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Oxidation-reduction properties of the hemoglobin of the opossum, Didelphis virginiana
Comp. Biochem. Physiol. Vol. 73B, No. 3, pp. 585 to 590, 1982 Printed in Great Britain.
0305-0491/82/110585-06503.00/0 Pergamon Press Ltd
O X I D A T I O N - R E D U C T I O N PROPERTIES OF THE HEMOGLOBIN OF THE OPOSSUM, DIDELPHIS VIRGINIANA M. E. JOHN,* N. C. BETHLENFALVAY~and MICHAEL R. WATERMAN* * Department of Biochemistry, University of Texas Health Science Center, Dallas, TX 75235 and t Department of Primary Care, Fitzsimons Army Medical Center, Aurora, CO 80045, U.S.A. (Received 16 February 1982)
Abstract--l. Isolated opossum hemoglobin is found to be more susceptible to oxidation by several oxidizing agents than is human hemoglobin A. 2. Opossum methemoglobin is found to be more susceptible to reduction by several reducing agents than is human hemoglobin A. 3. These results are due to amino acid differences between the two hemoglobins, including the replacement of the distal histidine residue in the alpha chains of hemoglobin A by a glutamine residue in these subunits of opossum hemoglobin. 4. Such differences also appear to be responsible for the ability of the opossum red cell NADH methemoglobin reductase to maintain opossum hemoglobin in the functional ferrous form,
INTRODUCTION In normal human erythrocytes, spontaneously oxidized hemoglobin (about 3% per day) is reduced back to the oxy form by methemoglobin reductase systems (Bodansky, 1951; Jaffe & Neumann, 1964; Jaffe et al., 1966). In the case of human mutant hemoglobins having amino acid substitutions in and around the heme pockets, the ability of these reductase systems to function normally can be altered (Winterbourn et al., 1976; Winterbourn & Carrell, 1974; White & Dacie, 1971). Throughout the evolutionary scale, certain critical amino acid residues have been preserved in most hemoglobins (Lehmann & Huntsman, 1974). These include the proximal and distal histidine residues, amino acids in the cq f12 contact regions and those in the heme contact regions. One exception to this rule is opossum (Didelphis viryiniana) hemoglobin which is the only normally occurring tetrameric animal hemoglobin known to lack a distal histidine (~58(E7)) in its alpha subunits (Stenzel et al., 1979). Although there are 83 additional substitutions when this hemoglobin is compared to human hemoglobin, the replacement of histidine by glutamine at (ct58(E7)) is unique and has prompted detailed studies on functional and conformational properties of opossum hemoglobin. These studies have led to the general conclusion that in the unligated (deoxy) state, the low affinity conformation (T) is much more predominant in opossum hemoglobin than in hemoglobin A. Thus, even in the carbonmonoxy derivative, the R - T equilibrium can be tipped toward the Tstate by addition of allostreic effectors such as organic phosphates. In opossum hemoglobin, the heme environment in the (~-chains) and probably also in the (fl-chains), is clearly different from that in human hemoglobin (Caughey et al., 1977; John & Waterman, 1979; John & Waterman, 1981; Imai et al., 1980; John et al., 1981).
Substitutions at the E7 or E l l (isoleucine in place of valine in opossum alpha chains) positions in human hemoglobin are often accompanied by severe clinical manifestations such as hemolytic anemia, autoxidation of hemoglobin, heme loss and abnormal ligand affinity. In the opossum, although both of these positions are altered in the alpha subunits, the hemoglobin is physiologically normal to the animal and methemoglobin is not present at a significant level in freshly drawn red cells or freshly prepared hemolysates (Waterman & Stenzel, 1974). In this same study it was shown that opossum hemoglobin is much more readily oxidized by Fe(CN) 3- than is human hemoglobin A. Opossum red cells have been shown to contain normal (as compared to human red cells) levels of NADH and NADPH methemoglobin reductase, but these enzymes have isoelectric points which differ from the human enzymes (Bethlenfalvay et aI., 1982). In this communication we present a comprehensive view of the oxidation-reduction properties of opossum hemoglobin and the comparison of these properties with those of hemoglobin A. These studies lead to a possible explanation as to why opossum hemoglobin is readily maintained in the reduced form in the erythrocyte.
MATERIALS AND METHODS Blood was drawn into EDTA from anesthetized adult opossums by cardiac puncture. Human blood was drawn into EDTA from healthy volunteers by veinapuncture. The erythrocytes were washed several times with isotonic saline by centrifugation and lysed with cold water. When necessary, organic phosphates were removed according to Berman et al. (1971). Methemoglobin samples were prepared by the addition of K 3 Fe(CN)6 and excess reagent was removed by gel filtration on Sephadex G-25 columns equalibrated with 0.1 M bis-tris buffer, pH 7.0. Concentrations of all hemoglobin samples were measured at 419 nm after
585
M. E, JOHN et al.
586
Methemoglobin formation was achieved b~ incubation of erythrocytes with 1.25';, sodium nitrite for 1 hr at 37C. The erythrocytes were washed 7 times with 50 vol phosphate buffered saline (PBS) pH 7.4 and then rcsuspcnded in 1.5vol PBS. The samples were incubated at 37 C and methemoglobin was determined at 30-rain intervals in aliquots by the method of Evelyn & Malloy 11938L Reduction of ferricyanide oxidized henloglobin 10.16 m M heine) by NADH methemoglobin reductase was measured in 0.05 M his tris, pH 7.0, al roonq temperature according to the procedure of Taketa & Chcn (1977). NADH was added to a final concentration of 0.1 mM and the reaction was monitored at 575 n m Control samples contained no NADH.
100 0
0
E
80 " 41%. 4P
~ , A
60
00"
"i"
x 0 ~e
Hb-A 41.
40
" ~ AOpossum
20
H b
\,,
2FO 4'0
6ro
8'0
10 0
' 20
TIME (hours)
Fig. 1. Autoxidation of opossum hemoglobin and hemoglobin A in 0.1 M phosphate buffer, pH 7.0, at 37°C. Heine concentration in both samples were 3.5 raM. Fresh hemolysates were incubated in closed tubes and methemoglobin was measured at various time intervals.
reduction with Na2 SzO~ and bubbling with CO (e = 191 M- 1 c m I t. Measurement of the rate of autoxidation of hemoglobin was carried out by incubating fresh hemoglobin (3.5 mM heme) in 0.1 M phosphate buffer, pH 7.0, at 3 7 C and determining methemoglobin at various time intervals by the method of Evelyn & Malloy (1938). Measurement of oxidation of hemoglobins by H20 2 was carried out using catalase- and superoxide dismutase-free hemoglobin solutions (Huisman & Dozy, 1965). Such samples were obtained by chromatography of hemoglobin solutions on DEAE Sephadex (Pharmacia) equalibrated with 50raM Tris-HCl, pH 8.0. Incubation of 441~M hemoglobin was carried out with 240 #M H202 in 50 mM bis-tris, pH 7.0, containing 0.1 M NaCI. The optical change of 578 nm was monitored at 23'C (Tomoda et a[., 1978). Control samples with added catalase showed no absorbance change under the same conditions. Reduction of methemoglobin in human and opossum erythrocytes was measured in the presence and absence of methylene blue, with or without glucose as substrate.
RESI_ UI'S Figure 1 shows the autoxidation profiles of opossum hemoglobin and hemoglobin A. O p o s s u m hemoglobin undergoes complete oxidation in a b o u t 110 hr while hemoglobin A is only half oxidized during this time period. Although the initial rates of autoxidation are not very different, b o t h subunits are oxidized in opossum hemoglobin while only one (probably alpha) is oxidized in hemoglobin A. Figure 2 shows, on the other hand, that H20~mediated oxidation of opossum hemoglobin is not very different from thai of hemoglobin A. Hydrogen peroxide is known to oxidize isolated hemoglobin subunits more readily than the tetrameric form (Eyer et al., 1975: T o m a d a et al,, 1978) and some mutant hemoglobins with amino acid substitutions in the heme region are k n o w n to dissociate more readil3 into m o n o m e r s (Godeau et al., 1976: Carrell et al., 1967). Following production of methemoglobin in opossum and h u m a n red cells, the reduction of this hemoglobin was followed in situ. Results obtained in the presence of methylene blue are shown in Fig. 3. The reduction is twice as fast in opossum red cells as in h u m a n red cells. In the absence of methylene blue a t½
0.6
-IHP
-t- IH P
0.5
Hb Op
Hb Op
¢: co I'.-
0.4
0.4
LU L) Z ,,~ co rr 0
0.3
02-
I /
,,n ~
03
"
02.
OI .
'~ 2 I H202
.
.
4 s TIME
.
.
s ~o (min)
.
°"
~
~4
~s
,
,
,
1
2
4
6
8
TIME
,
T
,
r
IO 12 14 16
(rnin)
H202
Fig. 2. Methemoglobin formation by H202. Catalase- and superoxide dismutase-ffee hemoglobin solutions (44#M) were treated with H20 2 (240#M) in 0.05 M bis tris buffer, pH 7.0, containing 0.1 M NaC1. Reactions were monitored at 578 nm and in control samples catalase was added at the beginning of the experiment, lnositol hexaphosphate, when added, was in a l :1 (heine :IHP) ratio.
Opossum hemoglobin oxidation-reduction IOC
9(3
8(
7(:
6(
z
5c
m 0
0 :E
4C
t~ "r-
ibJ :S
3C
I0 0
t
2
I
2
HOURS AT 37" C A
B
Fig. 3. Methemoglobin reduction in opossum (A) and human (B) erythrocytes. Following nitrite treatment of red cells, 3.9 ml of a 40~ suspension was incubated with 0.6 ml 10-4methylene blue. Incubation was carried out aerobically at 37°C, (e) in the presence of 0.9 ml, 0.17 M D-glucose; (O) in the absence of D-glucose (0.9ml isotonic saline).
of 3 hr was observed for reduction in opossum red cells and a t½ of 6.5 hr in human red cells. Interestingly, as can be seen in Fig. 3, opossum methemoglobin can be reduced in the absence of added glucose while human methemoglobin cannot. Figure 4 shows the reduction of ferricyanide oxidized hemoglobin by NADH methemoglobin reductase. The initial rate of reduction of methemoglobin A by the NADH reductase is very low (0.0006 min-1). Addition of inositol hexaphosphate causes an 8-fold increase in this reduction rate (0.005 min- 1). By comparison, opossum methemoglobin shows a much greater rate and extent of reduction. The initial rate of reduction of opossum methemoglobin is 0.024 min-1, a 40-fold increase from the methemoglobin A reduction rate. Addition of IHP increases this rate only marginally to 0.028 min- 1. DISCUSSION
Earlier studies have shown that opossum hemoglobin is oxidized more rapidly than hemoglobin A in the presence of ferricyanide (t½ = 75 sec and 3 rain respectively) and hydroquinone (k = 2.8 M -1 rain -1 and k = 10.1 M - 1 rain- 1 respectively) (Waterman &
587
Stenzel, 1974; Wallace et al., 1977). Figure 1 shows that isolated opossum hemoglobin is also more autoxidizable than hemoglobin A. The higher susceptibility to oxidation of opossum hemoglobin could be due to either electronic or steric factors or to a combination of both. The absence of the 58(E7) histidine in the a-chains will clearly cause both steric and electronic disturbance around the heine, as shown by changes in the C-O stretch frequency for opossum hemoglobin (Wallace et al., 1977) and by X-ray diffraction studies for other hemoglobins (Tucker et al., 1978). In addition, there are a number of amino acid alterations in both the ~- and/~-subunits of opossum hemoglobin which could cause structural instability (Wallace et al., 1977; Stenzel et al., 1979). Such instability could lead to higher rates of hemoglobin oxidation. Heinz body formation and isopropanol precipitation are both indicators of an unstable hemoglobin. We did not detect any performed Heinz bodies in freshly drawn opossum red cells, though such inclusions were found in samples incubated at 37°C for 30 min. Also a flocculent precipitate was observed with opossum hemoglobin in the isopropanol test while hemoglobin A solutions under the same conditions remained clear. These results indicate that there may he some structural instability in opossum hemoglobin compared to human hemoglobins, which could lead to a greater propensity for oxidation. However, the results in Fig. 2 indicate that the tendency of opossum hemoglobin toward instability and its increased autoxidizability are not related to subunit dissociation. The oxidation properties of opossum hemoglobin are summarized in Table 1 and compared to hemoglobin A. With the exception of H20 2, opossum hemoglobin is found to be more readily oxidized in all cases. However, only very small amounts of methemoglobin are observed in freshly drawn opossum red cells (Waterman & Stenzel, 1974). For this reason the
0.7 Met Opossum .I.IHP "~
0.6
t,-
0.5
W
0.4
c) z m n"
Ar
• A~ A
-A~
A A Met Opossum
A/. "/"
0.3
Met A 4.IHP
0.2
/A
m 0,0
o - 4 ~4r~ ~e,4r 0~
0.1
~t "t
Met A
I~..~s~t~'*'7"~*~* 0
1rO
2T0
30
....
40
. SO
- Control 60
T ( M E (rain.)
Fig. 4. Reduction of opossum and human methemoglobins by their respective NADH methemoglobin reductases. Ferricyanide oxidized hemoglobins (heine concentration = 1.6 × 10-4 M) which had been stripped of organic phosphates were incubated in 0.05 M bis-tris buffer pH 7.0 at room temperature. NADH was added to a final concentration of 0.1 mM and the reaction monitored at 575 nm. Controls contained no NADH. IHP when added was in a ratio of I :5 (heine :IHP).
5~8
M.E. JOHN et al.
reduction of opossum methemoglobin was examined. Ascorbic acid reduces opossum methemoglobin much more readily than it reduces methemoglobin A (John & Waterman, 1981). As seen in Fig. 3, in the presence of methylene blue and glucose the reduction of methemoglobin produced in red cells by nitrite treatment is about twice as fast in opossum red cells as in human red cells. Opossum red cells contain approximately the same level of both the N A D H and N A D P H methemoglobin reductases although these reductases differ in their isoelectric focusing patterns (Bethlenfalw~y et ul., 1982). Among the red cell methemoglobin reductase systems, N A D H methemoglobin reductase is proposed to account for about 60°,o of the reduction of ferric hemoglobin to the ferrous form in man (Scott et al., 1965). Therefore we have examined the reduction of ferricyanide oxidized hemoglobin by N A D H methemoglobin reductase (Fig. 4), As can be seen, methe-
moglobin A is a very poor substrate in the absence of organic phosphate. In the presence of inositol hexaphosphate the redution of methemoglobin A is considerably enhanced. The resulting change in conformation, a shift in allosteric equilibrium towards the 7 state, is postulated to cause the increase in the rate of reduction (Taketa & Chen, 1977}. Opossum methemoglobin is reduced at a much faster rate and to :~ greater extent and unlike methemoglobin A, inositol hexaphosphate is ineffective in increasing the rate of reduction. The significant differences in the rate and extent of opossum methemoglobin reduction by N A D H methemoglobin reductase compared to that taking place with methemoglobin A, could be due to (1) differences between the enzymes in opossum red cells and human red cells, (2) differences between the relative substrates or (3) a combination of these factors. The observation that methemoglobin reduction in intact, glucose-depleted, opossum erythrocytes
Table 1. Oxidation-reduction properties
oxidizing or Agellts
NADH
t~educilb"
I~Ulll
metllemoglobin
k
=
reductase
+
IMP
k
.
.
opossum hemoglobin
of
l-:a[c:~
][[3
0.024
-]
mill =
]][~
0.028
-1 rain
k
=
+
IHI'
,"\
(J.O000 k
-
( /{~' I a
1
rain 0.0(1%
-I
l'his;
3tud,
rain
pH 7.0, 23°C .
.
.
.
.
.
.
.
k x i0
.
.
.
.
.
.
.
.
.
.
~ -]
Ascorbie acid* pH 7.0, 2 3 ~ C
.
k I = 21 m i n , k2 _ :15.0 + IHP k I = 2 4 . 0 m m
mm
-1
k. = 2.1 + I [ H P k.
-1 rain = . 9 . 3• m L n -I
John
& Wat~,rma~1 ]981
k 2 = b.~ ma_n Nitric Oxide pH 7.0, 2 3 ° C
-1
**
-I
k I = 29.06 seEl k2 0 . 8 8 sec
k I : 20.7 sec t k~ 5.2 sec
5 h a r m a e_t :~i. ~unpublished results)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R e d u c t i o n in Erythrocytes
t i/2 = 3 hrs.
Autoxidation***
Complete
pH
ii0
K3Fe(CN)6
7.4,
k = 2.8 bl-lmin -I
in
i~r>
50 Y o x i d a t i o n 110
'I'hts S t u d \
in
This
23°C
Study
hrs
t 1/2 = ] rain
W a t e r m a n ¢, 5 t e n z e l , 1974
k : 5.2 M - i m t n -I
Wallace
25°C
et_ al,
1977
Azide **** pH 7.4, 2 5 ° C
H909 p~1 ?.0,
hrs
t 1/2 = 75 sec
Hydroquinone pH
oxidation
t 1/2 = 6.5
k = 0 . 1 8 M - i m i n -I
+
-i k = 0 . 0 0 4 3 sec -i IHP k = 0 . 0 0 6 sec
k = 0.069
M - i m i n -I
-1 k = 0 . 0 0 6 sec + IHP k = 0 . 0 0 6
W a l l a c e e t a l, 1977
_1
Thi:o; S t u d y
sec
* Ascorbic acid reduces both subunits of opossum Hb while only the fl-subunits of HbA are reduced. ** Nitric oxide reduces the fl-subunits of opossum Hb to form the hybrid Hb ( ~ "~fl~o): while x~o fl~n results from HhA. *** Autoxidation of opossum Hb is complete while HbA is only 50!~, oxidized. **** The rate constant is for the c~-subunits of opossum Hb.
Opossum hemoglobin oxidation-reduction does not seem to depend on glucose as substrate is intriguing. The availability of NADH, required for NADH methemoglobin reductase, presumably depends on the glycolytic pathway in erythrocytes. However, the source of NADH in opossum erythrocytes is unknown at the present time and alternative substrates to glucose are currently under investigation (Bethlenfalvay, unpublished observations). As noted above, it is found that level of NADH methemoglobin reductase is similar between the two types of red cells. When methemoglobin A, following treatment with DEAL cellulose to remove NADH methemoglobin reductase (Hegesh et al., 1968) was used as a substrate for the enzyme from opossum red cells, no significant change in methemoglobin A reduction rate from that shown in Fig. 4 was observed. This suggests that structural differences between methemoglobins opossum and A play an important role in the rate differences observed in Fig. 4. The inability of inositol hexaphosphate to enhance the reduction of opossum methemoglobin may be due to several different factors. In the case of methemoglobin A it has been suggested that stabilization of the T state by inositol hexaphosphate is responsible for the enhancement of the reduction reaction (Taketa & Chert, 1977). Studies on the kinetics of ligand binding to opossum hemoglobin (Sharma et al., 1982) and on the conformation of opossum nitrosylhemoglobin (John & Waterman, 1979) indicate that the allosteric equilibrium in opossum hemoglobin is shifted toward the Tstate, Therefore, at pH 7.0 opossum methemoglobin tends to assume a tertiary 't' structure accounting for both the faster reduction and the absence of enhancement of reduction by phosphate. Alternatively, the tertiary structural changes brought about by inositol hexaphosphate could be ineffective as far as the reductase reaction is concerned. We have shown from several pieces of evidence that the tertiary structural changes due to inositol hexaphosphate binding to opossum methemoglobin are very different from those observed in methemoglobin A (John & Waterman, 1981). Table 1 summarizes the oxidation-reduction properties of opossum hemoglobin. The general trend is for opossum hemoglobin to be more rapidly oxidized and more rapidly reduced than hemoglobin A, by a variety of agents. The absence of the distal histidine at position 58 (E7) could increase the accessibility of oxidizing or reducing agents to the heme pockets of opossum hemoglobin. Additional amino acid differences may play a role in the NADH methemoglobin reductase reaction, It is interesting to consider, however, that despite seemingly critical substitutions of positions E7 and E11 of the alpha subunits and a lowered oxygen affinity, opossum hemoglobin cannot be considered an abnormal hemoglobin since the species have survived over a very long period of time. Particularly interesting in this regard is the observation that glucose-depleted opossum erythrocytes, unlike human erythrocytes, can maintain hemoglobin in the reduced state even at an acid pH (Bethlenfalray, unpublished observation). Physiological systems within the opossum erythrocyte must have evolved to counter, i.e. to protect against, the various inherent anomalies of its oxygen transport system. As has been shown here, the reductase systems of the opossum red
589
cell are capable of maintaining its hemoglobin in a functional ferrous form despite its increased susceptibility to oxidation. The ability to accomplish this resides, most likely, in the particulars of opossum red cell metabolism, the reductase enzymes themselves, and the ability of this hemoglobin to be reduced. Thus, studies of animal hemoglobins may bring forth an understanding of yet unknown mechanisms operating in erythrocytes of animals as has recently been demonstrated in the case of the function of bicarbonate ions in crocodile hemoglobin (Perutz et al., 1981). Acknowledqements--This research was supported in part by Grant No. 1-624 (MRW) from The Robert A. Welch Foundation and by Grant No. 80-650 (NCB) from the Department of Clinical Investigation, Fitzsimons Army Medical Center.
REFERENCES
BERMANM., BENESCHR. & BENESCHR. E. (1971) The removal of organic phosphates from hemoglobin. Archs Biochem. Biophys. 145, 236-239. BETHLENFALVAYN. C., WATERMANM. R., LIMA J. E. & WALDRUP T. (1982) Cytosolic and membrane-bound methemoglobin reductases in erythrocytes of the opossum, Didelphis virginiana. Comp. biochem. Physiol. 73B, 591-594. BODANSKYO. (1951) Methemoglobinemia and methemoglobin-producing compounds. Pharmac. Rev. 3, 144-196. CAUGHEYW. S., HOUTCHENSR. A., LANIa A., MAXWELLJ. C. & CHARACHE S. 0977) Ligand binding to hemoglobins: effects of globin structure. In Biochemical and Clinical Aspects of Hemoglobin Abnormalities (Edited by CAU~nEV,W. S.), pp. 29-56. Academic Press, New York. CARRELL R. W., LEHMANNH., LORKIN P. A., RAIK E. HUNTER E. (1967) Hemoglobin Sydney fl67 (Ell) valine--* alanine: an emerging pattern of unstable hemoglobins. Nature, Lond. 215, 626-628. EVELYNK. A. & MALLOYH. T. (1938) Microdetermination of oxyhemoglobin, methemoglobin and sulfhemoglobin in a single sample of blood. J. biol. Chem. 126, 655-662. EVER P., HERTLEH., KIESEM. & KLEING. (1975) Kinetics of ferrihemoglobin formation by some reducing agents and the role of hydrogen peroxide. Molec. Pharmac. ll, 326-334. GODEAUJ. F., BEUZARDY. G., CACHELEUXJ., BRIZAROC. P., GIBANDA. & ROSAJ. (1976) Association of hemoglobin St Etienne with hemoglobin A and hemoglobin F. J. biol. Chem. 251, 4346--4354. HEGESH E., CALMANOV1CIN. & AVRON M. (1968) New method for determining ferrihemoglobin reductase (NADH-methemoglobin reductase) in erythrocytes. J. Lab, olin. Med. 72, 339-344. •UISMAN T. H. J. & DOZY A. M. (1965) Studies on the heterogeneity of hemoglobin IX. The use of Tris-HCl buffers in the anion exchange chromatography of hemoglobin. J. Chromat. 19, 160-169. l~al K., IKEOA-SAITOM. & YONETANIT. (1980) Studies on cobalt myoglobins and hemoglobins. J. molec. Biol. 144, 551-565. JAFFE E. R. & NEWMANYG. (1964) A comparison of the effect of the effect of menedione, methylene blue and ascorbic acid on the reduction of methemoglobin in vivo. Nature, Lond. 202, 607-608. JAFFE E. R., NEUMANNG., ROTHBERGH., WILSON F. T., WEBSTERR. M. & WOLFFJ. A. (1966) Hereditary methemoglobinemia with and without mental retardation: a study of 3 families. Am. J. Med. 41, 42-55.
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JOHN M. E. & WATERMAN M. R. (1979) Nitric oxide induced conformational changes in opossum hemoglobin. J. biol. Chem. 254, 11953 11957. JOHN M. E. & WATERMAN M. R. (1981) Nitric oxide induced conformational changes in opossum hemoglobin. Eur. J. Biochem. 115, 1-6. JOHN M, E., DuBots R. N. & WATERMAN M. R. (1981) Altered heine environments in opossum and rabbit methemoglobins. Z. Natu([i C. 36, 964 967. LEHMANN H. & HUNSTMANN R. G. (1974), In Man's Hemoglobins, p. 138. J. B. Lippincott, Philadelphia. PERUTZ M. F., BAUER C.. GROSS G., El ('I,ERGE F., VANDk('ASSERIE O., SCIIENK A. g., BR~UNIqZER G., FRIDAY A. E. & JOSEY K, A. (1981) Allosteric regulation of crocodilian hemoglobin. Nature, Lond. 291,682 684. SCOTT E. M., DUNCAN I. W. & EKS[RAND V. 11965) The reduced pyridine nucleotide dehydrogenases of human erythrocytes. J. biol. Chem. 240, 481 485. SHARMA V. S.. JOHN M. E. & WAltlkMgN M. R, (19821 Functional studies of a hemoglobin opossum: conch> sions drawn regarding the role of distal histidine. J. biol. Chem. (in press). STENZEL P,, BRIMHALL B., JONES R. T., BLACK J. K., McLACHLAN A. & GIBSON D. (1979) Opossum hemoglobin: the amino acid sequences of the ~ and fl chains. J. biol. Chem, 254, 2071 2076. rAK~TA F. & CHEN J. Y. (1977) Activation of the NADHmethemoglobin reductase reaction by inositol hexaphosphate. Bioehem. biophys. Res. Commun. 75, 38% 393.
TOMADA A., TsuJl A., MATSUKAWA S,. TAKESH1TA M. &, YONEYAMA Y. (1978) Mechanism of methemoglobin reduction by ascorbic acid under anaerobic conditions. J. biol. Chem. 253, 7420, 7423. TUCKER P. W,. PHILLIPS S. E. V., PI'RVI'/, M, F., I-It,Jr rCHENS R. A, & CAUGHEY W. S. (19781 Structure of hemoglobins Zurich [His E7(63)-+Arg] and Sydney[Val E11(671-+ Ala] and the role of the distal histidine in ligand binding. Proc, hath. 4cad. Sci. U.S.A. 7ft. 1076 -~1080. WALLAC'EW. J., HOUTCHENS R, A,, HOLF J. M. & C,\I.!GHI y W. S. (1977) Mechanisms for hemoglobin oxidation: the response of abnormal human and other hemoglobins to different oxidative pathways. In Biochemical and Clinical Aspects of Hemoglobin Abnormalities (Edited by CAt!CiHEY W. S), pp. 475--494. Academic Press, New York. WATERMAN M. R. & ST~NZEL P. (1974) Physical and chemical properties of opossum hemoglobin: a hemoglobin containing glutamine at position 58(E7) in the subunit. Biochim. Biophys. Acta. 359, 401 410. Wren: J. M. & DACn J. V. (1971) The unstable hemoglobins: molecular and clinical features. Proo, Hemal. 7. 69 109, WINTFRBOtIRN ('. C. & CAI~,R[I+L R. W. (1974) Studies of hemoglobin denaturation and Heinz body formation in the unstable hemoglobins. J. olin. lnrest. 54, 678 689, WINTERUOURN C. C.. MCGRATH B. M. & CARRELI_ R. W. (1976) Reactions involving superoxide and normal and unstable hemoglobins. Biochem. J. 155, 493 502.
0305-0491/82/110585-06503.00/0 Pergamon Press Ltd
O X I D A T I O N - R E D U C T I O N PROPERTIES OF THE HEMOGLOBIN OF THE OPOSSUM, DIDELPHIS VIRGINIANA M. E. JOHN,* N. C. BETHLENFALVAY~and MICHAEL R. WATERMAN* * Department of Biochemistry, University of Texas Health Science Center, Dallas, TX 75235 and t Department of Primary Care, Fitzsimons Army Medical Center, Aurora, CO 80045, U.S.A. (Received 16 February 1982)
Abstract--l. Isolated opossum hemoglobin is found to be more susceptible to oxidation by several oxidizing agents than is human hemoglobin A. 2. Opossum methemoglobin is found to be more susceptible to reduction by several reducing agents than is human hemoglobin A. 3. These results are due to amino acid differences between the two hemoglobins, including the replacement of the distal histidine residue in the alpha chains of hemoglobin A by a glutamine residue in these subunits of opossum hemoglobin. 4. Such differences also appear to be responsible for the ability of the opossum red cell NADH methemoglobin reductase to maintain opossum hemoglobin in the functional ferrous form,
INTRODUCTION In normal human erythrocytes, spontaneously oxidized hemoglobin (about 3% per day) is reduced back to the oxy form by methemoglobin reductase systems (Bodansky, 1951; Jaffe & Neumann, 1964; Jaffe et al., 1966). In the case of human mutant hemoglobins having amino acid substitutions in and around the heme pockets, the ability of these reductase systems to function normally can be altered (Winterbourn et al., 1976; Winterbourn & Carrell, 1974; White & Dacie, 1971). Throughout the evolutionary scale, certain critical amino acid residues have been preserved in most hemoglobins (Lehmann & Huntsman, 1974). These include the proximal and distal histidine residues, amino acids in the cq f12 contact regions and those in the heme contact regions. One exception to this rule is opossum (Didelphis viryiniana) hemoglobin which is the only normally occurring tetrameric animal hemoglobin known to lack a distal histidine (~58(E7)) in its alpha subunits (Stenzel et al., 1979). Although there are 83 additional substitutions when this hemoglobin is compared to human hemoglobin, the replacement of histidine by glutamine at (ct58(E7)) is unique and has prompted detailed studies on functional and conformational properties of opossum hemoglobin. These studies have led to the general conclusion that in the unligated (deoxy) state, the low affinity conformation (T) is much more predominant in opossum hemoglobin than in hemoglobin A. Thus, even in the carbonmonoxy derivative, the R - T equilibrium can be tipped toward the Tstate by addition of allostreic effectors such as organic phosphates. In opossum hemoglobin, the heme environment in the (~-chains) and probably also in the (fl-chains), is clearly different from that in human hemoglobin (Caughey et al., 1977; John & Waterman, 1979; John & Waterman, 1981; Imai et al., 1980; John et al., 1981).
Substitutions at the E7 or E l l (isoleucine in place of valine in opossum alpha chains) positions in human hemoglobin are often accompanied by severe clinical manifestations such as hemolytic anemia, autoxidation of hemoglobin, heme loss and abnormal ligand affinity. In the opossum, although both of these positions are altered in the alpha subunits, the hemoglobin is physiologically normal to the animal and methemoglobin is not present at a significant level in freshly drawn red cells or freshly prepared hemolysates (Waterman & Stenzel, 1974). In this same study it was shown that opossum hemoglobin is much more readily oxidized by Fe(CN) 3- than is human hemoglobin A. Opossum red cells have been shown to contain normal (as compared to human red cells) levels of NADH and NADPH methemoglobin reductase, but these enzymes have isoelectric points which differ from the human enzymes (Bethlenfalvay et aI., 1982). In this communication we present a comprehensive view of the oxidation-reduction properties of opossum hemoglobin and the comparison of these properties with those of hemoglobin A. These studies lead to a possible explanation as to why opossum hemoglobin is readily maintained in the reduced form in the erythrocyte.
MATERIALS AND METHODS Blood was drawn into EDTA from anesthetized adult opossums by cardiac puncture. Human blood was drawn into EDTA from healthy volunteers by veinapuncture. The erythrocytes were washed several times with isotonic saline by centrifugation and lysed with cold water. When necessary, organic phosphates were removed according to Berman et al. (1971). Methemoglobin samples were prepared by the addition of K 3 Fe(CN)6 and excess reagent was removed by gel filtration on Sephadex G-25 columns equalibrated with 0.1 M bis-tris buffer, pH 7.0. Concentrations of all hemoglobin samples were measured at 419 nm after
585
M. E, JOHN et al.
586
Methemoglobin formation was achieved b~ incubation of erythrocytes with 1.25';, sodium nitrite for 1 hr at 37C. The erythrocytes were washed 7 times with 50 vol phosphate buffered saline (PBS) pH 7.4 and then rcsuspcnded in 1.5vol PBS. The samples were incubated at 37 C and methemoglobin was determined at 30-rain intervals in aliquots by the method of Evelyn & Malloy 11938L Reduction of ferricyanide oxidized henloglobin 10.16 m M heine) by NADH methemoglobin reductase was measured in 0.05 M his tris, pH 7.0, al roonq temperature according to the procedure of Taketa & Chcn (1977). NADH was added to a final concentration of 0.1 mM and the reaction was monitored at 575 n m Control samples contained no NADH.
100 0
0
E
80 " 41%. 4P
~ , A
60
00"
"i"
x 0 ~e
Hb-A 41.
40
" ~ AOpossum
20
H b
\,,
2FO 4'0
6ro
8'0
10 0
' 20
TIME (hours)
Fig. 1. Autoxidation of opossum hemoglobin and hemoglobin A in 0.1 M phosphate buffer, pH 7.0, at 37°C. Heine concentration in both samples were 3.5 raM. Fresh hemolysates were incubated in closed tubes and methemoglobin was measured at various time intervals.
reduction with Na2 SzO~ and bubbling with CO (e = 191 M- 1 c m I t. Measurement of the rate of autoxidation of hemoglobin was carried out by incubating fresh hemoglobin (3.5 mM heme) in 0.1 M phosphate buffer, pH 7.0, at 3 7 C and determining methemoglobin at various time intervals by the method of Evelyn & Malloy (1938). Measurement of oxidation of hemoglobins by H20 2 was carried out using catalase- and superoxide dismutase-free hemoglobin solutions (Huisman & Dozy, 1965). Such samples were obtained by chromatography of hemoglobin solutions on DEAE Sephadex (Pharmacia) equalibrated with 50raM Tris-HCl, pH 8.0. Incubation of 441~M hemoglobin was carried out with 240 #M H202 in 50 mM bis-tris, pH 7.0, containing 0.1 M NaCI. The optical change of 578 nm was monitored at 23'C (Tomoda et a[., 1978). Control samples with added catalase showed no absorbance change under the same conditions. Reduction of methemoglobin in human and opossum erythrocytes was measured in the presence and absence of methylene blue, with or without glucose as substrate.
RESI_ UI'S Figure 1 shows the autoxidation profiles of opossum hemoglobin and hemoglobin A. O p o s s u m hemoglobin undergoes complete oxidation in a b o u t 110 hr while hemoglobin A is only half oxidized during this time period. Although the initial rates of autoxidation are not very different, b o t h subunits are oxidized in opossum hemoglobin while only one (probably alpha) is oxidized in hemoglobin A. Figure 2 shows, on the other hand, that H20~mediated oxidation of opossum hemoglobin is not very different from thai of hemoglobin A. Hydrogen peroxide is known to oxidize isolated hemoglobin subunits more readily than the tetrameric form (Eyer et al., 1975: T o m a d a et al,, 1978) and some mutant hemoglobins with amino acid substitutions in the heme region are k n o w n to dissociate more readil3 into m o n o m e r s (Godeau et al., 1976: Carrell et al., 1967). Following production of methemoglobin in opossum and h u m a n red cells, the reduction of this hemoglobin was followed in situ. Results obtained in the presence of methylene blue are shown in Fig. 3. The reduction is twice as fast in opossum red cells as in h u m a n red cells. In the absence of methylene blue a t½
0.6
-IHP
-t- IH P
0.5
Hb Op
Hb Op
¢: co I'.-
0.4
0.4
LU L) Z ,,~ co rr 0
0.3
02-
I /
,,n ~
03
"
02.
OI .
'~ 2 I H202
.
.
4 s TIME
.
.
s ~o (min)
.
°"
~
~4
~s
,
,
,
1
2
4
6
8
TIME
,
T
,
r
IO 12 14 16
(rnin)
H202
Fig. 2. Methemoglobin formation by H202. Catalase- and superoxide dismutase-ffee hemoglobin solutions (44#M) were treated with H20 2 (240#M) in 0.05 M bis tris buffer, pH 7.0, containing 0.1 M NaC1. Reactions were monitored at 578 nm and in control samples catalase was added at the beginning of the experiment, lnositol hexaphosphate, when added, was in a l :1 (heine :IHP) ratio.
Opossum hemoglobin oxidation-reduction IOC
9(3
8(
7(:
6(
z
5c
m 0
0 :E
4C
t~ "r-
ibJ :S
3C
I0 0
t
2
I
2
HOURS AT 37" C A
B
Fig. 3. Methemoglobin reduction in opossum (A) and human (B) erythrocytes. Following nitrite treatment of red cells, 3.9 ml of a 40~ suspension was incubated with 0.6 ml 10-4methylene blue. Incubation was carried out aerobically at 37°C, (e) in the presence of 0.9 ml, 0.17 M D-glucose; (O) in the absence of D-glucose (0.9ml isotonic saline).
of 3 hr was observed for reduction in opossum red cells and a t½ of 6.5 hr in human red cells. Interestingly, as can be seen in Fig. 3, opossum methemoglobin can be reduced in the absence of added glucose while human methemoglobin cannot. Figure 4 shows the reduction of ferricyanide oxidized hemoglobin by NADH methemoglobin reductase. The initial rate of reduction of methemoglobin A by the NADH reductase is very low (0.0006 min-1). Addition of inositol hexaphosphate causes an 8-fold increase in this reduction rate (0.005 min- 1). By comparison, opossum methemoglobin shows a much greater rate and extent of reduction. The initial rate of reduction of opossum methemoglobin is 0.024 min-1, a 40-fold increase from the methemoglobin A reduction rate. Addition of IHP increases this rate only marginally to 0.028 min- 1. DISCUSSION
Earlier studies have shown that opossum hemoglobin is oxidized more rapidly than hemoglobin A in the presence of ferricyanide (t½ = 75 sec and 3 rain respectively) and hydroquinone (k = 2.8 M -1 rain -1 and k = 10.1 M - 1 rain- 1 respectively) (Waterman &
587
Stenzel, 1974; Wallace et al., 1977). Figure 1 shows that isolated opossum hemoglobin is also more autoxidizable than hemoglobin A. The higher susceptibility to oxidation of opossum hemoglobin could be due to either electronic or steric factors or to a combination of both. The absence of the 58(E7) histidine in the a-chains will clearly cause both steric and electronic disturbance around the heine, as shown by changes in the C-O stretch frequency for opossum hemoglobin (Wallace et al., 1977) and by X-ray diffraction studies for other hemoglobins (Tucker et al., 1978). In addition, there are a number of amino acid alterations in both the ~- and/~-subunits of opossum hemoglobin which could cause structural instability (Wallace et al., 1977; Stenzel et al., 1979). Such instability could lead to higher rates of hemoglobin oxidation. Heinz body formation and isopropanol precipitation are both indicators of an unstable hemoglobin. We did not detect any performed Heinz bodies in freshly drawn opossum red cells, though such inclusions were found in samples incubated at 37°C for 30 min. Also a flocculent precipitate was observed with opossum hemoglobin in the isopropanol test while hemoglobin A solutions under the same conditions remained clear. These results indicate that there may he some structural instability in opossum hemoglobin compared to human hemoglobins, which could lead to a greater propensity for oxidation. However, the results in Fig. 2 indicate that the tendency of opossum hemoglobin toward instability and its increased autoxidizability are not related to subunit dissociation. The oxidation properties of opossum hemoglobin are summarized in Table 1 and compared to hemoglobin A. With the exception of H20 2, opossum hemoglobin is found to be more readily oxidized in all cases. However, only very small amounts of methemoglobin are observed in freshly drawn opossum red cells (Waterman & Stenzel, 1974). For this reason the
0.7 Met Opossum .I.IHP "~
0.6
t,-
0.5
W
0.4
c) z m n"
Ar
• A~ A
-A~
A A Met Opossum
A/. "/"
0.3
Met A 4.IHP
0.2
/A
m 0,0
o - 4 ~4r~ ~e,4r 0~
0.1
~t "t
Met A
I~..~s~t~'*'7"~*~* 0
1rO
2T0
30
....
40
. SO
- Control 60
T ( M E (rain.)
Fig. 4. Reduction of opossum and human methemoglobins by their respective NADH methemoglobin reductases. Ferricyanide oxidized hemoglobins (heine concentration = 1.6 × 10-4 M) which had been stripped of organic phosphates were incubated in 0.05 M bis-tris buffer pH 7.0 at room temperature. NADH was added to a final concentration of 0.1 mM and the reaction monitored at 575 nm. Controls contained no NADH. IHP when added was in a ratio of I :5 (heine :IHP).
5~8
M.E. JOHN et al.
reduction of opossum methemoglobin was examined. Ascorbic acid reduces opossum methemoglobin much more readily than it reduces methemoglobin A (John & Waterman, 1981). As seen in Fig. 3, in the presence of methylene blue and glucose the reduction of methemoglobin produced in red cells by nitrite treatment is about twice as fast in opossum red cells as in human red cells. Opossum red cells contain approximately the same level of both the N A D H and N A D P H methemoglobin reductases although these reductases differ in their isoelectric focusing patterns (Bethlenfalw~y et ul., 1982). Among the red cell methemoglobin reductase systems, N A D H methemoglobin reductase is proposed to account for about 60°,o of the reduction of ferric hemoglobin to the ferrous form in man (Scott et al., 1965). Therefore we have examined the reduction of ferricyanide oxidized hemoglobin by N A D H methemoglobin reductase (Fig. 4), As can be seen, methe-
moglobin A is a very poor substrate in the absence of organic phosphate. In the presence of inositol hexaphosphate the redution of methemoglobin A is considerably enhanced. The resulting change in conformation, a shift in allosteric equilibrium towards the 7 state, is postulated to cause the increase in the rate of reduction (Taketa & Chen, 1977}. Opossum methemoglobin is reduced at a much faster rate and to :~ greater extent and unlike methemoglobin A, inositol hexaphosphate is ineffective in increasing the rate of reduction. The significant differences in the rate and extent of opossum methemoglobin reduction by N A D H methemoglobin reductase compared to that taking place with methemoglobin A, could be due to (1) differences between the enzymes in opossum red cells and human red cells, (2) differences between the relative substrates or (3) a combination of these factors. The observation that methemoglobin reduction in intact, glucose-depleted, opossum erythrocytes
Table 1. Oxidation-reduction properties
oxidizing or Agellts
NADH
t~educilb"
I
metllemoglobin
k
=
reductase
+
IMP
k
.
.
opossum hemoglobin
of
l-:a[c:~
][[3
0.024
-]
mill =
]][~
0.028
-1 rain
k
=
+
IHI'
,"\
(J.O000 k
-
( /{~' I a
1
rain 0.0(1%
-I
l'his;
3tud,
rain
pH 7.0, 23°C .
.
.
.
.
.
.
.
k x i0
.
.
.
.
.
.
.
.
.
.
~ -]
Ascorbie acid* pH 7.0, 2 3 ~ C
.
k I = 21 m i n , k2 _ :15.0 + IHP k I = 2 4 . 0 m m
mm
-1
k. = 2.1 + I [ H P k.
-1 rain = . 9 . 3• m L n -I
John
& Wat~,rma~1 ]981
k 2 = b.~ ma_n Nitric Oxide pH 7.0, 2 3 ° C
-1
**
-I
k I = 29.06 seEl k2 0 . 8 8 sec
k I : 20.7 sec t k~ 5.2 sec
5 h a r m a e_t :~i. ~unpublished results)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R e d u c t i o n in Erythrocytes
t i/2 = 3 hrs.
Autoxidation***
Complete
pH
ii0
K3Fe(CN)6
7.4,
k = 2.8 bl-lmin -I
in
i~r>
50 Y o x i d a t i o n 110
'I'hts S t u d \
in
This
23°C
Study
hrs
t 1/2 = ] rain
W a t e r m a n ¢, 5 t e n z e l , 1974
k : 5.2 M - i m t n -I
Wallace
25°C
et_ al,
1977
Azide **** pH 7.4, 2 5 ° C
H909 p~1 ?.0,
hrs
t 1/2 = 75 sec
Hydroquinone pH
oxidation
t 1/2 = 6.5
k = 0 . 1 8 M - i m i n -I
+
-i k = 0 . 0 0 4 3 sec -i IHP k = 0 . 0 0 6 sec
k = 0.069
M - i m i n -I
-1 k = 0 . 0 0 6 sec + IHP k = 0 . 0 0 6
W a l l a c e e t a l, 1977
_1
Thi:o; S t u d y
sec
* Ascorbic acid reduces both subunits of opossum Hb while only the fl-subunits of HbA are reduced. ** Nitric oxide reduces the fl-subunits of opossum Hb to form the hybrid Hb ( ~ "~fl~o): while x~o fl~n results from HhA. *** Autoxidation of opossum Hb is complete while HbA is only 50!~, oxidized. **** The rate constant is for the c~-subunits of opossum Hb.
Opossum hemoglobin oxidation-reduction does not seem to depend on glucose as substrate is intriguing. The availability of NADH, required for NADH methemoglobin reductase, presumably depends on the glycolytic pathway in erythrocytes. However, the source of NADH in opossum erythrocytes is unknown at the present time and alternative substrates to glucose are currently under investigation (Bethlenfalvay, unpublished observations). As noted above, it is found that level of NADH methemoglobin reductase is similar between the two types of red cells. When methemoglobin A, following treatment with DEAL cellulose to remove NADH methemoglobin reductase (Hegesh et al., 1968) was used as a substrate for the enzyme from opossum red cells, no significant change in methemoglobin A reduction rate from that shown in Fig. 4 was observed. This suggests that structural differences between methemoglobins opossum and A play an important role in the rate differences observed in Fig. 4. The inability of inositol hexaphosphate to enhance the reduction of opossum methemoglobin may be due to several different factors. In the case of methemoglobin A it has been suggested that stabilization of the T state by inositol hexaphosphate is responsible for the enhancement of the reduction reaction (Taketa & Chert, 1977). Studies on the kinetics of ligand binding to opossum hemoglobin (Sharma et al., 1982) and on the conformation of opossum nitrosylhemoglobin (John & Waterman, 1979) indicate that the allosteric equilibrium in opossum hemoglobin is shifted toward the Tstate, Therefore, at pH 7.0 opossum methemoglobin tends to assume a tertiary 't' structure accounting for both the faster reduction and the absence of enhancement of reduction by phosphate. Alternatively, the tertiary structural changes brought about by inositol hexaphosphate could be ineffective as far as the reductase reaction is concerned. We have shown from several pieces of evidence that the tertiary structural changes due to inositol hexaphosphate binding to opossum methemoglobin are very different from those observed in methemoglobin A (John & Waterman, 1981). Table 1 summarizes the oxidation-reduction properties of opossum hemoglobin. The general trend is for opossum hemoglobin to be more rapidly oxidized and more rapidly reduced than hemoglobin A, by a variety of agents. The absence of the distal histidine at position 58 (E7) could increase the accessibility of oxidizing or reducing agents to the heme pockets of opossum hemoglobin. Additional amino acid differences may play a role in the NADH methemoglobin reductase reaction, It is interesting to consider, however, that despite seemingly critical substitutions of positions E7 and E11 of the alpha subunits and a lowered oxygen affinity, opossum hemoglobin cannot be considered an abnormal hemoglobin since the species have survived over a very long period of time. Particularly interesting in this regard is the observation that glucose-depleted opossum erythrocytes, unlike human erythrocytes, can maintain hemoglobin in the reduced state even at an acid pH (Bethlenfalray, unpublished observation). Physiological systems within the opossum erythrocyte must have evolved to counter, i.e. to protect against, the various inherent anomalies of its oxygen transport system. As has been shown here, the reductase systems of the opossum red
589
cell are capable of maintaining its hemoglobin in a functional ferrous form despite its increased susceptibility to oxidation. The ability to accomplish this resides, most likely, in the particulars of opossum red cell metabolism, the reductase enzymes themselves, and the ability of this hemoglobin to be reduced. Thus, studies of animal hemoglobins may bring forth an understanding of yet unknown mechanisms operating in erythrocytes of animals as has recently been demonstrated in the case of the function of bicarbonate ions in crocodile hemoglobin (Perutz et al., 1981). Acknowledqements--This research was supported in part by Grant No. 1-624 (MRW) from The Robert A. Welch Foundation and by Grant No. 80-650 (NCB) from the Department of Clinical Investigation, Fitzsimons Army Medical Center.
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590
M . E . JOHN et al.
JOHN M. E. & WATERMAN M. R. (1979) Nitric oxide induced conformational changes in opossum hemoglobin. J. biol. Chem. 254, 11953 11957. JOHN M. E. & WATERMAN M. R. (1981) Nitric oxide induced conformational changes in opossum hemoglobin. Eur. J. Biochem. 115, 1-6. JOHN M, E., DuBots R. N. & WATERMAN M. R. (1981) Altered heine environments in opossum and rabbit methemoglobins. Z. Natu([i C. 36, 964 967. LEHMANN H. & HUNSTMANN R. G. (1974), In Man's Hemoglobins, p. 138. J. B. Lippincott, Philadelphia. PERUTZ M. F., BAUER C.. GROSS G., El ('I,ERGE F., VANDk('ASSERIE O., SCIIENK A. g., BR~UNIqZER G., FRIDAY A. E. & JOSEY K, A. (1981) Allosteric regulation of crocodilian hemoglobin. Nature, Lond. 291,682 684. SCOTT E. M., DUNCAN I. W. & EKS[RAND V. 11965) The reduced pyridine nucleotide dehydrogenases of human erythrocytes. J. biol. Chem. 240, 481 485. SHARMA V. S.. JOHN M. E. & WAltlkMgN M. R, (19821 Functional studies of a hemoglobin opossum: conch> sions drawn regarding the role of distal histidine. J. biol. Chem. (in press). STENZEL P,, BRIMHALL B., JONES R. T., BLACK J. K., McLACHLAN A. & GIBSON D. (1979) Opossum hemoglobin: the amino acid sequences of the ~ and fl chains. J. biol. Chem, 254, 2071 2076. rAK~TA F. & CHEN J. Y. (1977) Activation of the NADHmethemoglobin reductase reaction by inositol hexaphosphate. Bioehem. biophys. Res. Commun. 75, 38% 393.
TOMADA A., TsuJl A., MATSUKAWA S,. TAKESH1TA M. &, YONEYAMA Y. (1978) Mechanism of methemoglobin reduction by ascorbic acid under anaerobic conditions. J. biol. Chem. 253, 7420, 7423. TUCKER P. W,. PHILLIPS S. E. V., PI'RVI'/, M, F., I-It,Jr rCHENS R. A, & CAUGHEY W. S. (19781 Structure of hemoglobins Zurich [His E7(63)-+Arg] and Sydney[Val E11(671-+ Ala] and the role of the distal histidine in ligand binding. Proc, hath. 4cad. Sci. U.S.A. 7ft. 1076 -~1080. WALLAC'EW. J., HOUTCHENS R, A,, HOLF J. M. & C,\I.!GHI y W. S. (1977) Mechanisms for hemoglobin oxidation: the response of abnormal human and other hemoglobins to different oxidative pathways. In Biochemical and Clinical Aspects of Hemoglobin Abnormalities (Edited by CAt!CiHEY W. S), pp. 475--494. Academic Press, New York. WATERMAN M. R. & ST~NZEL P. (1974) Physical and chemical properties of opossum hemoglobin: a hemoglobin containing glutamine at position 58(E7) in the subunit. Biochim. Biophys. Acta. 359, 401 410. Wren: J. M. & DACn J. V. (1971) The unstable hemoglobins: molecular and clinical features. Proo, Hemal. 7. 69 109, WINTFRBOtIRN ('. C. & CAI~,R[I+L R. W. (1974) Studies of hemoglobin denaturation and Heinz body formation in the unstable hemoglobins. J. olin. lnrest. 54, 678 689, WINTERUOURN C. C.. MCGRATH B. M. & CARRELI_ R. W. (1976) Reactions involving superoxide and normal and unstable hemoglobins. Biochem. J. 155, 493 502.