water). After 5 min of incubation at room temperature, the absorbance was measured at 562 nm. A millimolar extinction coefficient of 27.9 was used for calculations of iron concentration. To measure the ghost-associated iron, the ghost suspension was spun down and the supernatant was discarded. The ghosts were incubated for 10 min at 37 °C with 1 ml of 0.5 mM DTPA in order to chelate all the free iron from the solution, were washed three times in 1 ml of Hepes buffer in order to remove the residual DTPA, and the iron content was measured as described above. Controls (ghosts not loaded with heme) were treated in the same way to measure the basal levels of iron and heme.

Degradation of Heme in Heme-loaded Intact RBC

GSH at alkaline pH has been shown previously to extract heme from RBC ghost membranes and to form a hemebulletGSH complex absorbing at 368 nm(10) . This technique has been used to determine the degradation of heme in heme-loaded intact RBC. Washed RBC in PBS (10 ml at 5% hematocrit) were incubated with 28 µM heme for 10 min at 37 °C. Cells were spun down and washed twice with 10 ml of PBS supplemented with 50 mM sucrose to reduce lysis. The cell pellet was resuspended in the same buffer ± 10 mM glucose and incubated at 37 °C. Aliquots of 1 ml were taken immediately and at different time intervals and centrifuged, and the cells were resuspended in ice-cold alkaline (pH 8.7) PBS supplemented with 50 mM sucrose and 2 mM GSH. After 7 min on ice, cells were spun down, and the absorption spectrum of the supernatant was determined. The contribution of hemoglobin (present due to spontaneous lysis) was subtracted from the absorbance at 368 nm, and the amount of heme was calculated using an extinction coefficient of 60.85 mMbulletcm.

Intracellular Hydrogen Peroxide Production in Heme-loaded RBC

Catalase bound to H(2)O(2) is irreversibly inhibited by 3-amino-1,2,4-triazole (3-AT; (17) ). Hence, the extent of inhibition is proportional to the concentration of H(2)O(2). RBC were suspended in PBS containing 40 µM heme and 40 mM 3-AT, and various additives as described under ``Results,'' at 5 times 10^8 cells/ml, and incubated at 37 °C. Samples of 1 ml were washed three times with 1 ml of PBS with 2-min intervals at 37 °C and then lysed in 1 ml of ice-cold 5P8. After centrifugation (10 min at 16,000 times g), catalase activity was measured as described before(18) . Briefly, to 10 µl of 4-fold PBS-diluted lysate (equivalent to 1.25 times 10^6 cells) were added 990 µl of 6 mM H(2)O(2), and the decomposition of the substrate was followed by monitoring the absorbance at 236 nm. A millimolar extinction coefficient of 0.071 was used for the calculation of catalase activity.

Effect of Heme and Iron on the Hexose Monophosphate Shunt of Human Red Blood Cells

Red blood cells (RBC) were separated from other cells and plasma by 3-4 washes in RPMI 1640 medium supplemented with 8 mM NaHCO(3) and 25 mM Hepes. The activity of the RBC hexose monophosphate shunt (HMS) was assayed by measuring the evolution of ^14CO(2) from [1-^14C]glucose as described before(19) . Normal RBC were suspended at 5 times 10^8 cells/ml in RPMI 1640 and preincubated with different concentrations of heme for 30 min at 37 °C. RBC were then washed 5 times in 5 ml of glucose-free RPMI 1640 supplemented as above and 5 mM glucose. To measure the effect of heme on HMS activity, RBC were incubated with the desired concentration of heme for 30 min at 37 °C, washed of nonassociated heme, and the HMS activity was assayed without or with 2 mM GSH and/or 1 mM DTPA. The combination between DTPA and GSH without heme or DTPA alone did not affect the HMS activity. On the other hand, GSH alone caused a significant activation of the RBC HMS. This was probably due to nonspecific oxidation of the GSH by the heavy metals which usually contaminates the solutions. This was abolished completely when DTPA was used at a 1 mM final concentration.

To measure the time-dependent activation of HMS by heme, 5 times 10^8 RBC/ml were incubated for different times with a 40 µM heme. At the end of each incubation period, the cells were treated by the same way as described above, and the HMS activity was measured.

The effect of iron on HMS activity of RBC was tested by preincubation of cells (5 times 10^8 cells/ml) for 30 min at 37 °C in RPMI 1640 supplemented with 10-100 µM of the following additives: Fe(NH(4))(2)(SO(4))(2), FeCl(3):PIH, and FeCl(3):SIH both at 1:1 molar ratios, and FeCl(3):Na-citrate (1:20). Fe and the various forms of chelated Fe are known to penetrate into cells(20, 21) . After the preincubation, cells were washed and resuspended in glucose-free RPMI 1640 supplemented with 5 mM glucose and the same concentration of iron additive, and HMS was measured as described above. Controls containing 100 µM PIH or SIH were run in parallel. To test the effect of iron released during the degradation of heme in heme-loaded RBC on HMS activity, RBC (5% hematocrit) were incubated for 30 min at 37 °C in RPMI 1640 containing 50 µM freshly prepared heme, in the absence or presence of 100 µM DFO, DFO + 100 µM PIH, or DFO + 100 µM SIH. Heme was washed off, and HMS activity was measured in the presence of the chelators. Control RBC were run in parallel.


RESULTS

Degradation of Heme by GSH

Mixing of heme with GSH results in a blue shift in the maximal absorbance of heme from 387.5 to 365 nm as observed previously(10) , probably due to the formation GSHbulletheme complexes, and a rapid decline in peak absorbance indicating the degradation of the heme molecule (Fig. 1). The absence of well-defined isosbestic points suggests that degradation succeeds through various intermediate products. The rate of heme degradation was fastest in 0.2 M Hepes and considerably slower in either PBS, 0.1 M Na(2)HPO(4), or 0.05 M Tris base titrated with H(2)SO(4), all at pH 7.0. As shown in the inset of Fig. 1, heme degradation follows a monoexponential decay with t of 70 ± 0.45 s. The final product of this reaction (lowest trace in Fig. 1) is obtained after substantially longer incubations (geq2 h). The chemical nature of the end product(s) has not been investigated although it has been ascertained that it is neither biliverdin nor bilirubin (data not shown). That heme destruction is not due to contaminating traces of iron was evidenced either by using buffer solutions that were passed through a Chelex 100 resin, or by adding DTPA to the reaction mixture. In both cases, there was no change in the rate of heme degradation. In spite of this fact, we included DTPA regularly in order to discard any nonspecific heavy metal-catalyzed GSH oxidation reactions.


Figure 1: Heme degradation by GSH. Heme (10 µM) was mixed with 2 mM GSH + 1 mM DTPA in Hepes buffer, pH 7.0, and incubated at 37 °C. Absorption spectra (300-500 nm) were taken at 100-s intervals starting immediately after mixing (5 upper continuous traces) and then after 2 h (dotted trace). Inset, the absorbance at 365 nm was regressed against time to fit the kinetics of single exponential decay + offset, yielding a rate constant of decay of 9.91 times 10 ± 6.00 times 10 s. The derived t was 70 ± 0.45 s.



Hematoporphyrin that does not contain iron was not degraded by GSH, even when exogenous Fe was added as FeCl(3) (data not shown). No degradation could be observed with Zn-protoporphyrin. Deferoxamine (DFO) that interacts with the heme iron as evidenced by a blue shift in the maximal absorbance (22) almost completely inhibited the degradation of heme by GSH (data not shown).

As shown in Fig. 2A, the rate of heme degradation was [GSH]-dependent displaying Michaelis-Menten-like kinetics with K = 1.59 ± 0.08 nmolbulletmin and an apparent K(m) = 0.49 ± 0.07 mM. Heme degradation is also [heme]-dependent with a K = 8.68 ± 0.92 nmolbulletmin and an apparent K(m) = 59.04 ± 9.28 µM (Fig. 2B). These kinetics are reminiscent of those described for heme degradation by H(2)O(2)(23, 24) , but no further attempts were made to determine the precise molecular mechanism. Heme degradation is pH-dependent in either 0.2 M Hepes or PBS, peaking at pH 7 (Fig. 3). The technique used for the assay of heme degradation precludes the possibility that the low rates observed at acid pH are due to precipitation of heme. Degradation also occurs when heme is bound nonspecifically to protein. Heme was complexed with defatted BSA in Hepes buffer by mixing the two compounds for 30 min at room temperature at a molar ratio of 70:1 (heme:BSA) and then subjected to the same spectrophotometric assay in the presence of GSH as done with free heme. The rate of degradation of BSA-bound heme was somewhat lower than that of free heme, e.g. 1.005 and 1.69 nmolbulletmin, respectively (Fig. 3).


Figure 2: The dependence of heme degradation on GSH and heme concentration. Heme and GSH were mixed + 1 mM DTPA at the desired concentrations in Hepes buffer, pH 7.0, at 37 °C, and heme degradation was monitored for 200 s at 365 nm. The initial rates were calculated and converted to nmol of heme/min. A, dependence on GSH concentration, [heme] = 10 µM. The continuous trace is the best fit to Michaelis-Menten kinetics, yielding k = 1.586 ± 0.079 nmol/min and K(m) = 0.492 ± 0.107 mM. B, dependence on heme concentration, [GSH] = 2 mM. K = 8.68 ± 0.92 nmol/min and K(m) = 59.04 ± 9.28 µM.




Figure 3: The dependence of heme degradation on pH. Heme (10 µM) was mixed with 2 mM GSH and 1 mM DTPA in buffers preset to the indicated pH. The rate of heme degradation was determined as described in the legend to Fig. 2. bullet, 0.2 M Hepes; , PBS; circle, defatted BSA at 70:1 (heme:BSA) molar ratio.



In absence of molecular oxygen from the reaction, or in the presence of catalase (128 units/ml), GSH-dependent degradation did not occur. Superoxide dismutase (90 units/ml) inhibited heme degradation by 91%. H(2)O(2) is known to destroy heme(23, 24) , and the absorption spectrum of heme degraded in presence of high (0.5 mM) H(2)O(2) concentration is similar to, but not identical with, the final product of GSH-dependent heme degradation (not shown).

The Liberation of Iron from Degraded Heme

The decomposition of the heme molecule leads to the liberation of the heme iron as determined by the Ferrozine method (Fig. 4). At the same time, GSH undergoes oxidation to GSSG: during the 8.3 min that are required for the destruction of most heme by GSH, 200 µM GSH (10% of the total amount of the GSH present) was oxidized. This amount exceeds the quantity of heme present (10 µM), indicating that oxidation of GSH did occur.


Figure 4: Iron release from heme in the presence of GSH in vitro. The reaction conditions were the same as described in Fig. 1. The concentration iron was determined by the Ferrozine method. Data were fitted by nonlinear least square regression to first order + offset kinetics (continuous trace), yielding a rate constant of 0.527 ± 0.1 min, t = 1.3 ± 0.25 min.



The destruction of heme was also studied fluorometrically. A fluorescent product is formed during GSH-dependent heme degradation having excitation and emission maxima at 473 and 550 nm, respectively. Since GSH can form a fluorescent adduct with Cu(25) , we have verified that the final product is not due to GSH:iron adduct formation. It is due neither to the mere release of iron nor to the production of bilirubin or biliverdin, as neither hematoporphyrin nor the latter compounds ± GSH, ± iron, yielded any specific fluorescence. The kinetics of end product formation shown in Fig. 5are rather complex, characterized by a lag phase, then a rapid rate of production followed by a slower one. The rapid rate is faster in Hepes buffer compared to PBS. Addition of DTPA reduces the lag time and increases the rapid rate of end product formation, but has no effect on the slower rate.


Figure 5: The evolution of the final degradation product of GSH-degraded heme. Heme (10 µM) was mixed with 2 mM GSH ± 1 mM DTPA (D), in either Hepes buffer (H) or in PBS (P), and the resulting fluorescence was monitored with time. = 473 nm; = 550 nm.



Effect of GSH on RBC Membrane-associated Heme

Since in RBC heme is released from denatured or oxidized hemoglobin into the cell membrane, and normal red cells contain millimolar GSH concentrations, it was interesting to study whether membrane-associated heme can also be degraded by GSH. Our results demonstrate that GSH interacts with heme associated with RBC ghost membranes and decompose the heme molecule thereby releasing iron. White ghosts loaded with heme do not lose the heme. However, when GSH was added, heme was degraded as assessed by dissolving the membranes in 1% SDS and measuring the absorption spectrum, from which the accurate amount of the heme in the membranes was calculated. The final spectrum after long incubations (geq6 h) resembles that obtained at the end of the direct reaction between GSH and heme in solution (not shown). During the degradation of heme, iron is liberated. As indicated in Fig. 6: 1) heme degradation and iron release were found to be linear with time up to 3 h; 2) the free iron detected in the whole system is stoichiometrically related to the amount of heme that decomposed; and 3) about 75% of liberated iron remains in the membrane compartment, while the rest is released into the aqueous phase of the assay system.


Figure 6: Degradation of membrane-associated heme by GSH. White RBC ghosts were loaded with 30 µM heme. After the removal of nonassociated heme, the membranes were incubated in the presence of 10 mM GSH, and membrane heme (bullet), total non-heme iron (up triangle), and membrane-associated non-heme iron () were determined in aliquots taken at various time intervals as described under ``Materials and Methods.''



To test if heme is also degraded in intact RBC, cells were loaded with heme and subsequently incubated in PBS + sucrose (sucrose was found to minimize spontaneous lysis of heme-loaded RBC). The cell-associated heme was determined after extraction in the cold with GSH in alkaline pH(10) . The amount of heme thus extracted correlated with the concentration of heme during the loading, hence validating this assay (data not shown). Incubation of heme-loaded RBC resulted in a time-dependent decrease of cell-associated heme (Fig. 7). The presence of glucose in the incubation medium had no effect on the rate of heme depletion, suggesting that intracellular GSH, even without replenishment, was sufficient to explain the disappearance of heme.


Figure 7: Time-dependent depletion of heme from heme-loaded intact RBC. RBC were loaded with heme, thoroughly washed, and incubated at 37 °C in PBS, with (circle) or without (up triangle) glucose. The heme content in the RBC was determined with incubation time, as described under ``Materials and Methods.'' The continuous line depicts the best fit of the data to a single monoexponential decay process. The derived t is 73.4 ± 3.8 min.



Depletion of Intracellular GSH in Heme-loaded RBC

Control RBC and RBC loaded with 20 µM heme were suspended in PBS with or without 5 mM glucose and were incubated at 37 °C. At different time intervals, samples were taken and assayed for their GSH content in the presence of 1 mM DTPA. As shown in Fig. 8, in the presence of glucose, control cells maintained a steady GSH level. The omission of glucose resulted in an expected decrease in GSH reflecting its normal endogenous utilization. In heme-loaded RBC, GSH decreased substantially even in the presence of glucose, but much more so in its absence, indicating that intracellular GSH is consumed by the degradation of heme.


Figure 8: Effect of heme load on intraerythrocytic GSH level. RBC were loaded for 30 min with 20 µM heme, and non-cell-associated heme was washed away. Unloaded and heme-loaded RBC were then incubated in PBS buffer (pH 7.4, 37 °C) ± 5 mM glucose, and the intracellular GSH concentration was determined as a function of incubation time as described under ``Materials and Methods.'' , control RBC + glucose; bullet, control RBC without glucose; up triangle, heme-loaded RBC + glucose; circle, heme-loaded RBC without glucose.



Heme and GSH Generate Hydrogen Peroxide Intracellularly

Heme can translocate across membranes (7, 8, 9) and thus can interact with both intracellular and exogenous GSH. During the GSH-mediated decomposition of membrane-associated heme, H(2)O(2) can be generated (as evidenced by the inhibitory effect of catalase, see above). To test this presumption, RBC were incubated in PBS ± heme and ± 3-AT, an irreversible inhibitor of H(2)O(2)-associated catalase(17) . Hence, the extent of inhibition of catalase is used as a measure of intracellular H(2)O(2). Results shown in Table 1indicate that incubation with heme alone resulted in a time-dependent inactivation of catalase, indicating that H(2)O(2) was generated. In the presence of 2 mM GSH (+ 1 mM DTPA), catalase was inhibited to a much greater extent, indicating that H(2)O(2) was generated, and at least part of it was released to the cellular compartment during the degradation of heme by GSH. Extracellular GSH (+1 mM DTPA) in absence of heme did not affect catalase activity.



Heme and GSH Activate the Hexose Monophosphate Shunt in RBC

The effect of heme on the hexose monophosphate shunt (HMS) activity was utilized as an indicator for the interaction of the endogenous GSH and heme in intact RBC. Since part of the GSH is oxidized and H(2)O(2) was shown to be produced during this process, both effects should increase the activity of the HMS due to the consumption of NADPH during the reduction of oxidized glutathione by glutathione reductase. To test the effect of heme on HMS activity, RBC were loaded with 40 µM heme for various lengths of time, and HMS activity was measured. The relation between HMS activity and time of incubation with heme was analyzed by nonlinear regression of the data to fit first order kinetics + offset (the basal HMS activity). The basal HMS activity thus calculated was 0.153 ± 0.015 µmol of glucose consumed/10 cells/h, and it was maximally increased by 2.3-fold. The heme-dependent activity was 0.199 ± 0.024 µmol of glucose consumed/10 cells/h. The half-time of HMS activation was 24.8 ± 7.8 min. To test the effect of heme concentration on HMS, RBC were preincubated with increasing heme concentrations for 30 min, and HMS activity was measured. Data were analyzed by nonlinear regression to fit the Michaelis-Menten equation. The activation of HMS is saturable with [heme], the derived K(m) and k increases in HMS activity are 27.05 ± 9.48 µM and 0.193 ± 0.028 µmol of glucose consumed/10 cells/h. In the presence of externally added GSH, the respective values were 64.22 ± 24.02 µM and 0.685 ± 0.144 µmol/10 cells/h. Since GSH does not enter into RBC, these results suggest that extracellular GSH can degrade membrane-associated heme with consequent activation of HMS. Catalase (128 units/ml) and superoxide dismutase (90 units/ml) added to the bathing medium did not affect the HMS activation by GSH and heme (data not shown).

Activation of HMS by Iron

Since iron derived from degraded heme can enter into redox cycling and generate H(2)O(2), it could in itself activate HMS. To test this possibility, RBC were preincubated in the presence of increasing concentrations of Fe or membrane-permeable forms of Fe, and HMS was measured. Fe-citrate had no effect, and Fe or Fe:PIH at 100 µM increased HMS activity only slightly (0.067 over a basal activity of 0.165 µmol of glucose consumed/10 cells/h). In presence of Fe:SIH, HMS increased by 0.155 at 10 µM and 0.333 at 100 µM, suggesting that this chelator was the best vehicle for the introduction of iron into RBC. Indeed, the 1-octanol:water partition coefficient of SIH is 1 order of magnitude higher than that of PIH(25) .

In order to test whether the activation of HMS is due to H(2)O(2) generated during the degradation of heme or due to the release of iron which may enter into redox cycling and the consequent generation of the peroxide, RBC were loaded with heme in the absence or presence of DFO alone, DFO + PIH, or DFO + SIH. Thereafter, cells were washed and HMS was measured in the presence of chelators alone. Results shown in Table 2indicate that none of the chelators had any effect on the HMS activity of control RBC. DFO alone or DFO + PIH had no effect on the activity of heme-loaded cells, but in presence of DFO + SIH, HMS activity was considerably reduced, although the remaining activity was higher than control levels. These results suggest that SIH can chelate intracellular iron that is released during the degradation of heme and translocate it to the extracellular medium where it is chelated by DFO (the stability constant of Fe:DFO is substantially higher than that of Fe:SIH). (^2)Consequently, iron freed from heme is the major culprit responsible for HMS activation.




DISCUSSION

Abnormal hemoglobins readily autooxidize to give methemoglobin and hemichromes which lead to the formation of Heinz bodies. Those were shown to bind preferentially to the membranes of RBC(1, 26) . Structural changes in the globin molecule may lead to serious modifications of the hydrophobic heme binding pocket(1, 4) , ensuing in the loss of heme to other cell components, mainly to the membrane compartment of the RBC. High heme levels were detected in the membrane of abnormal RBC, such as beta-thalassemic (27) and sickle cells(11, 12) . In parallel, a significant phospholipid-bound fraction of non-heme iron was detected in the membranes of these cells(13, 28) . Iron decompartmentalization was recently suggested to be an important feature of abnormal RBC and an important cause for cell lysis(29) . This membrane-bound iron may play an important role in RBC disorders and may be the causative factor for oxidative membrane damage. Lipid peroxidation and oxidation of thiol groups were shown to be characteristic features of the abnormal RBC(3) .

Knowledge about the mechanism which leads to the release of free iron detected in abnormal RBC is scarce. Iron can be released from hemoglobin or heme molecules by hydroperoxides and cause lipid peroxidation(29) . The parallel increase of membrane heme and non-heme iron concentrations suggests that iron derives from heme(13) , but the mechanism responsible for this phenomenon, or the elimination of excess intracellular heme altogether, remains an enigma.

The interaction between heme and GSH has been known for some time(10) , but here we show directly, and for the first time, that this interaction leads to a destruction of the tetrapyrrole ring of heme. This is evidenced by the unique absorbance and fluorescent spectra of the final product of the reaction, the [GSH] and [heme] dependence of this process (Fig. 2), and the release of iron from the destroyed tetrapyrrole ring (Fig. 4). Heme degradation was not observed previously because experiments were done in 0.14 M phosphate buffer at pH 8.0. Indeed, we have observed that the degradation of heme is maximal at pH 7 and almost undetectable in PBS at pH 8 (Fig. 3). The conclusion of these experiments is that GSH-dependent heme degradation occurs at physiological conditions, even when heme is bound nonspecifically to protein.

The decomposition of heme by GSH was not affected by traces of free iron which usually contaminate various chemicals, since Chelex 100 treatment of buffers did not alter the kinetics of heme breakdown. No effect was seen in the presence of the iron chelators DTPA and EDTA, suggesting that iron released due to heme decomposition does not participate in the destruction of the tetrapyrrole ring. However, DFO completely abolished the destruction of heme, probably due to its ability to bind to heme iron(22) . (^3)

At the present time, one can only speculate about the mechanism of GSH-dependent heme degradation. The fact that it does not occur in anaerobic conditions implies that O(2) is involved. Since heme degradation is not observed with hematoporphyrin or Zn-protoporphyrin, heme iron is essential for the complexation of heme and GSH, as previously suggested(10) , and this iron is essential for the destruction of heme by GSH. The heme iron is most probably in the Fe state and can be reduced to Fe by GSH while the latter is oxidized to GSSG. The following reactions are then bound to occur (even if iron is still bound to the porphyrin ring, but not to DTPA):

displays the formation of O(2), whereas reactions 2 and 3 show the nonenzymatic dismutation of O(2) which does occur at physiological pH, although 4 orders of magnitude slower than the superoxide dismutase-mediated reaction(30) . Once O(2) and H(2)O(2) are present in the system, the following reactions could take place:

The efficient inhibition of heme degradation in presence of either superoxide dismutase, cytochrome c, or catalase suggests that the process depends on the oxidative effect of the perferryl radical which is an intermediate of reaction 1. A suitable proportion of FeO(2) and OFeO(2) in this radical may be necessary for the destruction of heme, as has been suggested for the mechanisms of iron-mediated lipid peroxidation(31) . and could alter this proportion, thus explaining the inhibitory effect of superoxide dismutase and catalase which could prevent this alteration. OH radicals which can be formed from the oxidation of Fe and H(2)O(2) are probably not involved in heme destruction, as the rate of destruction in the presence of OH radicals scavenger Hepes is faster than in any other buffer tested.

The pH dependence of GSH-mediated heme degradation suggests that heme binds preferentially to the nonprotonated GSH. Indeed, no spectral changes upon mixing of heme with GSH could be observed at pH 4.4 but were conspicuous at pH > 6.5 increasing at higher pH values (not shown). The decreased ability of GSH to bind heme at low pH will prevent the reduction of heme Fe. At the high pH range, binding does occur but the spontaneous dismutation of O(2) to H(2)O(2) is considerably decreased, thus potentially altering the composition of the perferryl intermediate.

In discussing the GSH-dependent mechanism of heme destruction, one should also consider the possible involvement of thiyl radicals (GS) which could be formed by the reaction of GSH with O(2), HO(2) and/or OH radicals. As previously reviewed(32) , the following reactions can take place:

Hence, in presence of O(2), GSH can form a GS radical and H(2)O(2), and the radical can then undergo a series of reactions that lead to the formation of GSSG and O(2). These reactions may explain the greater than stoichiometric oxidation of GSH observed in the present and other studies (14, 32, 33) and insinuates the involvement of the GS, GSOO, and GSS]G radicals in the destruction of the tetrapyrrole ring of heme. Further precise investigations on the mechanism of heme degradation by GSH are needed to clarify the details of this important phenomenon.

During the destruction of heme, a fluorescent product is formed showing complex kinetics (Fig. 5). Since the fluorescence measurement monitors the final product, the simplest explanation is that the lag time is required for the generation of the final product from the various intermediates that are insinuated from the lack of distinct isosbestic points in the time-dependent changes in the absorption spectra. Accordingly, the t for the generation of the final product is considerably longer (approximately 180 s or longer in the absence of DTPA or in PBS, Fig. 5) than that of heme destruction monitored by the decline of absorbance (70 s, Fig. 1, inset). Apparently, the precise mechanism of heme destruction and iron release from it is rather complex, as well as the effect of Hepes which accelerates both heme destruction and the formation of the final fluorescent product, and the expediting effect of DTPA on the formation of the final product. The precise sequence of events obviously requires further investigation, but it does not preclude us from pondering the biological implications of GSH-dependent heme destruction.

The GSH-dependent decomposition of heme also occurs when it is dissolved in RBC membranes. White RBC ghosts retain loaded heme for long times (18 h). When GSH is added to this system, heme disappears with the concomitant emergence of free iron, most of which (75%) remains associated with the membrane (Fig. 6). The rate of production of iron, however, was slower (t approx 180 min) than that observed in aqueous solutions (t = 1.3 min, Fig. 4), probably due to limited accessibility of GSH to membrane-associated heme. Heme has also been shown to be depleted from heme-loaded intact RBC with a t of 73.4 ± 3.8 min (Fig. 7), indicating that heme is also degraded in intact RBC. In parallel, GSH is oxidized (in heme-loaded RBC and in the absence of glucose, t = 67.9 ± 11.4 min), implicating GSH in heme depletion.

The present results reveal for the first time a mechanism that could account for the observed high levels of non-heme iron in sickle cell membranes that initially display high levels of heme(12, 13, 34) . Furthermore, the increased consumption of GSH in this process is compatible with higher levels of oxidized sulfhydryl groups found in abnormal red blood cell membranes(3) . This and the prooxidant activity of iron in the membrane(2, 26, 30, 34, 35, 36, 37) connect the thiol status, the endogenous generation of oxidative stress, and the sensitivity to prooxidants in cells with abnormal hemoglobins and cell injury.

The oxidation of GSH and the production of oxidative radicals during the decomposition of membrane-associated heme is expected to increase the HMS activity in intact RBC. We have clearly demonstrated that higher levels of H(2)O(2) are produced in RBC incubated in the presence of heme and more so when GSH was added to the medium (Table 1). That neither extracellular catalase nor superoxide dismutase influenced this process suggests that oxidative radicals are delivered to the intracellular compartment. In the absence of extracellular GSH, intracellular GSH fulfills the task, as evidenced by the decline of GSH levels in heme-loaded RBC (Fig. 8). This decrease can be accounted for by direct oxidation of GSH to GSSG through thiyl radical formation (see above) or due to the detoxification of H(2)O(2) by GSH peroxidase. That this effect is exacerbated in the absence of glucose suggests that the amplification of HMS could provide much of the reducing equivalents (in the form of NADPH) to reduce GSSG back to GSH by glutathione reductase. Indeed, considerably higher HMS activity was detected in heme-loaded RBC and more so when GSH was added to the bathing medium. The activation of HMS was rapid (t of 25 min), in agreement with the fast translocation of heme across membranes(7, 8) , and depended on the heme concentration. It could not be ascertained whether the rate-limiting step in HMS activation is the translocation of heme through the membrane or the interaction of heme with intracellular GSH. Most of the activation of HMS is due to iron liberated from heme and dissociating from the membrane into the intracellular compartment, which enters into redox cycling to generate H(2)O(2). Indeed, introduction of Fe into the cells increased HMS activity. In the presence of chelators that can trap the iron released during the degradation of heme in heme-loaded RBC, the heme-dependent HMS activity is reduced by 77% (Table 2). Thus, H(2)O(2) produced by the redox cycling of released iron seems to play a major role in the activation of HMS.

In conclusion, the present investigation indicates that heme is decomposed by GSH, either when heme is free, bound to proteins, or dissolved in membranes. During this process, GSH is oxidized, oxidative radicals are produced, and iron is released, and most of it remains associated with the membrane. It seems, however, that it is the redox cycling of the released iron which is the dominant factor in the activation of HMS. It is quite plausible that in sickle or thalassemic RBC, the transformation of the unstable hemoglobin into hemichromes that release their heme to the cell membrane, the oxidant damage due to heme degradation, surpasses the antioxidant defense mechanisms in these cells. This would explain their lower GSH and increased HMS activity. Further research is needed in order to verify the nature of the degradation products of the tetrapyrrole ring, and the types of the free radicals generated during GSH-dependent heme degradation. However, the elucidation of this mechanism and the identification of the reaction products, are not pertinent to the present work which seeks to investigate the biochemical source of membrane iron and some of the consequences of heme degradation by GSH to RBC physiology.


FOOTNOTES

*
This investigation received financial support from the United Nations Developmental Program/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases (TDR) and from the United States-Israel Binational Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 972-2-6585-539; Fax: 972-2-6585-440; hagai@vms.huji.ac.il.

(^1)
The abbreviations used are: DTPA, diethylenetriaminepentaacetic acid; BSA, bovine serum albumin; DFO, deferoxamine; 3-AT, 3-amino-1,2,4-triazole; PIH, pyridoxal isonicotinoyl hydrazone; SIH, salicylaldehyde isonicotinoyl hydrazone; PBS, phosphate-buffered saline; RBC, red blood cell(s); HMS, hexose monophosphate shunt.

(^2)
Z. I. Cabantchik, personal communication.

(^3)
H. Atamna and H. Ginsburg, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. P. Ponka for the generous gift of PIH and SIH and Professor Z. I. Cabantchik for helpful discussions.


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