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Structural and functional studies of hemoglobins isolated from Amazon stingrays of the genusPotamotrygon
Comp Bin(hem Phtsml Iol 62A pp 131 to 138 ~) Pt,rqut)loll Pt('~ Ltd 1979 Prltll~'d u) Great B)'uat)l
0300-9629'79~0101-0131S02i)ii/11
STRUCTURAL AND FUNCTIONAL STUDIES OF HEMOGLOBINS ISOLATED FROM AMAZON STINGRAYS O F THE G E N U S P O T A M O T R Y G O N * JOSEPH P. MARTIN, I t JOSEPH BONAVENTURA,2~ HANS J. FYHN, 3 UNNI E. H. FYHN, 3 ROBERT L. GARLICK,4 and DENNIS A. POWERS~ IDepartment of Zoology, Duke University Marine Laboratory, Beaufort, NC 28516, U.S.A.: 2Department of Biochemistry, Duke University Marine Laboratory and Duke University Medical Center, Beaufort, NC 28516, U.S.A.: 3Institute of Zoophysiology, University of Oslo, P.O. Box 1051, Bhndern, Oslo 3, Norway: 4Department of Zoology, University of Texas at Austin, Austin, TX 78712, U.S.A.: SDepartment of Biology, Johns Hopkins Umversity, Baltimore, MD 21218, U.S.A. (Recewed 5 April 1978)
Abstrae(--1. The hemolysates isolated from Amazonian stingrays, Potamotrygon sp. are heterogeneous. Their apparent molecular weights, determined by gel filtration, vary from 54,000 to 58,000. Gel filtration experiments of ferro- and ferrihemoglobin show that all of the hemolysates are tetramers which do not polymerize upon oxidation. 2. A Bohr effect is evident in oxygen equilibrium experiments of whole blood and of hemolysates. The pt of stripped hemolysates decreases 33-fold between pH 5.95 and 9.0. Netther NaCI nor urea in concentrations up to 4 M significantly affect the oxygen affinity of the stripped hemolysate. 3. The hemoglobin shows cooperative behavior. The n values, determined by the Hill equation, fall within the range 1.0-1.6 between pH 6.0 and 9.0. 4. The 02 dissociation and CO combination kinetics were measured by stopped flow spectrophotometry and flash photolysis, respectively. The oxygen dissociation rate decreases and carbon monoxide combination rate increases with increasing pH, both changing over four-fold between pH 6.3 and 8.8. The oxygen dissociation rate is also phosphate sensitive, increasing approx 22°,/, in the presence of 1 mM ATP at pH 6.3. 5. Urea denaturation experiments indicate that Potamotrygonid hemoglobin is more stable at high urea concentrations than those of other elasmobranchs and teleosts, being only 50,"',, denatured at urea concentrations greater than 9 M. 6. The oxygen affinity of the blood is higher than those previously reported for many species of marine rays and skates (Pt = 12 mm Hg at 30~C in the absence of CO_~).
INTRODUCTION E l a s m o b r a n c h hemoglobins exhibit many properties unlike those of higher vertebrates. Fyhn & Sullivan (1975) demonstrated that many elasmobranch hemoglobins readily polymerize under oxidizing conditions to form aggregates of tetramers, a characteristic shared by reptilian and a m p h i b i a n hemoglobins (Sullivan, 1974a, b) but not generally by teleost or mammalian hemoglobins. H e m e - h e m e interactions have not evolved in elasmobranch hemoglobins to as great a degree as in teleost and m a m m a l i a n respiratory pigments (Manwell, 1958, 1963; Riggs, 1970; Bonaventura et al., 1974a, b). E l a s m o b r a n c h hemoglobins also differ from teleost and m a m m a l i a n hemoglobins in their response to salt. The oxygen affinity of various ray hemoglobins increases at high salt concentrations while the oxygen affinity of fish and h u m a n hemoglobins is reduced-under similar conditions (Bonaventura et al., 1974a, b; Brunori et al., 1975; Weber et
* A Portuguese translation of this article will appear in Acta Amazonica. t To whom reprint requests should be addressed. .+An established investigator of the American Heart Association. 131
al., 1977). Moreover, the blood of elasmobranchs contains urea, a protein denaturant, and it has been proposed (Bonaventura et al., 1974) that elasmobranch hemoglobins have evolved an increased structural stability in consequence of their exposure to urea, though this hypothesis has been contested (Edelstein et al., 1976). Clearly, elasmobranch hemoglobins further demonstrate the range of functional flexibility of the hemoglobin molecule already exhibited in other vertebrate organisms. We report here structural and functional features evinced by hemoglobins isolated from freshwater stingrays of the genus Potamotrygon. This group is one of few elasmobranch taxa to have secondarily evolved a freshwater habit (Nelson, 1976). Two considerations prompted our investigation of these hemoglobins. First: rays of this family reside in the relatively hypoxic waters of the upper Amazon River and its tributaries. Hence adaptations in the hemoglobins of these animals to low environmental oxygen concentrations may have occurred. Second: life in fresh water obviates the requirement for high urea concentrations in blood as it is no longer required for osmoregulation (Smith, 1931; Goldstein & Foster, 1971; Griffith et al., 1973). Consequently, P o t a m o t r y - " gon blood has a low urea content (Thorsen et al.,
132
001 E
JOSEPH P. MARTIN et al.
0.80 .
_
0
0.60
>
0.40
"--
.¢.-
0 020
a
b
c
d
Fig. 1 Disc gel patterns of representative Potamotryoon hemolysates. Stippled areas represent blurred bands. The ordinate gives the mobility of the hemoglobin relative to bovine serum albumin. 1967) a n d its h e m o g l o b i n s m a y h a v e lost t h e u r e a stability a t t r i b u t e d to e l a s m o b r a n c h h e m o g l o b i n s . E v i d e n c e o f s u c h m o l e c u l a r a l t e r a t i o n s in f u n c t i o n s h o u l d s t r e n g t h e n t h e idea that differences a m o n g h e m o g l o b i n s o f v a r i o u s species r e p r e s e n t a d a p t a t i o n s to the exigencies o f their respective e n v i r o n m e n t s . MATERIALS AND METHODS
Isolation Specimens of Potamotrygon species were collected In November and December, 1976 during an expedition on the R.V. Alpha Helix on the Amazon River about 5 0 k m upstream on the Rio Solim6es from the junction with the Rio Negro. The rays were caught by beach seine and transported to the Alpl, a Helix for study. Blood was obtained by cardiac puncture and drawn into cold heparinized glass syringes [100pl of s o d m m heparin (5000 t.u./ml) in 1.7°,, NaCI/5 ml of blood]. Small samples were taken up in 50,ul capdlary tubes and spun for 3 mln in a serum centrifuge for hematocrlt estimations. The red blood cells were washed 3 times in 10 volumes of cold I m M Tris.* pH 8.0, 1.7°o NaCI and then lysed in 3 volumes of 1 m M Tris, p H 8 . 0 for l h r at 0:C. One tenth volume of 1 M NaCI was added to the hemolysate and the mixture was centrifuged at 28,000 0 for 15 min to remove cellular debris. The supernatant was then stripped of salt and organic phosphates by passage through a 2.5 × 50cm column of Sephadex G-25 resin equilibrated in 0.1 m M Tns, pH 8.5 followed by treatment on a delonizmg column with the following resins from top to bottom: Dowex-50W a m m o n i u m form (2 cm), Dowex-1 acetate form (2 cm) and Bio Rad AG 501-X8(D) mixed bed resin'(20 cm). The purified hemoglobin was stored at 5"C until required. Samples of ray hemolysates were frozen at - 7 0 " C , transported on dry ice to Beaufort, NC, and stored at - 2 0 ~ C for 2 months. In Beaufort, the hemolysates were thawed and used ,n gel filtration experiments and rapid kinetic studies. * Abbreviations: Bis-tris, bis (2-hydroxyethyl)iminotris (hydroxymethyl)methane: Tris, tris(hydroxymethyl)aminomethane: ATP, adenosine triphosphate: EDTA, ethylenediaminetetraacetic acid: 1', second order carbon monoxide combination velocity constant: k, first order oxygen dissoclation velocity constant: P~., the partial pressure of oxygen at whmh half of the available heme sites have bound oxygen.
Electrophoresis Vertical polyacrylamlde gel electrophoresis (pH 8.9, 7.5~, gelsJ was done at room temperature according to Ornstein (1964) and Davis (1964). Hemoglobin samples (1 mg/ml) were put into upper buffer containing 0.1 M fl-mercaptoethanol and a small a m o u n t of dithionite, bubbled with carbon monoxide, and applied to the gels. Bovine serum albumin was used as a mobility standard. The gels were stained for 3 hr in 0.25°',', Coomassie Brilliant Blue R m an acetic acid-methanol-water solution (1:2:4) and destained by diffusion. The gels were scanned at 560 nm using a Gllford gel scanner attached to a Beckman D U m o n o chromator. Individual hemolysates were identified and c o m m o n phenotypes pooled. To avoid difficulties in interpretation of the ligand binding results only one phenotype (Fig, ld) which corresponds to species IV, phenotype 2, of Fyhn e t a / . (1979), was employed in the kinetic and e q u i h b r m m experiments. Urea determinations Serum and hemolysate urea concentrations were measured in triplicate using urea nitrogen kits (Clay Adams, Inc.). lntracellular urea concentrations were estimated by determining the urea content of hemolysates treated with 10°~ trlchloroacetic acid and corrected for dilution. Molecular weight studies Gel filtration experiments of oxidized and reduced hemoglobin solutions from three rays were carried out according to Martin eta/. (1979). Oxygen equdtbrium studies Whole blood oxygen equilibria were performed at 30:C by the method of Powers et aL (1979) using a Heme-O-Scan oxygen dissociation analyzer (American Instruments Corp ). Oxygen equilibria of stripped hemolysates were carried out at 20~C as described by Riggs & Wolbach (1956). The hemoglobin solutions (60,uM) were brought to 0.05 ionic strength in Tris or bis-Tris buffers. P~ values were determined under varying conditions of pH, NaCI and urea concentration. The urea used in these experiments was deionized with Bio Rad AG 501-X8(D) mixed bed resin. The pH dependence experiments were performed in the presence and in the absence of 1 m M ATP. Rapid kinetw expermwnts Oxygen dissociation rates of ray hemoglobin in buffered solutions containing dithionite were determined using a Gibson-Durrum stopped flow spectrophotometer equipped with a pneumatic drive and a 2 c m observation chamber by a previously described method (Bonaventura et a/,, 1974c). Air-equilibrated hemoglobin solutions were rapidly mixed with degassed buffer solutions containing excess dithionite and the rate of absorbance change of the hemoglobin solution was followed at 437.5 nm. Dissociation rates were measured as a function of pH, urea and organic phosphate concentration. C O combination rates of Potamotrygon hemoglobin were examined by flash photolysis using the method and apparatus of Bonaventura et al. (1974c), and the influence or urea, ATP and pH on the combination rates were ascertained. The reaction was studied at one C O concentration and analyzed as a pseudo first order process. Analysis of the data was facilitated by use of a P D P II/E computer (Digital Equipment Corporation) and a data acquisition and storage device (DASAR, American Instruments Corp.). All experiments were performed in Tris or Bis-Tris buffers, 0.05 ionic strength after mixing. The heme concentration after mixing was 4,uM.
Structural and functional studies of hemoglobins
Detltttto'ttlion sttldies
133
I
Experiments testing the denaturing effects of urea on the hemoglobin of Pottinlotr)'qon sp. and other fish species were performed essentmlly according to Herskowts et al. {19701. The denaturing effect of urea at various concentrattons was determined by observing the decrease m absorbance m the Soret band of the oxyhemoglobin after 2 hr mcubanons at 23 C in urea solutions. No further change m the degree of denaturation was observed after 2 hr. The hcmoglobm was mixed m buffered urea solutions (0.05 M Tns, pH 7 8) to gtve a final concentration of 8 ItM heine m 1 ml. Fully denaturing conditmns were obtained by incubating thc hemoglobin m buffered 6 M guanidine hydrochloride. Absorbance readings were made at the wavelength of maximum protein absorbance for each species. The data were plotted according to the following equation'
0.8
A 0 --
-
-
---
Reduced Oxidized
m 0.7 E o
~ o6 u.l 0 5
t•
O.4
o
L
o3
O.2
i I
I i
OI
A , , - A# x 100, Itt~ --
I
!
I00
A~j
150
200
250
where n, is the percentage of hemoglobin nanve structure remaining after a 2 hr incubanon m a solt.tion with urea concentratton I,: A, is the absorbance of the hemoglobin solutton after incubation in a solution of urea concentration t~: A, is the absorbance of the hemoglobin solution in 6 M guanidine hydrochlonde, Ao is the absorbance of the hemoglobin solution after incubation m 0.05 Trts pH 7.8 without urea or guanidine hydrochlonde. The hemoglobin solutions whtch were used as fresh samples m this study were obtained from the clear nosed skate. Rt(la eglanterla: the spot, Leiostomu.s Aalllhlll'tl.',~ and the toadfish, Op.saml.s tam Samples which had been stored as frozen hemolysates at - 2 0 C were those of the tiger shark, Galeocerdo sp., the trout. Salmo n'ldell.s: the South American catfish, Hvpo,stonut~ sp. and the Amazon stingray Potamotrrgon sp.
bands in each case. Perststent streaking in the stained gels complicates the interpretation. Although it is clear that differences m the relative m o b i l i t y of the bands exist among gels, it was difficult to obtain consistent differences among phenotypes.
Gel hltration The h e m o g l o b i n s eluted with apparent molecular weights between 54,000 and 58,000. Both the oxidized [ K 3 F e ( C N ) 6 treated] and reduced (incubated in 0.1 M fl-mercaptoethanol) forms of three p h e n o t y p e s were examined. Within phenotypes, both oxidized and reduced hemolysates eluted in the same position. Spot fish hemoglobin, whtch was found to be tetrameric under the same conditions by centrifugation (s20... = 4.46S) eluted in a position identtcal to that of one of the ray phenotypes. Both K s F e ( C N ) 6 and m e r c a p t a n treated hemolysates eluted as single symmetrical peaks a m o n g all p h e n o t y p e s (Fig. 2). Neither polymerization of tetramers nor the dissociatton o f tetramers into dtmers occurred in these experiments.
RESU LTS
Characteristic,s of the blood of P o t a m o t r y g o n .sp. H e m a t o c r i t readings taken for the blood of stx mdivtduals revealed an erythrocyte p r o p o r t i o n of 24 + 5°. of the total blood. Urea d e t e r m i n a t i o n s of p l a s m a and erythrocytes gave values of 0.83 and 0.81 m M of urea, respectively. The hemolysates from four rays, five replicates per individual, were submitted to disc gel electrophoresls. Representative gel patterns appear in Fig. 1. All h e m o g l o b i n p h e n o t y p e s are complex, with two or three anodally migrating
Oxygen equilihrtum .studies Whole blood oxygenation curves, in the presence
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./
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c:
o5
0
i
i
i
i
05
i0
15
20
LOG P(Oz)
Figure 3. O',,ygen saturation curves of Potttntotr)'goll sp. whole blood wtthout CO_,( ) and with 5.6", ('02 ( - I m the eqtuhbration gases at 30 C. The ordinatc, f. ~s the fracnonal saturation, the absos.sa is the logarithm of tile partial pressure of oxygen in mm Hg. { It I' 62 I "~ I
300
ELUTION VOLUME ( m l ) Fig. 2. (]el chromatography of oxidized ( - - - - ) and reduced ( ) Potamotrygon sp. hemolysates,
134
JOSEPH P. MARTIN el al. i
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Hemoglobin + Imm ATP Hemoglobin
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Fig. 5(a, b) The dependence of the p.~ of stripped Potamotrygon sp. hemolysate on (A) chloride and (B) urea concentration at pH 7.5. Conditions are as described in text. The ordinates are expressed in mm Hg.
0
90
pH
Fig. 4. A plot of log p~, the log of the partial pressure of oxygen at which the hemoglobin is half saturated, and n vs pH for stripped (OI and stripped + 1 mM ATP (QI hemolysates taken from a single specimen of Potamotry.qon sp. Conditions sire sis described in text. and absence of 5.6°,, CO2, are represented in Fig. 3. The blood possesses a Bohr effect and there are homotropic interactions between subunits. The Pi is 12.3mm Hg at 30'C and pH 7.7 and 17.0mm Hg in the presence of 5.6% CO2. The log p~ vs pH plots of stripped ray hemolysate and stripped hemolysate + 1 mm A T P are illustrated in Fig. 4. P~ and n values were obtained by a least squares fit of the oxygenation data to the Hill equation. The Pi estimates of stripped and A T P containing hemolysates decrease approx 33-fold between pH 5.95 and 9.0. The Bohr effect, calculated as Alog P~/A pH is about - 0 . 4 in stripped ray hemoglobin. The p_~ of stripped hemoglobin at 20"C and pH 7.6 is about
1 mm Hg while that of hemoglobin + 1 mm A T P is approx 3 mm Hg. At pH 6.0 in 0.05 M bis-Tris buffer we obtained a depression in the degree of saturation of Potamotry9on hemoglobins of about 420. F a r m e r et al. (1979) observed a depression of approx 10% in experiments done in 0.05 M citrate buffer, pH 5.5. The difference between these values is small and undoubtedly arises because of differences in experimental conditions. Extrapolation of the P! data of Fig. 4 to pH 5.5 and using an n value of 1 and fitting the values to the Hill equation yields a saturation level approximately equal to that of Farmer et aL (1979). The n values vary from 1 to 1.6 between pH 5.95 and 9.0. The stripped and stripped + l m M ATP equdibrium data were analyzed by linear regression analysis. The correlation coefficients for each of the experimental treatments were r = -0.948, p < 0.001 and r = -0.968, p < 0.001, respectively. The good fit of the equihbrium data to a linear model is consistent with the observations of M u m m et al. (1978) on the
I
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6.0
7. 0
8.0
I00
'O
6O
x,"
2O
90
pH Fig. 6. The dependence on pH of the tirst order dlssocmtlon constant of Potamotry(jon sp. hcmolysate for oxygen (k) taken from initial rate data. Conditions are sis described in text. Stripped hcmolysate (O). stripped hemolysatc + I mM ATP (O), stripped hemolysate + I mM ATP + 5 M urea (11).
Structural and functional studies of hemoglobins Table 1. Rate constants for the combination of Potamotrygon hemoglobin with carbon monoxide* I' x 10-S/M per sec pH 6.2 pH 8.8
Conditions Stripped
2.44 2.22 2.44 2.48 --
3 M urea 1 mM ATP
10.4 10.3 10.6 10.3 10.3
--
10.3
hemoglobin of the sting ray Dasyatis sabina. This hemoglobin displays an almost linear relationship between its log Pi and pH. The oxygen affinity of stripped hemolysates was examined at pH 7.6 and 20~C as a function of NaCI and urea concentrations (Fig. 5a, b). Neither compound appears to exert a significant effect at up to 4 M: the P L varied little more than 1.5 mm Hg over the entire range of concentrations in each case.
Kinetics of oxygen dissociation The rate constants for oxygen dissociation of ray hemolysates, with and without 1 m M ATP, are presented as a function of pH (Fig. 6). Air equilibrated hemoglobin solutions, 8,uM, were rapidly mixed with buffer solutions containing dithionite. The kinetics of dissociation were heterogeneous at all pH values studied. The dissociation process was analyzed in terms of first order kinetics and only the initial rates of dissociation were plotted vs pH. The O2 ,,tr constant varies approx 4.3-fold in the absence and 5.2-fold in the presence of 1 mM ATP within the pH range of 6.3-8.8. At pH 6.3, O2 ,,. equals about 85/sec I
I
and 0 2 ,,,, (1 m M ATP) equals 104/sec. The presence of l m M A T P increases the O2,,,, rate approx 22.°/,, at pH 6.3. Dissociation rates of hemolysates + 1 m M ATP and 5 M urea were inspected at pH 8.8. No significant effect of urea on the O2 ,,,, rate could be discerned.
Carbon monoxide combination by flash photolysis
* Values are for the second order rate constant for CO binding calculated from the tmtlal rate data. All experiments performed in duplicate. Conditions are as described in text.
I00
135
I
Buffered hemoglobin solutions (8-10,uM in heme before mixing) containing dlthionite were examined in the absence and presence of urea under various conditions. The C O combination process was multiphasic under all conditions. Second order rate constants, calcnlated from initial reaction rates observed under varions conditions, appear in Table I. Littlc effect of 5 M urea is seen on the CO,,,, rate at either low or high pH. However, the C O .... constant is affectcd by pH increasing approx 4.4-fold between pH 6.2 and 8.8. No cffcct of A T P is observed at high pH.
Effects of urea on the Soret spectra offish hemoglobins The effects of denatnratlon by urea on the spectra in the Soret region of the oxyhemoglobins from seven species of fish is shown in Fig. 7. The urea concentration at which the n, value of each of the hemoglobins is 50°, differs considerably a m o n g species. It is evident that low concentrations of urea ( < 3 M) have little effect on the molecular environment of the heme m most of the hemoglobins inspected. At the higher urea concentrations the protein presumably unfolds, the heme pocket of the globins opens and differences in the degrees of unfolding a m o n g species become pronounced. The teleost hemoglobins denature more readily than the elasmobranch hemoglobins tested in this study. However, the hemoglobin of Potamotryyon sp., a freshwater species with little urea in its blood, possesses the greatest stablhty in urea solutions, its n value being only 50", at urea concentrations greater than 9 M.
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50
40
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6.0
7.0
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Urea Concentrahon (M) Fig 7. The pcrcentage of hemoglobin nativc struclure (n) rcmaining aftcr 2 hr incubations at 23 C in 0.05 M Tris, pH 7.8 vs urea concemral]on Hvposlomu.~sp (catfish) IO) Leur, tomu:, .\amhuru.~ (spot fish) (O): Trout I. Salmo wideus (El): Op.~anll.~ tau (toadlish) [+): Galeocerdo sp. (tiger shark) (m): Rala t'ylaJllerla (clear-nosed skate) (A): Potamotrj'qon sp. (Amazon stingray) (/x). Conditmns arc as de,;cribed in tcxt.
[36
JOSEPH P. MARTIN el al Table 2 Summary of P~ data for hemoglobins and bloods for various rays and skates Assay temperature l0 CI
pH
P! (ram Hg)
G vtmnlra mtcrm'a'~ butterfly ray Rhiuoptera hona~u,~+ cow-nosed ray Da,syati.s ,say sting ray Da.s)'atl,S ceutltra sting ray Dasyatls anlericana sting ray DasvUtlS sahma sting ray Raja chn ata thornback ray Raja hiuocuhtta barndoor skate Torpedo nohiliana Atlantic torpedo
25
7.4
14.5
25
74
14
25
7.4
14.5
25
74
15
25
7.4
15
Raja e.qhmterta clear-nosed skate Raja o.sctlhlta winter skate Raju Oscllhtta winter skate Potanlotrrqon sp. Amazon ray Potanlotr)'gon sp. Amazon ray
Species
-
Conditions
Dilute hemoErythro- globin cytes solution
Source
+
McCutcheon (1947)
+
McCutcheon (1947)
+
McCutcheon (1947)
+
McCutcheon (1947)
+
McCutcheon (1947)
+
McCutcheon (1947)
0.033 M phosphate buffer 0.033 M phosphate buffer 0.033 M phosphate buffer 0.033 M phosphate buffer 0.033 M phosphate buffer 0.033 M phosphate buffer Whole blood
-
+
Hughes & Wood (1974)
+
Manwell (1958")
25
7.4
]5
15
7.7
30.2
10
7.5
20
20
7.5
16
20
7.4
19.9
20
7.6
30
buffered Ringer's solution 0.05 M Trts 0.1 M NaCI 0.001 M ATP 0.05 M Tris 0.1 M NaCI Whole blood
30
7.6
50
Whole blood
+
-
20
75
3.4
--
+
Martin el al. (this paper)
30
--
12.2
0.05 M Trts 0.001 M ATP Whole blood 0.2 M THs
+
--
Martin et al. (this paper)
+
+
Bonaventura et al. (1974a)
--
Bonaventura et al. (1974b) Dill et al. (1932)
+
Dill et al. (1932)
t McCulcheon refers to this ,~pccics as Pterol~lttlt,a micrltra. McCutchcon refers to this species as R. quadriloha DISCUSSION Potmnotrygon sp. live in waters which are relatively hypoxic in comparison to the habitats of marine rays and skates. Although the hematocrits of the freshwater rays do not differ significantly from that reported in a marine species, Raja oscilhlta (Dill et al., 1932), the oxygen affinity of their hemoglobin is substantially higher than those reported for various species of marine rajiformes (Table 21. Admittedly, the experimental conditions reported in Table 2 differ from one study to another and these differences may accentuate minor differences in hemoglobin oxygen affinity. Nonetheless, the magnitude of the difference between the P~ of Potantotrvgon hemoglobin and those reported for the hemoglobins of other species are apparent. Thus Potamotry.qon hemoglobin may have evolved a higher oxygen affimty in response to hypoxic stress, The data from this study indicate that a considerable difference exists in the oxygen affinity measured in whole blood and the hemoglobin oxygen affinity measured in the presence of 1 mM ATP. This discrepancy may have several sources. Whole blood affinities were measured at 30 C rather than at 20 C as in the case of hemolysates: increases in temperature generally reduce hemoglobin oxygen affinity. Also the intracellular pH of the whole blood was not determined and may have been below pH 7. Inspection of Fig. 4 shows that stripped hemolysates + I mM ATP
do approach the lower affinity state seen in the whole blood when experiments are performed in the lower pH range. Also, potent modulators such as guanosine triphosphate or inositol pentaphosphate, whose effects on the hemolysates were not examined, may influence hemoglobin oxygen affinity within the ceil. The seemingly linear response of log p~ to pH is an interesting feature of Potamotrygon hemoglobin. At least two interpretations of this phenomenon are possible. The molecule may possess many Bohr groups, each of small effect. Alternatively, and more likely, the hemolysate may consist of a mixture of hemoglobins with different oxygen affinities and differential responses to pH (Antonini & Brunori, 1971). This view is supported by the multiphasic binding and dissociation rate of ligands noted in kinetic experiments. Potmnotry.qon hemoglobin is insensitive to changes in salt concentration, a behavior quite different from that evinced by hemoglobins of teleosts and mammals, whose affinities decrease with increasing salt concentration, and of those observed in the hemoglobins of many marine Rajlformes, where the oxygen affinities increase with an increase in salt concentration. It is of interest that Mumm et al. (1978) have recently reported that the oxygen affinity of hemoglobin isolated from the sting ray, Dasyatis sabinu is also insensitive to NaCI. Bonaventura et al. (1974b) suggests that dissociation of tetramers to more reactive dimers may be responsible for the increased
Structural and functional studies of hemoglobins affinity of ray and skate hemoglobin at high salt concentrations. They infer from this phenomenon that electrostatic interactions may be responsible for the great stability of these hemoglobins. If so, other mechanisms must be suggested to account for the extraordinary stability observed of Potomatryyon hemoglobin. Potamotryyon hemoglobin also differs from most elasmobranch hemoglobins in its inability to polymerize. Probably sulfhydryl residues are not available at the surface of these proteins for intermolecular disulfide bridge formation. Potamotryyon hemoglobin resembles the hemoglobins of other elasmobranchs and of teleosts In its kinetic behavior in that the organic phosphate effect is manifested in its influence on the O2,,,, constant. Also, this hemoglobin possesses a Bohr effect as do most fish hemoglobins. However, the relative contribution of the CO,,,, constant to the pH effect seems greater than that characteristic of other fish species. The p.~ of stripped Potamotryyon hemoglobin decreases approx 33-fold between pH 5.95 and 9.0. Over the pH range 6.3-8.8 the CO .... constant increases 4.4-fold and the O2,,,, constant decreases 4.2-fold. Both the direction and magnitude of the changes in the on and off constants are in qualitative agreement with the equilibrium results. Subunit dissociation, as measured by the increase in proportion of fast reacting material after flash photolysis, appears negligible in urea concentrations up to 5 M. These results are further confirmed by the outcome of oxygen equilibrium and oxygen dissociation experiments performed m urea. Thus, while Potamotrygon species do not maintain high blood urea concentrations their hemoglobins nevertheless retain a remarkable stability in urea. Further proof of this stability comes from the urea denaturation studies. Although the urea concentrations in these experiments were far in excess of the concentrations found in marine elasmobranchs, a definite trend is discernible. It appears that the teleost hemoglobins were more easily denatured that the elasmobranch hemoglobins. What physiological advantage is to be derived from this added stability is obscure. It seems that rather than having lost the extra stability generally exhibited by elasmobranch hemoglobins, that of Potamotrygon has maintained whatever stability its marine phylogenetic ancestors may have possessed.
Acl, nowledyements--We would like to thank Dr Celia Bonaventura for her assistance in performing the kinetic experiments, Dr Emilia Pandolfelh for her advice and suggestions, the crew of the Alpha Helix, and the Brazilian government for the permission to work in Amazon waters, Hans J. Fyhn and Unni E. H. Fyhn acknowledge support from the Norwegian Research Council for Science and the Humanities. This work was supported m part by the National Science Foundation under Grant PCM 75-06451 to the Scripps Institute of Oceanography for support of the Alpha Helix Program and by National Institutes of Health Grant HL15460. Joseph Martin was supported by National Institutes of Health Grant GM07184 to Dt'kc University. REFERENCES ANTONINI E. & BRUNORI M. (1971) Hemot.llobin and Myot.llobin ul Their Reactton.~ wath Li.qand.s. 436 pp. NorthHolland, Amsterdam.
137
BONAVENTURAJ., BONAVENTURAC. & SULLIVANB. (1974a) Hemoglobin of the electric Atlantic torpedo, Torpedo nohdiana: a cooperative hemoglobin without Bohr effects. Biochim. hlophy.s. A cta 37 I, 147-154. BONAVENTURAJ., BONAVEN1URAC. & SULt.lVANB. (1974b) Urea tolerance as a molecular adaptation of elasmobranch hemoglobins. Science 186, 57-59. BONAVENTURAC., SULLIVANB. & BONAVENTURAJ. (1974c) The effects of pH and anions on functional properties of hemoglobin from Lemur fidrus fidt'us. J. biol. Chem. 249, 3768-3775. BRtJNORI M. FALCIONI G.. FORTtrNI G. & GIAROINA B. (1975) Effects of amons on the oxygen binding properties of the hemoglobin components from trout (Sahno irtdeltx}. Archs. Btochem. Biophys. 168, 512 519 DAVIS B. J. (1964) D,sc electrophoresis II. method and application to human serum proteins, Ann N.Y. Aead. Sci. 121,404-427. DILL D. B., EDWARDS H. T. & FLORgIN M. A. (1932) Properties of the blood of skate (Rt(/a o.setllata). Biol. Bldl. 62, 23-36. ED~LSTE,N S. J., McEwEN B. & GmSON Q. H. (19761 Subumt dissociation of fish hemoglobins. J. biol. Chem. 251, 7632-7637. FARMER M,, FYHN H.J., FVHN U. E. H. & NOBLr: R. (19791 Occurrence of Root effect hemoglobins in Amazonian fishes. Comp. Bioehenl. Ph)'xiol. 62, 115 124. FVHN U. E. H. & SULLIVANB. (1975) Elasmobranch hemoglobins dimerJzation and polymerization in various species. Comp. Bioehem. Physiol. 50B, 119-129. FYHN U. E. H., FYHN H. J.. DAVIS B. J., PDWERS D. A., FINK W. L. & GARLtCK R. L (1979) Hemoglobin heterogeneity in Amazon,an fishes. Comp. Btochem. Phl'.~iol. 62, 39 66. GOLDSTr~IN L. & FOSTER R. P. (1971) Urea biosynthesis and excretion in freshwater and marine elasmobrancbs Comp. Biochem. PhyMol, 39B, 415 421. GRIFFITH R W.. PANG P.. SRIVASTAVAA, 6~ PICKFORDG. (19731 Serum composition of fresh water stmgrays (Potamotrygonidae) adapted to fresh and dilute seawater. Biol. Bull 144, 304-320. HI-:RSKOVITST. T., JAILLET H. & GADEGBKU B. (1970) On the structural stability and solvent denaturation of proteins. I1. Denaturation by ureas. J. biol. Chem. 245, 4544-4560. HUC;HESG. M. & WOODS. C. (1974) Respiratory properties of the blood of the thornback ray (Rata clarata). Experientia 30/2, 167-168. McCuTcHEON F. (1947) Specific oxygen affinity of hemoglobin m elasmobranchs and turtles. J. Cell. Comp. Phy.siol. 29, 333-338. MANWELL C. (1958) Ontogeny of hemoglobin in the skate Rata hinoculata. Science 128, 419 420. MANWELt. C. (19631 Fetal and adult hemoglobins of the spray dogfish Sq,ahs s,ckle)'l. ,4rch.s Btochem. Biophys. 101, 504-51 I. MARtIN J. P., BONAVENTURA J., BRUNORI M., F'YHN H. J.. FVHN U. E. H., GAgLICK R. L, POWERS D. A. & WILSON M. T. (1979) The isolation and characterization of the hemoglobin components of Mylos.soma sp., an Amazonian teleost. Comp. Biochem. Physiol. (this issue). MOMM D. P., ATHA D. H. & RIGC3S A. (1978) The hemoglobin of the common sting ray, DtLsyuti.s .sabma: structural and functional properties. Comp. Biochem. Physiol. (in press). NELSON J. S. (1976) Fi.she.s of the |,Vorhl. 415 pp. John Wdey, New York. ORNST,':tN L. (1964) Disc electrophores~s--l. Background and theory. Ann. N.Y. Acad. Scl. 121, 321 349. POWERS D. A.. FVHN H J.. FVHN U. E. H.. MARTIN J. P., GARL,CK R. L & WOOD S. C. (1979) A comparative study of the oxygen equilibria of blood from 40 genera of Amazonian fishes Comp. Bioehem. PIn'siol. (this issue).
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RIGGS A (1970) Properties of fish hemoglobins. In Fish Phy.~iohu.ly, Vol. 4 (Edited by HOAR W. S. & RANDALL D. J.), pp. 209 251. Academic Press, New York RIGGS A. & WOLBACH R. A. (1956) Sulfhydryl groups and the structure of hemoglobin. J qen. Ph)'siol 39, 585-605. SMITH H. W. (1931) The absorption and excretion of water and salts by the elasmobranch fishes I. Freshwater elasmobranchs. Am J. Physiol. 98, 279-295. SULLIVAN B, (1974a) Amphibian hemoglobins. In Chemical Zoolo.qy, Vol 9 (Edfled by FLORKIN M. & SCHF.ER B. T ), pp. 77 118. Academic Press, New York.
SULLIVAN B. (1974b) Reptilian hemoglobins. In Chemical Zoolo~jy, Vol. 9 (Edited by FLORKIN M. & SCHEER B. T), pp. 353-374. Academic Press, New York. THORSON T. V., COWAN C. M. & WATSON D. E. (1967) Potamotry#on sp.: elasmobranchs with low urea content. Science 158, 375-377. WEBER R E., JOHANSEN K., LYKKEBOE G & MALOIY G. O. (1977) The oxygen binding properties of hemoglobins from aestivating and active African lungfish. J. exp. Zool. 199, 85 99.
0300-9629'79~0101-0131S02i)ii/11
STRUCTURAL AND FUNCTIONAL STUDIES OF HEMOGLOBINS ISOLATED FROM AMAZON STINGRAYS O F THE G E N U S P O T A M O T R Y G O N * JOSEPH P. MARTIN, I t JOSEPH BONAVENTURA,2~ HANS J. FYHN, 3 UNNI E. H. FYHN, 3 ROBERT L. GARLICK,4 and DENNIS A. POWERS~ IDepartment of Zoology, Duke University Marine Laboratory, Beaufort, NC 28516, U.S.A.: 2Department of Biochemistry, Duke University Marine Laboratory and Duke University Medical Center, Beaufort, NC 28516, U.S.A.: 3Institute of Zoophysiology, University of Oslo, P.O. Box 1051, Bhndern, Oslo 3, Norway: 4Department of Zoology, University of Texas at Austin, Austin, TX 78712, U.S.A.: SDepartment of Biology, Johns Hopkins Umversity, Baltimore, MD 21218, U.S.A. (Recewed 5 April 1978)
Abstrae(--1. The hemolysates isolated from Amazonian stingrays, Potamotrygon sp. are heterogeneous. Their apparent molecular weights, determined by gel filtration, vary from 54,000 to 58,000. Gel filtration experiments of ferro- and ferrihemoglobin show that all of the hemolysates are tetramers which do not polymerize upon oxidation. 2. A Bohr effect is evident in oxygen equilibrium experiments of whole blood and of hemolysates. The pt of stripped hemolysates decreases 33-fold between pH 5.95 and 9.0. Netther NaCI nor urea in concentrations up to 4 M significantly affect the oxygen affinity of the stripped hemolysate. 3. The hemoglobin shows cooperative behavior. The n values, determined by the Hill equation, fall within the range 1.0-1.6 between pH 6.0 and 9.0. 4. The 02 dissociation and CO combination kinetics were measured by stopped flow spectrophotometry and flash photolysis, respectively. The oxygen dissociation rate decreases and carbon monoxide combination rate increases with increasing pH, both changing over four-fold between pH 6.3 and 8.8. The oxygen dissociation rate is also phosphate sensitive, increasing approx 22°,/, in the presence of 1 mM ATP at pH 6.3. 5. Urea denaturation experiments indicate that Potamotrygonid hemoglobin is more stable at high urea concentrations than those of other elasmobranchs and teleosts, being only 50,"',, denatured at urea concentrations greater than 9 M. 6. The oxygen affinity of the blood is higher than those previously reported for many species of marine rays and skates (Pt = 12 mm Hg at 30~C in the absence of CO_~).
INTRODUCTION E l a s m o b r a n c h hemoglobins exhibit many properties unlike those of higher vertebrates. Fyhn & Sullivan (1975) demonstrated that many elasmobranch hemoglobins readily polymerize under oxidizing conditions to form aggregates of tetramers, a characteristic shared by reptilian and a m p h i b i a n hemoglobins (Sullivan, 1974a, b) but not generally by teleost or mammalian hemoglobins. H e m e - h e m e interactions have not evolved in elasmobranch hemoglobins to as great a degree as in teleost and m a m m a l i a n respiratory pigments (Manwell, 1958, 1963; Riggs, 1970; Bonaventura et al., 1974a, b). E l a s m o b r a n c h hemoglobins also differ from teleost and m a m m a l i a n hemoglobins in their response to salt. The oxygen affinity of various ray hemoglobins increases at high salt concentrations while the oxygen affinity of fish and h u m a n hemoglobins is reduced-under similar conditions (Bonaventura et al., 1974a, b; Brunori et al., 1975; Weber et
* A Portuguese translation of this article will appear in Acta Amazonica. t To whom reprint requests should be addressed. .+An established investigator of the American Heart Association. 131
al., 1977). Moreover, the blood of elasmobranchs contains urea, a protein denaturant, and it has been proposed (Bonaventura et al., 1974) that elasmobranch hemoglobins have evolved an increased structural stability in consequence of their exposure to urea, though this hypothesis has been contested (Edelstein et al., 1976). Clearly, elasmobranch hemoglobins further demonstrate the range of functional flexibility of the hemoglobin molecule already exhibited in other vertebrate organisms. We report here structural and functional features evinced by hemoglobins isolated from freshwater stingrays of the genus Potamotrygon. This group is one of few elasmobranch taxa to have secondarily evolved a freshwater habit (Nelson, 1976). Two considerations prompted our investigation of these hemoglobins. First: rays of this family reside in the relatively hypoxic waters of the upper Amazon River and its tributaries. Hence adaptations in the hemoglobins of these animals to low environmental oxygen concentrations may have occurred. Second: life in fresh water obviates the requirement for high urea concentrations in blood as it is no longer required for osmoregulation (Smith, 1931; Goldstein & Foster, 1971; Griffith et al., 1973). Consequently, P o t a m o t r y - " gon blood has a low urea content (Thorsen et al.,
132
001 E
JOSEPH P. MARTIN et al.
0.80 .
_
0
0.60
>
0.40
"--
.¢.-
0 020
a
b
c
d
Fig. 1 Disc gel patterns of representative Potamotryoon hemolysates. Stippled areas represent blurred bands. The ordinate gives the mobility of the hemoglobin relative to bovine serum albumin. 1967) a n d its h e m o g l o b i n s m a y h a v e lost t h e u r e a stability a t t r i b u t e d to e l a s m o b r a n c h h e m o g l o b i n s . E v i d e n c e o f s u c h m o l e c u l a r a l t e r a t i o n s in f u n c t i o n s h o u l d s t r e n g t h e n t h e idea that differences a m o n g h e m o g l o b i n s o f v a r i o u s species r e p r e s e n t a d a p t a t i o n s to the exigencies o f their respective e n v i r o n m e n t s . MATERIALS AND METHODS
Isolation Specimens of Potamotrygon species were collected In November and December, 1976 during an expedition on the R.V. Alpha Helix on the Amazon River about 5 0 k m upstream on the Rio Solim6es from the junction with the Rio Negro. The rays were caught by beach seine and transported to the Alpl, a Helix for study. Blood was obtained by cardiac puncture and drawn into cold heparinized glass syringes [100pl of s o d m m heparin (5000 t.u./ml) in 1.7°,, NaCI/5 ml of blood]. Small samples were taken up in 50,ul capdlary tubes and spun for 3 mln in a serum centrifuge for hematocrlt estimations. The red blood cells were washed 3 times in 10 volumes of cold I m M Tris.* pH 8.0, 1.7°o NaCI and then lysed in 3 volumes of 1 m M Tris, p H 8 . 0 for l h r at 0:C. One tenth volume of 1 M NaCI was added to the hemolysate and the mixture was centrifuged at 28,000 0 for 15 min to remove cellular debris. The supernatant was then stripped of salt and organic phosphates by passage through a 2.5 × 50cm column of Sephadex G-25 resin equilibrated in 0.1 m M Tns, pH 8.5 followed by treatment on a delonizmg column with the following resins from top to bottom: Dowex-50W a m m o n i u m form (2 cm), Dowex-1 acetate form (2 cm) and Bio Rad AG 501-X8(D) mixed bed resin'(20 cm). The purified hemoglobin was stored at 5"C until required. Samples of ray hemolysates were frozen at - 7 0 " C , transported on dry ice to Beaufort, NC, and stored at - 2 0 ~ C for 2 months. In Beaufort, the hemolysates were thawed and used ,n gel filtration experiments and rapid kinetic studies. * Abbreviations: Bis-tris, bis (2-hydroxyethyl)iminotris (hydroxymethyl)methane: Tris, tris(hydroxymethyl)aminomethane: ATP, adenosine triphosphate: EDTA, ethylenediaminetetraacetic acid: 1', second order carbon monoxide combination velocity constant: k, first order oxygen dissoclation velocity constant: P~., the partial pressure of oxygen at whmh half of the available heme sites have bound oxygen.
Electrophoresis Vertical polyacrylamlde gel electrophoresis (pH 8.9, 7.5~, gelsJ was done at room temperature according to Ornstein (1964) and Davis (1964). Hemoglobin samples (1 mg/ml) were put into upper buffer containing 0.1 M fl-mercaptoethanol and a small a m o u n t of dithionite, bubbled with carbon monoxide, and applied to the gels. Bovine serum albumin was used as a mobility standard. The gels were stained for 3 hr in 0.25°',', Coomassie Brilliant Blue R m an acetic acid-methanol-water solution (1:2:4) and destained by diffusion. The gels were scanned at 560 nm using a Gllford gel scanner attached to a Beckman D U m o n o chromator. Individual hemolysates were identified and c o m m o n phenotypes pooled. To avoid difficulties in interpretation of the ligand binding results only one phenotype (Fig, ld) which corresponds to species IV, phenotype 2, of Fyhn e t a / . (1979), was employed in the kinetic and e q u i h b r m m experiments. Urea determinations Serum and hemolysate urea concentrations were measured in triplicate using urea nitrogen kits (Clay Adams, Inc.). lntracellular urea concentrations were estimated by determining the urea content of hemolysates treated with 10°~ trlchloroacetic acid and corrected for dilution. Molecular weight studies Gel filtration experiments of oxidized and reduced hemoglobin solutions from three rays were carried out according to Martin eta/. (1979). Oxygen equdtbrium studies Whole blood oxygen equilibria were performed at 30:C by the method of Powers et aL (1979) using a Heme-O-Scan oxygen dissociation analyzer (American Instruments Corp ). Oxygen equilibria of stripped hemolysates were carried out at 20~C as described by Riggs & Wolbach (1956). The hemoglobin solutions (60,uM) were brought to 0.05 ionic strength in Tris or bis-Tris buffers. P~ values were determined under varying conditions of pH, NaCI and urea concentration. The urea used in these experiments was deionized with Bio Rad AG 501-X8(D) mixed bed resin. The pH dependence experiments were performed in the presence and in the absence of 1 m M ATP. Rapid kinetw expermwnts Oxygen dissociation rates of ray hemoglobin in buffered solutions containing dithionite were determined using a Gibson-Durrum stopped flow spectrophotometer equipped with a pneumatic drive and a 2 c m observation chamber by a previously described method (Bonaventura et a/,, 1974c). Air-equilibrated hemoglobin solutions were rapidly mixed with degassed buffer solutions containing excess dithionite and the rate of absorbance change of the hemoglobin solution was followed at 437.5 nm. Dissociation rates were measured as a function of pH, urea and organic phosphate concentration. C O combination rates of Potamotrygon hemoglobin were examined by flash photolysis using the method and apparatus of Bonaventura et al. (1974c), and the influence or urea, ATP and pH on the combination rates were ascertained. The reaction was studied at one C O concentration and analyzed as a pseudo first order process. Analysis of the data was facilitated by use of a P D P II/E computer (Digital Equipment Corporation) and a data acquisition and storage device (DASAR, American Instruments Corp.). All experiments were performed in Tris or Bis-Tris buffers, 0.05 ionic strength after mixing. The heme concentration after mixing was 4,uM.
Structural and functional studies of hemoglobins
Detltttto'ttlion sttldies
133
I
Experiments testing the denaturing effects of urea on the hemoglobin of Pottinlotr)'qon sp. and other fish species were performed essentmlly according to Herskowts et al. {19701. The denaturing effect of urea at various concentrattons was determined by observing the decrease m absorbance m the Soret band of the oxyhemoglobin after 2 hr mcubanons at 23 C in urea solutions. No further change m the degree of denaturation was observed after 2 hr. The hcmoglobm was mixed m buffered urea solutions (0.05 M Tns, pH 7 8) to gtve a final concentration of 8 ItM heine m 1 ml. Fully denaturing conditmns were obtained by incubating thc hemoglobin m buffered 6 M guanidine hydrochloride. Absorbance readings were made at the wavelength of maximum protein absorbance for each species. The data were plotted according to the following equation'
0.8
A 0 --
-
-
---
Reduced Oxidized
m 0.7 E o
~ o6 u.l 0 5
t•
O.4
o
L
o3
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i I
I i
OI
A , , - A# x 100, Itt~ --
I
!
I00
A~j
150
200
250
where n, is the percentage of hemoglobin nanve structure remaining after a 2 hr incubanon m a solt.tion with urea concentratton I,: A, is the absorbance of the hemoglobin solutton after incubation in a solution of urea concentration t~: A, is the absorbance of the hemoglobin solution in 6 M guanidine hydrochlonde, Ao is the absorbance of the hemoglobin solution after incubation m 0.05 Trts pH 7.8 without urea or guanidine hydrochlonde. The hemoglobin solutions whtch were used as fresh samples m this study were obtained from the clear nosed skate. Rt(la eglanterla: the spot, Leiostomu.s Aalllhlll'tl.',~ and the toadfish, Op.saml.s tam Samples which had been stored as frozen hemolysates at - 2 0 C were those of the tiger shark, Galeocerdo sp., the trout. Salmo n'ldell.s: the South American catfish, Hvpo,stonut~ sp. and the Amazon stingray Potamotrrgon sp.
bands in each case. Perststent streaking in the stained gels complicates the interpretation. Although it is clear that differences m the relative m o b i l i t y of the bands exist among gels, it was difficult to obtain consistent differences among phenotypes.
Gel hltration The h e m o g l o b i n s eluted with apparent molecular weights between 54,000 and 58,000. Both the oxidized [ K 3 F e ( C N ) 6 treated] and reduced (incubated in 0.1 M fl-mercaptoethanol) forms of three p h e n o t y p e s were examined. Within phenotypes, both oxidized and reduced hemolysates eluted in the same position. Spot fish hemoglobin, whtch was found to be tetrameric under the same conditions by centrifugation (s20... = 4.46S) eluted in a position identtcal to that of one of the ray phenotypes. Both K s F e ( C N ) 6 and m e r c a p t a n treated hemolysates eluted as single symmetrical peaks a m o n g all p h e n o t y p e s (Fig. 2). Neither polymerization of tetramers nor the dissociatton o f tetramers into dtmers occurred in these experiments.
RESU LTS
Characteristic,s of the blood of P o t a m o t r y g o n .sp. H e m a t o c r i t readings taken for the blood of stx mdivtduals revealed an erythrocyte p r o p o r t i o n of 24 + 5°. of the total blood. Urea d e t e r m i n a t i o n s of p l a s m a and erythrocytes gave values of 0.83 and 0.81 m M of urea, respectively. The hemolysates from four rays, five replicates per individual, were submitted to disc gel electrophoresls. Representative gel patterns appear in Fig. 1. All h e m o g l o b i n p h e n o t y p e s are complex, with two or three anodally migrating
Oxygen equilihrtum .studies Whole blood oxygenation curves, in the presence
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./
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o5
0
i
i
i
i
05
i0
15
20
LOG P(Oz)
Figure 3. O',,ygen saturation curves of Potttntotr)'goll sp. whole blood wtthout CO_,( ) and with 5.6", ('02 ( - I m the eqtuhbration gases at 30 C. The ordinatc, f. ~s the fracnonal saturation, the absos.sa is the logarithm of tile partial pressure of oxygen in mm Hg. { It I' 62 I "~ I
300
ELUTION VOLUME ( m l ) Fig. 2. (]el chromatography of oxidized ( - - - - ) and reduced ( ) Potamotrygon sp. hemolysates,
134
JOSEPH P. MARTIN el al. i
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Fig. 5(a, b) The dependence of the p.~ of stripped Potamotrygon sp. hemolysate on (A) chloride and (B) urea concentration at pH 7.5. Conditions are as described in text. The ordinates are expressed in mm Hg.
0
90
pH
Fig. 4. A plot of log p~, the log of the partial pressure of oxygen at which the hemoglobin is half saturated, and n vs pH for stripped (OI and stripped + 1 mM ATP (QI hemolysates taken from a single specimen of Potamotry.qon sp. Conditions sire sis described in text. and absence of 5.6°,, CO2, are represented in Fig. 3. The blood possesses a Bohr effect and there are homotropic interactions between subunits. The Pi is 12.3mm Hg at 30'C and pH 7.7 and 17.0mm Hg in the presence of 5.6% CO2. The log p~ vs pH plots of stripped ray hemolysate and stripped hemolysate + 1 mm A T P are illustrated in Fig. 4. P~ and n values were obtained by a least squares fit of the oxygenation data to the Hill equation. The Pi estimates of stripped and A T P containing hemolysates decrease approx 33-fold between pH 5.95 and 9.0. The Bohr effect, calculated as Alog P~/A pH is about - 0 . 4 in stripped ray hemoglobin. The p_~ of stripped hemoglobin at 20"C and pH 7.6 is about
1 mm Hg while that of hemoglobin + 1 mm A T P is approx 3 mm Hg. At pH 6.0 in 0.05 M bis-Tris buffer we obtained a depression in the degree of saturation of Potamotry9on hemoglobins of about 420. F a r m e r et al. (1979) observed a depression of approx 10% in experiments done in 0.05 M citrate buffer, pH 5.5. The difference between these values is small and undoubtedly arises because of differences in experimental conditions. Extrapolation of the P! data of Fig. 4 to pH 5.5 and using an n value of 1 and fitting the values to the Hill equation yields a saturation level approximately equal to that of Farmer et aL (1979). The n values vary from 1 to 1.6 between pH 5.95 and 9.0. The stripped and stripped + l m M ATP equdibrium data were analyzed by linear regression analysis. The correlation coefficients for each of the experimental treatments were r = -0.948, p < 0.001 and r = -0.968, p < 0.001, respectively. The good fit of the equihbrium data to a linear model is consistent with the observations of M u m m et al. (1978) on the
I
I
I
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I
I
6.0
7. 0
8.0
I00
'O
6O
x,"
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pH Fig. 6. The dependence on pH of the tirst order dlssocmtlon constant of Potamotry(jon sp. hcmolysate for oxygen (k) taken from initial rate data. Conditions are sis described in text. Stripped hcmolysate (O). stripped hemolysatc + I mM ATP (O), stripped hemolysate + I mM ATP + 5 M urea (11).
Structural and functional studies of hemoglobins Table 1. Rate constants for the combination of Potamotrygon hemoglobin with carbon monoxide* I' x 10-S/M per sec pH 6.2 pH 8.8
Conditions Stripped
2.44 2.22 2.44 2.48 --
3 M urea 1 mM ATP
10.4 10.3 10.6 10.3 10.3
--
10.3
hemoglobin of the sting ray Dasyatis sabina. This hemoglobin displays an almost linear relationship between its log Pi and pH. The oxygen affinity of stripped hemolysates was examined at pH 7.6 and 20~C as a function of NaCI and urea concentrations (Fig. 5a, b). Neither compound appears to exert a significant effect at up to 4 M: the P L varied little more than 1.5 mm Hg over the entire range of concentrations in each case.
Kinetics of oxygen dissociation The rate constants for oxygen dissociation of ray hemolysates, with and without 1 m M ATP, are presented as a function of pH (Fig. 6). Air equilibrated hemoglobin solutions, 8,uM, were rapidly mixed with buffer solutions containing dithionite. The kinetics of dissociation were heterogeneous at all pH values studied. The dissociation process was analyzed in terms of first order kinetics and only the initial rates of dissociation were plotted vs pH. The O2 ,,tr constant varies approx 4.3-fold in the absence and 5.2-fold in the presence of 1 mM ATP within the pH range of 6.3-8.8. At pH 6.3, O2 ,,. equals about 85/sec I
I
and 0 2 ,,,, (1 m M ATP) equals 104/sec. The presence of l m M A T P increases the O2,,,, rate approx 22.°/,, at pH 6.3. Dissociation rates of hemolysates + 1 m M ATP and 5 M urea were inspected at pH 8.8. No significant effect of urea on the O2 ,,,, rate could be discerned.
Carbon monoxide combination by flash photolysis
* Values are for the second order rate constant for CO binding calculated from the tmtlal rate data. All experiments performed in duplicate. Conditions are as described in text.
I00
135
I
Buffered hemoglobin solutions (8-10,uM in heme before mixing) containing dlthionite were examined in the absence and presence of urea under various conditions. The C O combination process was multiphasic under all conditions. Second order rate constants, calcnlated from initial reaction rates observed under varions conditions, appear in Table I. Littlc effect of 5 M urea is seen on the CO,,,, rate at either low or high pH. However, the C O .... constant is affectcd by pH increasing approx 4.4-fold between pH 6.2 and 8.8. No cffcct of A T P is observed at high pH.
Effects of urea on the Soret spectra offish hemoglobins The effects of denatnratlon by urea on the spectra in the Soret region of the oxyhemoglobins from seven species of fish is shown in Fig. 7. The urea concentration at which the n, value of each of the hemoglobins is 50°, differs considerably a m o n g species. It is evident that low concentrations of urea ( < 3 M) have little effect on the molecular environment of the heme m most of the hemoglobins inspected. At the higher urea concentrations the protein presumably unfolds, the heme pocket of the globins opens and differences in the degrees of unfolding a m o n g species become pronounced. The teleost hemoglobins denature more readily than the elasmobranch hemoglobins tested in this study. However, the hemoglobin of Potamotryyon sp., a freshwater species with little urea in its blood, possesses the greatest stablhty in urea solutions, its n value being only 50", at urea concentrations greater than 9 M.
I
I
[
I
I
I
ID °~
z 50 (..9
tl.
0
I
1.0
I
I
I
I
I
I
I
I
20
50
40
5.0
6.0
7.0
80
9.0
Urea Concentrahon (M) Fig 7. The pcrcentage of hemoglobin nativc struclure (n) rcmaining aftcr 2 hr incubations at 23 C in 0.05 M Tris, pH 7.8 vs urea concemral]on Hvposlomu.~sp (catfish) IO) Leur, tomu:, .\amhuru.~ (spot fish) (O): Trout I. Salmo wideus (El): Op.~anll.~ tau (toadlish) [+): Galeocerdo sp. (tiger shark) (m): Rala t'ylaJllerla (clear-nosed skate) (A): Potamotrj'qon sp. (Amazon stingray) (/x). Conditmns arc as de,;cribed in tcxt.
[36
JOSEPH P. MARTIN el al Table 2 Summary of P~ data for hemoglobins and bloods for various rays and skates Assay temperature l0 CI
pH
P! (ram Hg)
G vtmnlra mtcrm'a'~ butterfly ray Rhiuoptera hona~u,~+ cow-nosed ray Da,syati.s ,say sting ray Da.s)'atl,S ceutltra sting ray Dasyatls anlericana sting ray DasvUtlS sahma sting ray Raja chn ata thornback ray Raja hiuocuhtta barndoor skate Torpedo nohiliana Atlantic torpedo
25
7.4
14.5
25
74
14
25
7.4
14.5
25
74
15
25
7.4
15
Raja e.qhmterta clear-nosed skate Raja o.sctlhlta winter skate Raju Oscllhtta winter skate Potanlotrrqon sp. Amazon ray Potanlotr)'gon sp. Amazon ray
Species
-
Conditions
Dilute hemoErythro- globin cytes solution
Source
+
McCutcheon (1947)
+
McCutcheon (1947)
+
McCutcheon (1947)
+
McCutcheon (1947)
+
McCutcheon (1947)
+
McCutcheon (1947)
0.033 M phosphate buffer 0.033 M phosphate buffer 0.033 M phosphate buffer 0.033 M phosphate buffer 0.033 M phosphate buffer 0.033 M phosphate buffer Whole blood
-
+
Hughes & Wood (1974)
+
Manwell (1958")
25
7.4
]5
15
7.7
30.2
10
7.5
20
20
7.5
16
20
7.4
19.9
20
7.6
30
buffered Ringer's solution 0.05 M Trts 0.1 M NaCI 0.001 M ATP 0.05 M Tris 0.1 M NaCI Whole blood
30
7.6
50
Whole blood
+
-
20
75
3.4
--
+
Martin el al. (this paper)
30
--
12.2
0.05 M Trts 0.001 M ATP Whole blood 0.2 M THs
+
--
Martin et al. (this paper)
+
+
Bonaventura et al. (1974a)
--
Bonaventura et al. (1974b) Dill et al. (1932)
+
Dill et al. (1932)
t McCulcheon refers to this ,~pccics as Pterol~lttlt,a micrltra. McCutchcon refers to this species as R. quadriloha DISCUSSION Potmnotrygon sp. live in waters which are relatively hypoxic in comparison to the habitats of marine rays and skates. Although the hematocrits of the freshwater rays do not differ significantly from that reported in a marine species, Raja oscilhlta (Dill et al., 1932), the oxygen affinity of their hemoglobin is substantially higher than those reported for various species of marine rajiformes (Table 21. Admittedly, the experimental conditions reported in Table 2 differ from one study to another and these differences may accentuate minor differences in hemoglobin oxygen affinity. Nonetheless, the magnitude of the difference between the P~ of Potantotrvgon hemoglobin and those reported for the hemoglobins of other species are apparent. Thus Potamotry.qon hemoglobin may have evolved a higher oxygen affimty in response to hypoxic stress, The data from this study indicate that a considerable difference exists in the oxygen affinity measured in whole blood and the hemoglobin oxygen affinity measured in the presence of 1 mM ATP. This discrepancy may have several sources. Whole blood affinities were measured at 30 C rather than at 20 C as in the case of hemolysates: increases in temperature generally reduce hemoglobin oxygen affinity. Also the intracellular pH of the whole blood was not determined and may have been below pH 7. Inspection of Fig. 4 shows that stripped hemolysates + I mM ATP
do approach the lower affinity state seen in the whole blood when experiments are performed in the lower pH range. Also, potent modulators such as guanosine triphosphate or inositol pentaphosphate, whose effects on the hemolysates were not examined, may influence hemoglobin oxygen affinity within the ceil. The seemingly linear response of log p~ to pH is an interesting feature of Potamotrygon hemoglobin. At least two interpretations of this phenomenon are possible. The molecule may possess many Bohr groups, each of small effect. Alternatively, and more likely, the hemolysate may consist of a mixture of hemoglobins with different oxygen affinities and differential responses to pH (Antonini & Brunori, 1971). This view is supported by the multiphasic binding and dissociation rate of ligands noted in kinetic experiments. Potmnotry.qon hemoglobin is insensitive to changes in salt concentration, a behavior quite different from that evinced by hemoglobins of teleosts and mammals, whose affinities decrease with increasing salt concentration, and of those observed in the hemoglobins of many marine Rajlformes, where the oxygen affinities increase with an increase in salt concentration. It is of interest that Mumm et al. (1978) have recently reported that the oxygen affinity of hemoglobin isolated from the sting ray, Dasyatis sabinu is also insensitive to NaCI. Bonaventura et al. (1974b) suggests that dissociation of tetramers to more reactive dimers may be responsible for the increased
Structural and functional studies of hemoglobins affinity of ray and skate hemoglobin at high salt concentrations. They infer from this phenomenon that electrostatic interactions may be responsible for the great stability of these hemoglobins. If so, other mechanisms must be suggested to account for the extraordinary stability observed of Potomatryyon hemoglobin. Potamotryyon hemoglobin also differs from most elasmobranch hemoglobins in its inability to polymerize. Probably sulfhydryl residues are not available at the surface of these proteins for intermolecular disulfide bridge formation. Potamotryyon hemoglobin resembles the hemoglobins of other elasmobranchs and of teleosts In its kinetic behavior in that the organic phosphate effect is manifested in its influence on the O2,,,, constant. Also, this hemoglobin possesses a Bohr effect as do most fish hemoglobins. However, the relative contribution of the CO,,,, constant to the pH effect seems greater than that characteristic of other fish species. The p.~ of stripped Potamotryyon hemoglobin decreases approx 33-fold between pH 5.95 and 9.0. Over the pH range 6.3-8.8 the CO .... constant increases 4.4-fold and the O2,,,, constant decreases 4.2-fold. Both the direction and magnitude of the changes in the on and off constants are in qualitative agreement with the equilibrium results. Subunit dissociation, as measured by the increase in proportion of fast reacting material after flash photolysis, appears negligible in urea concentrations up to 5 M. These results are further confirmed by the outcome of oxygen equilibrium and oxygen dissociation experiments performed m urea. Thus, while Potamotrygon species do not maintain high blood urea concentrations their hemoglobins nevertheless retain a remarkable stability in urea. Further proof of this stability comes from the urea denaturation studies. Although the urea concentrations in these experiments were far in excess of the concentrations found in marine elasmobranchs, a definite trend is discernible. It appears that the teleost hemoglobins were more easily denatured that the elasmobranch hemoglobins. What physiological advantage is to be derived from this added stability is obscure. It seems that rather than having lost the extra stability generally exhibited by elasmobranch hemoglobins, that of Potamotrygon has maintained whatever stability its marine phylogenetic ancestors may have possessed.
Acl, nowledyements--We would like to thank Dr Celia Bonaventura for her assistance in performing the kinetic experiments, Dr Emilia Pandolfelh for her advice and suggestions, the crew of the Alpha Helix, and the Brazilian government for the permission to work in Amazon waters, Hans J. Fyhn and Unni E. H. Fyhn acknowledge support from the Norwegian Research Council for Science and the Humanities. This work was supported m part by the National Science Foundation under Grant PCM 75-06451 to the Scripps Institute of Oceanography for support of the Alpha Helix Program and by National Institutes of Health Grant HL15460. Joseph Martin was supported by National Institutes of Health Grant GM07184 to Dt'kc University. REFERENCES ANTONINI E. & BRUNORI M. (1971) Hemot.llobin and Myot.llobin ul Their Reactton.~ wath Li.qand.s. 436 pp. NorthHolland, Amsterdam.
137
BONAVENTURAJ., BONAVENTURAC. & SULLIVANB. (1974a) Hemoglobin of the electric Atlantic torpedo, Torpedo nohdiana: a cooperative hemoglobin without Bohr effects. Biochim. hlophy.s. A cta 37 I, 147-154. BONAVENTURAJ., BONAVEN1URAC. & SULt.lVANB. (1974b) Urea tolerance as a molecular adaptation of elasmobranch hemoglobins. Science 186, 57-59. BONAVENTURAC., SULLIVANB. & BONAVENTURAJ. (1974c) The effects of pH and anions on functional properties of hemoglobin from Lemur fidrus fidt'us. J. biol. Chem. 249, 3768-3775. BRtJNORI M. FALCIONI G.. FORTtrNI G. & GIAROINA B. (1975) Effects of amons on the oxygen binding properties of the hemoglobin components from trout (Sahno irtdeltx}. Archs. Btochem. Biophys. 168, 512 519 DAVIS B. J. (1964) D,sc electrophoresis II. method and application to human serum proteins, Ann N.Y. Aead. Sci. 121,404-427. DILL D. B., EDWARDS H. T. & FLORgIN M. A. (1932) Properties of the blood of skate (Rt(/a o.setllata). Biol. Bldl. 62, 23-36. ED~LSTE,N S. J., McEwEN B. & GmSON Q. H. (19761 Subumt dissociation of fish hemoglobins. J. biol. Chem. 251, 7632-7637. FARMER M,, FYHN H.J., FVHN U. E. H. & NOBLr: R. (19791 Occurrence of Root effect hemoglobins in Amazonian fishes. Comp. Bioehenl. Ph)'xiol. 62, 115 124. FVHN U. E. H. & SULLIVANB. (1975) Elasmobranch hemoglobins dimerJzation and polymerization in various species. Comp. Bioehem. Physiol. 50B, 119-129. FYHN U. E. H., FYHN H. J.. DAVIS B. J., PDWERS D. A., FINK W. L. & GARLtCK R. L (1979) Hemoglobin heterogeneity in Amazon,an fishes. Comp. Btochem. Phl'.~iol. 62, 39 66. GOLDSTr~IN L. & FOSTER R. P. (1971) Urea biosynthesis and excretion in freshwater and marine elasmobrancbs Comp. Biochem. PhyMol, 39B, 415 421. GRIFFITH R W.. PANG P.. SRIVASTAVAA, 6~ PICKFORDG. (19731 Serum composition of fresh water stmgrays (Potamotrygonidae) adapted to fresh and dilute seawater. Biol. Bull 144, 304-320. HI-:RSKOVITST. T., JAILLET H. & GADEGBKU B. (1970) On the structural stability and solvent denaturation of proteins. I1. Denaturation by ureas. J. biol. Chem. 245, 4544-4560. HUC;HESG. M. & WOODS. C. (1974) Respiratory properties of the blood of the thornback ray (Rata clarata). Experientia 30/2, 167-168. McCuTcHEON F. (1947) Specific oxygen affinity of hemoglobin m elasmobranchs and turtles. J. Cell. Comp. Phy.siol. 29, 333-338. MANWELL C. (1958) Ontogeny of hemoglobin in the skate Rata hinoculata. Science 128, 419 420. MANWELt. C. (19631 Fetal and adult hemoglobins of the spray dogfish Sq,ahs s,ckle)'l. ,4rch.s Btochem. Biophys. 101, 504-51 I. MARtIN J. P., BONAVENTURA J., BRUNORI M., F'YHN H. J.. FVHN U. E. H., GAgLICK R. L, POWERS D. A. & WILSON M. T. (1979) The isolation and characterization of the hemoglobin components of Mylos.soma sp., an Amazonian teleost. Comp. Biochem. Physiol. (this issue). MOMM D. P., ATHA D. H. & RIGC3S A. (1978) The hemoglobin of the common sting ray, DtLsyuti.s .sabma: structural and functional properties. Comp. Biochem. Physiol. (in press). NELSON J. S. (1976) Fi.she.s of the |,Vorhl. 415 pp. John Wdey, New York. ORNST,':tN L. (1964) Disc electrophores~s--l. Background and theory. Ann. N.Y. Acad. Scl. 121, 321 349. POWERS D. A.. FVHN H J.. FVHN U. E. H.. MARTIN J. P., GARL,CK R. L & WOOD S. C. (1979) A comparative study of the oxygen equilibria of blood from 40 genera of Amazonian fishes Comp. Bioehem. PIn'siol. (this issue).
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JDSL:PII P. MARTIN el al.
RIGGS A (1970) Properties of fish hemoglobins. In Fish Phy.~iohu.ly, Vol. 4 (Edited by HOAR W. S. & RANDALL D. J.), pp. 209 251. Academic Press, New York RIGGS A. & WOLBACH R. A. (1956) Sulfhydryl groups and the structure of hemoglobin. J qen. Ph)'siol 39, 585-605. SMITH H. W. (1931) The absorption and excretion of water and salts by the elasmobranch fishes I. Freshwater elasmobranchs. Am J. Physiol. 98, 279-295. SULLIVAN B, (1974a) Amphibian hemoglobins. In Chemical Zoolo.qy, Vol 9 (Edfled by FLORKIN M. & SCHF.ER B. T ), pp. 77 118. Academic Press, New York.
SULLIVAN B. (1974b) Reptilian hemoglobins. In Chemical Zoolo~jy, Vol. 9 (Edited by FLORKIN M. & SCHEER B. T), pp. 353-374. Academic Press, New York. THORSON T. V., COWAN C. M. & WATSON D. E. (1967) Potamotry#on sp.: elasmobranchs with low urea content. Science 158, 375-377. WEBER R E., JOHANSEN K., LYKKEBOE G & MALOIY G. O. (1977) The oxygen binding properties of hemoglobins from aestivating and active African lungfish. J. exp. Zool. 199, 85 99.