(Received for publication, May 8, 1995)
From the
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
O
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.
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
O
; 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 were obtained from the following sources: GSH,
diethylenetriaminepentaacetic acid (DTPA), ()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-
C]Glucose was from Amersham, United
Kingdom, Chelex 100 from Bio-Rad, FeCl
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.
To measure the
time-dependent activation of HMS by heme, 5 10
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 10
cells/ml) for 30 min
at 37 °C in RPMI 1640 supplemented with 10-100 µM of the following additives:
Fe(NH
)
(SO
)
,
FeCl
:PIH, and FeCl
:SIH both at 1:1 molar
ratios, and FeCl
: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.
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 10
± 6.00
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
(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
nmol
min
and an apparent K
= 0.49 ± 0.07 mM. Heme degradation is also
[heme]-dependent with a K
=
8.68 ± 0.92 nmol
min
and an apparent K
= 59.04 ± 9.28 µM (Fig. 2B). These kinetics are reminiscent of those
described for heme degradation by
H
O
(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
nmol
min
, 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
= 0.492 ±
0.107 mM. B, dependence on heme concentration,
[GSH] = 2 mM. K
= 8.68 ± 0.92 nmol/min and K
= 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.
, 0.2 M Hepes;
, PBS;
, 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%.
HO
is known to destroy
heme(23, 24) , and the absorption spectrum of heme
degraded in presence of high (0.5 mM) H
O
concentration is similar to, but not identical with, the final
product of GSH-dependent heme degradation (not shown).
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.
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 (), total
non-heme iron (
), 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 () or without (
)
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.
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;
, control
RBC without glucose;
, heme-loaded RBC + glucose;
,
heme-loaded RBC without glucose.
In order to test whether the
activation of HMS is due to HO
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). (
)Consequently, iron freed from heme is the major culprit
responsible for HMS activation.
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
-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) . ()
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 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, whereas reactions 2 and 3 show the
nonenzymatic dismutation of O
which does
occur at physiological pH, although 4 orders of magnitude slower than
the superoxide dismutase-mediated reaction(30) . Once
O
and H
O
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
and
OFe
O
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
O
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
to
H
O
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
, HO
and/or
OH radicals. As previously reviewed(32) , the
following reactions can take place:
Hence, in presence of O, GSH can form a
GS
radical and H
O
, and the
radical can then undergo a series of reactions that lead to the
formation of GSSG and O
. 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
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 HO
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
O
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
O
. 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
O
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.