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The root effect hemoglobin of the jaraqui´, a teleost fish,Prochilodus sp.
( tmll~ IJlollt'm I)ht~t,,I I ,d 62 ~ pp 19"~ to 21)() cO I','J,I,mUm I'~, ,, 1.1,1 1979 I'lt.t,,d .r (,~,,,/t 13tit,..
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THE ROOT EFFECT HEMOGLOBIN OF THE JARAQUI, A TELEOST FISH, P R O C H I L O D U S SP.* JOSEPH P. MARTIN,~'I" JOSEPH BONAVENTURA, 2 MAURIZIO BRUNORI, 3 ROBERT L. GARLICK ¢ and DENNIS A. POWERSs )Department of Zoology, Duke UmversJty Marine Laboratory, Beaufort, NC 28516, U.S.A.: "Department of Biochemistry, Duke Umversity Medical Center and Duke University Marine Laboratory, Beaufort, NC 28516, U.S.A.; ~CNR Center for Molecular Bmlogy. Institute of Chemistry and Biochemistry, Faculty of Medicine, University of Rome, Rome, Italy: 4Department of Zoology, Umverslty of Texas at Austin, Austin, TX 78712, U.S.A.: ~Department of Biology, Johns Hopkins Umverslty. Balnmorc. MD 21218. U.S.A Abstrac!
I The hemoglobin system of the jaraqui, Prochtlodu.s sp., consists of multiple hemoglobin
components.
2. The oxidized denvauve elutes as a single molecular wetght spec,es m gel filtratmn experiments. The carbox.v denvauve has an apparent molecular weight of 60,000 as determined by gel filtratmn. 3 A 44-fold change m p, occurs between pH 6.4 and 8.6 in stripped hemoglobm solutions. This change increases to 388-fold over the same pH range m the presence of I m M ATP. 4. Cooperauwty in oxygen binding, as measured by , m the Hill equation, is greater than one tit pH ~,alues abovc 6.7 but less than one below this value.
5. The hemolysate displays a Root effect, being only 44°, saturated tit pH 6 4 in the presence of I mM ATP and eqtuhbrated with air at I atm. 6 At 30 C and pH 7.6 the whole blood possesses a low p,, 4.7 mm Hg, lower than that of many other Amazonian teleosts. 7. Both the carbon monoxide combination rate and the oxygen d~ssoctation rate are pH and ATP dependent. Between pH 6.2 and 8 8 the CO,,,, rate increases 10-fold. ATP reduces the rate at intermediate pH values. The O,.,. rate increases 2.4-fold between pH 8.8 and 6.7. Add)Iron of I mM ATP causes a 4.5-fold increase over the same pH range. 8. The low , values below pH 6.7 and the heterogeneity of the CO combmat|on and O , dissociation processes suggests that the hemoglobin components may be functionally dtfferentmted and/or intramolecular dilTerences exist m the kinetic properties of ~- and fl-hke chains. INTRODUCTION T h e family P r o c h i l o d o n t i d a e is a g r o u p of characold fish widely distributed m rivers t h r o u g h o u t tropical South America. Species of this widespread g r o u p conduct yearly s p a w n i n g mlgrattons often over distances exceeding 700 k and travel m large schools (i.e. greater than I x 105 individuals). They are mud eaters, digesting the bacteria and o t h e r small o r g a n i s m s living in b o t t o m detritus, and an ~mportant food item, being one of the fish species most c o m m o n l y cons u m e d by the riparmn peoples of m a n y South American countries. A l t h o u g h Prochflodus spp. regularly frequent fast-moving river habitats, they also inhabit the lower reaches of rivers, riverside pools, channels, small depressions and o x b o w lakes, often under dense beds of floating m a c r o p h y t e s (i.e. floating m e a d o w s ) ( L o w e - M c C o n n e l l , [975) O u r studies on Prochflodus sp. h e m o g l o b i n were c a m e d out for two reasons: first, Prochilodus sp. are a cultivated food source: consequently, they are readily obtained. Second, these species inhabit a * This work will also appear as a Portuguese translation i n .'~CI~I
4III~IZOIIIC~I
"I"To whom reprint requests should be addressed ++Abbrevmtions used m this study arc the following: B~sTrJs, (2hydroxyethyl)mfino trts (hydroxymethylJmethane: Tns. tris(hydroxynlethyljammomethane: ATP. adettosinc tnphosphate: EDTA. ethylenediammctetraacet~c acid: I', second order carbon monoxide combination velocity constant; A, tirsl order oxygen dissocmtton constant: p,., the oxygen partial pressure at which half the awfilable heme sites tire occupied 195
remarkable range of aquatic environments. Thus we h o p e d that their h e m o g l o b i n s would exhibit molecular properties characteristic of fast water species (e.g. Bohr effect, Root effect) such as trout (Salmo irideus, Brunori, 1975) and salmon (O,corhy,chus keta, Hashimoto et aL, 1960) and yet display properties (e.g. high oxygen affinity) typical of the h e m o g l o b i n of species such as carp Cyprimts carpio (Krogh & Leitch, 1919; Noble et al., 1970), the bowfin, Amia cah'a (Weber et al., 1976) and Hoplias malabaricus (Powers et al., 1979), fish that periodically experience hypoxic conditions. MATERIALS
AND
METHODS
Isolation Specimens of Prochilodu~ sp. were collected m November and December, 1976 during an expedition on lhe R.V. 41pEa Heh\ on the Amazon Rwer about 50km upstream on the Rio Solim6es from the junction with the Rio Negro The fish were captured with gdl nets and transported to the Alpha Heh\ for study. Blood was obtained by cardmc puncture and drawn into cold heparinized glass syringes [1001d of sodium heparin (5000 i.u./ml)in 1.7",. NaCI/5 ml of blood]. The red blood cells were washed 3 tmtes m 10 ,,olumes of cold I mM Tris, pH 8.0,:~ 1.7", NaCI and lysed in 3 volumes of I mM Tris, pH 8.0 for I hr at 0 C. One tenth volume of I M NaCI was added to the hemolysate and the mixture was centrifuged at 28.000 y for 15 mm to remove red cell ghosts arid cellular debris The supernatant was then stripped of salts and organic phosphates by passage through a 2.5 x 50cm column of Scphadex G-25 followed by treatment on a detomzing cohntm with the following resins from top to
JOSFPII P MARTIN el al.
196
botlorn 2 cm Dowex-50(w) a m m o r n u m form. 2 c m Dowex-I acetate form, 20cm BioRad AG 501-X8 (D) mixed bed resin. The purified hemoglobin was stored at 5 C unhl reqtnred. A hemolysate sample was frozen at - 7 0 C and transported on dry ice to Beaufort, NC, and stored at - 2 0 C for 2 months. Molecular wmght determmattons by gel filtrahon of a carboxy derivattve, and all rapid kinetic studies were perforrned on this sample The thawed sample was reduced by treatment with a small amount of dnhaomte, after which the hemoglobin was purified b.,, passage through a I x 25cm Sephadex G-25 column eqmhbrated with I m M Trls, pH 8.0 and used immedmtel,, m the 02 dlssocmtJon and CO combinatzon experiments
Electt'opltoresls Vertical polyacrylamtde gel electrophorests (pH 8.9. 7 5". gels) was done at room remperature according to Davts (I 964) and Ornstem (1964). Hemoglobin samples ( I mg/ml) m upper buffer containing 0. l M fl-mercaptoethanol and a small amount of dlthlonite were bubbled with cltrbon monoxide and apphed to the gels. Bovine serum albumin ,,,,its used as a mobdlty standard The gels were stained for 3 hr nl 0 25". Coomasste Brilhant Blue R m acettc acid nlethanol water solution ( 1 : 2 : 4 ) a n d destamed by diffusion ~lole~ ltlar weLqht ~ludle.s Gel liltrat~on e \ p e n m e n t s wnh oxidized Proelnlodus hemolysates \vere carried out on a Sepharose 4B c o h m m (2 × 90cm) m 0.05 M TNs. pH 7 5, I m M EDTA according Io F.,,hn & Sulh,.art (1975). In addition, the molecular weight of Prochdodu.s carboxy hemoglobin was determined according to Martin el al. (1979).
Root effect studie~ The hemolysate was examined for the presence of a Root effect The absorbances of mr equdibrated buffered hemoglobin solutions + I mM ATP were monitored its a runetton of pH Absorbance readings lit two wavelengths (560 and 576 ran) were converted to fracttonal saturatton by the following e q u a h o n . -1,~, - A,. .....
= fract;onal saturation.
4 .... is the absorbance of the oxygenated solution at pH 8. ,-I,,t...... ~ is the absorb:moo of the oxygenated solution at the pH under study, ,4, ..... is the absorbance of the dnh~omtc treated solution ilt pH 8, The data were plotted its fi'acttonal saturahon vs pH.
O\vqen eqmhhrnmt expertnwnls Oxygen eqmhbrm of Proehdodu,s whole blood were measured at 30 C as described by Powers et al. (1979) using a Hem-O-Scan oxygen dissocmtlon analyzer (American Instruments Corporation). Oxygen equihbrm of pur~fled hemoglobins were carried out at 20 C as described b.,, R~ggs & Wolbach (1956). The hernoglobin solutions (6(.) ItM m heme)were brought to 0 05 iomc strength in Tris or Bis Tr~s buffers, p, values v, ere estnnated under various pH conditions. These expcrnnents were performed m the presence and absence of I m M ATP Rapul kllletl(' e\po'iments Oxygen dtssoc~atlon rates of the laemoglobm m buffered soltmons containing d~thtonite were determined in a Gtbson Durrum stopped flow spectrophotometer equipped wtth a pneumatic drwe and a 2 crn observauon chamber by a prewously descrtbed method (Bonaventura et a/., 1974). Atr eqtuhbrated degassed hemoglobin soluttons (7 ItM heme) were rapidly mixed w~th buffer solutions contaming excess dlthtomte and tile absorbance change of the
mixed sohltton was followed at 437.5 nrn Dissocmtton rates were measured its a function of pH v,,ith and without I mM ATP. The Jomc strength of the buffer after mixing was 0.05 I, and the concentration of ATP was I m M Carbon monoxide combnlatlorl rates of Prochdodus hemoglobn3s were examined b~ stopped-Ilow spectrophoIometry according to Bonaventura el al (1974) and the influence of pH :rod orgamc phosphates on the rates were ascertained at 437 5 nm. The heme concentration after mixmg was 3 51tM All experiments were performed at 20 C m TNs or Bis TrJs buffers The ionic strength of the buffer after mixing was 0 0 5 I . Analysis of all the kinetic data wits facilitated by use of a P D P I I,E Computer (Digital Equipment Corporation) and a data acqtnsil~on and storage device (DASAR, American Instruments Corporahon).
R ES U LTS
Characterization o l hemolysate,~ Prochdodus h e m o g l o b i n s exh~bn s t r u c t u r a l features similar to t h o s e of o t h e r teleost h e m o g l o b i n s . T h e h e m o l y s a t c s c o n t a i n e d two h e m o g l o b i n s , p h e n o t y p e II a c c o r d i n g to F y h n et al. (1979): o t h e r Prochilodus p h e n o t y p e s consist of up to three c o m p o n e n t s ( F y h n et al., 1979). T h e hemol.~sate did not p o l y m e r i z e after o x i d a t i o n by p o t a s s m m ferricyanide a l t h o u g h polymerization o f Hoplias malaharicus hemoglobin ( a n o t h e r A m a z o n teleost) h a s been reported by Reischl (1976). T h e c a r b o x y d e r w a t i v e h a s an a p p a r ent m o l e c u l a r weight of 60,000. Ligaml hireling data Prochilodus h e m o l y s a t e evinces a R o o t effect (Fig. I) w h i c h is e n h a n c e d in the presence of I m M A T P . At p H 7.0 the h e m o g l o b i n s a t u r a t i o n level is lowered to 87"o at a t m o s p h e r i c o x y g e n tension. Below p H 7.0 the s a t u r a h o n level d r o p s further, r e a c h i n g a m i n i m u m of 4 4 " . at p H 6 4 . O x y g e n equilibria of w h o l e blood (Fig. 2) s u p p o r t the f o r e g o i n g Root effect d a t a in that in b l o o d equilib r a t e d with 5.6",, CO., the 100",. s a t u r a h o n is n o t a t t a i n e d at a t m o s p h e r i c o,~ygen p a r t m l pressure. A large B o h r effect is evident in e q u i l i b r i u m expertm e n t s with blood (Fig. 2) a n d stripped h e m o l y s a t e s (Fig. 3). At p H 7.6 a n d 30 C the e r y t h r o c y t e p, is
I
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pH Fig. I. Tile dependence of the fractional saturation+ Y, of air equdibrated Ilemoglobnl solutions as a funehon of pH. The experm~ent was performed m T n s and Bis-Tns buffers 005 / + I m M ATP.
Tile root effect hemoglobin of tile jaraqui I0
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Fig 2 Tile fractional saturatton. Y. of Prochflodtts whole blood vs tile log of the oxygen partial pressure at various pH values and 30 C. Curve I ( -) the mean curve of the oxygen equihbria of the bloods of four mdwlduals at pH 76 with no CO2 Curve 2 ( --)--the mean curve of the ox)gcn cquihbrvl of the bloods of two indwlduals at pH 70 m the presence of 5.6% CO, Curve 3 ( - ) the mean curve of the oxygen equihbria of tile blood of two mdtviduals at pH 6.7 in the presence of 5.6". CO, 4.7 mm Hg, substantmlly lower than those reported for other Amazonian species known to possess Root effect hemoglobins (t,e. Osteoglo.ssum hwirrhosum, Hoplostermm~ httorale and M.vlo.s.soma sp., Powers et aL, 1979). The/)~ of stripped hemolysate at 20 C, pH 7.6 is 0.63 mm Hg wdhout and 0 . 7 6 m m Hg with I mm A T P present m soltmon. The p, values increase as pH is lowered and the inhlbnory effects of ATP increase, until at pH 6.4 they become 16.22 and 182ram Hg, respcctwely. Thus the p~ changes approximately 44-fold in stripped and 388-fold in stripped hemoglobin + I m M ATP between 6.4 and 8.6. At pH 5.9 the stripped hemoglobin soltmon attains a p! of 34.7 mm Hg, about a 94-fold change over the p~ at pH 86. The Bohr effect, measured between pH 7.0 and 7.5, was Alog F:/ApH = - 0 . 4 6 for stripped hemoglobin solution and Alog
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p d A p H = - 0 . 6 0 for stripped hemoglobin + I m M ATP. At pH 7.0, A T P addition causes a 4.8-fold increase in p.: at pH 6.4 it causes an 15.7-fold increase m p.. Reference to F~gs 2 and 3 indicates that oxygen binding is cooperative (n = 1.3 at pH 7.0 in stripped hemoglobin solution). However, at pH values below 6.7 n decreases to less than one, suggesting either intra- or mtermolecular heterogeneity of oxygen binding (Antoninl & Brunori, 1971). The addition of I m M ATP does not seem to affect the n value significantly at any pH.
Rapid l, inettc resuhs Figure 4 represents the fraction of the maximal absorbance change obtained at pH 8.8, observed at zero time of the oxygen dissociation process, attained at various pH values. These data are the results of experiments performed in the presence of I mM ATP.
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Fig. 3 Tile dependence of p, and II of Prochilodu.s hen]oglobms on pH Stripped (©) and stripped + I mM ATP (0) henlolvsatcs Experimental condmons arc as described
In le ".,I
Fig. 4 A plot of A.4/A 4 ...... at 0 time for the oxygen dissoclation reachon of Proclulodus hemoglobins in the presence of dlthlonitc and I m M A T P at various pH values. The
heine concentration after mixing was 3.51tM. A.4 =s the absorbance change noted at the pH trader study. AA...... is tile absorbanee change measured at pH 8.8. Conditions arc as described m text
198
JOShPH P. MARTIN
As the pH is reduced the rate increases and a progressively greater percentage of the dissociation process occurs within the dead time of the apparatus, 2.3 msec. Thus the percentage of the total reaction which is observed decreases as the pH is lowered. The incrcasc in the 02 .... rate suggested in Fig. 4 parallels the reduction in per cent saturation of hemoglobin solutions at low pH recorded in Fig. I. Indeed, a large lncrcase in the O,,., rate at low pH is characteristic of Root effect hemoglobins. The hemoglobin of spot (Leio.stomus ramhuru.q exceeds 300/see below pH 6.0 as measured by stopped flow spectrophotomerry (Bonaventura et al, 1976), and that of the Root effect hemoglobin of trout (Salmo irideu.s), determined by temperature jump studies, attains a rate of l l00/sec at pH 7 0 (Glardina et aL, 1963). Of course, the reduction in the rnaximal absorbance difference at low pH may also arise as a conseqtlence of a pH influence on the O _ , rate which was not determined in this study. The oxygen dissociation data arc snmmarized in Fig. 5. The 02 .... rate was heterogeneous at all pH values tested. The plotted rates represent those obtained during the second quarter of the reaction as the initial rate could not be ewduated at low pH values. These dissociation rates are both pH and ATP sensitive. Between pH 8.8 and 6.2 the 02 .... rate increases approx 5.5-fold in stripped solutions, while in the presence of I mM ATP the change is 4.5-fold between pH 8.8 and 6.7. At pH 7.4 addition of ATP causes a 2.2-fold increase in the dissociation rate. Data for the CO combination reaction are plotted as a function of pH and ATP concentration in Fig. 6. The combination process was multiphasic at all pH values studied. The difference between the initial and final rates increased with decreasing pH The plotted rates represent the initial rates of repletion. The second order rate constant for CO binding is pH dependent,
e l tt].
9.0
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'0
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Fig. 6. The second order combination veloaty constant of carbon monoxide binding to Prochtlodu,s hemoglobin (l') at various pH values. Conditions are as described in text. The concentration of berne after mixing was 3.5 HM Stripped hemolysate (O), stripped hemolysate + ImM ATP (Q) changing 10-fold between pH 6.2 and 8.8 in stripped hemoglobin solutions. At low pH, 6.2, I' equals about 5.7 × 104/M per sec as compared to 20 x 104/M per sec at pH 6.5 and 5 x 104/M per sec at pH 6.2 obtained for pure hemoglobin solutions of carp (Noble et al., 19701 and spot fish (Bonaventura et al., 1976), respectively. The maximum CO .... rate, obtained at pH 8.8, is 6.4 × 10~/M per sec. I mM ATP exerts an effect on the CO,,.. rate at intermediate pH values, reducing the combination rate by 41",, at pH 7.4. DISCUSSION
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pH Fig 5 The first order dissoemtion constant (k) for oxygen vs pH. The plotted rates represent the second quarter of the reacuon. The fieme concentration after mixing was 3 5 ItM. Condmons are as described m text (O) stripped Prochflodus hernoglobins, (O) stripped fiemoglobms + I mM ATP.
The genus Prochilodus consists of open river, mudeating specms (Fink & Fink, 1979). These fish spend much of their time foraging on river bottoms. In other eplbenthic Amazon species (e.g. members of various catfish famihes), which require a slight negative buoyancy to remain stationary on the river bed, the swim bladder has secondarily evolved into a sound sensing and emitting organ and is no longer employed as a hydrostatic device (Alexander, 1967). Although Prochilodus possesses a functional swim bladder and hemoglobins that display considerable Bohr and Root effects, it lacks the oxygen-secreting apparatus of the swim bladder, the fete mirabile (Bridge & Haddon, 1893). However, as pointed out by Farmer et aL (1979), it apparently does have the related apparatus of the eye, the choroid fete, and this is often associated with the occurrence of a Root effect. The large Bohr and Root effects probably reflect the diverse habitat distribution of the species. ProchiIodus sp. whole blood displays a high oxygen affinity at pH 7.6 and 3 0 C (/3, = 4 . T r a m Hg), a higher affinity than that reported for 44 out of 47 Amazonian fish species by Powers et aL (1979), especially higher than those of species whose hemo-
199
The root effect hemoglobin of the jaraqui globins display Root effects (i.e. M3'h),~,~oma sp., Martin et al., 1979; Hoplosternum littorale, Garlick et al., 19791. In fact, the affinity is comparable to those attained by certain aestivating fish species (i.e. Amia cah,a, Weber et al., 1976; Protopterus amtectans, Weber et al., 1977; LepMosiren paradoxa, Powers et td., 19791 which survive for long periods of time buried in mud. The high erythrocyte oxygen affimty may offer some physiological advantage in the hypoxic conditions found in various Prochilodus sp. habitats. The p~ of blood at pH 7.6 and 30~C is higher than that of the stripped hemoglobm solution + I m M ATP measured at 2 0 ' C under similar pH conditions. The difference undoubtedly arises in part because of the dtfferences in temperature and also because hemoglobin within fish erythrocytes may be modulated by c o m p o u n d s other than A T P (lsaacks et al., 19771. The heterogeneity of the kinetic data and the low n values, obtained in tonometric experiments below pH 6.7 indicate that the components of the ProchHodus hemoglobin system may be functionally differentiated. However, part of this heterogeneity may stem from a nonequivalence in ligand binding of ~t- and ,8-hke chains at low pH. lntramolecular functional heterogeneity is known in other fish hemoglobins having been reported for spot (Bonaventura et al., 1976), trout (Brunori, 1975), M y l o s s o m a (Martin et al., 19791 and carp (Tan et al., 19731. The results of oxygen dissociation and carbon monoxide combination experiments are in good agreement with those of oxygen equilibrium experiments in that changes induced in k and I" by pH and organic phosphates are paralleled by the influence of these modulators on the p., for oxygen. The Root effect of ProchHodus hemoglobins is chiefly mediated through the effects of pH on the Oz,,., constant, as is the case in spot, trout and carp hemoglobins, and variation in both the C O .... and 02,,, constants underlie the organic phosphate effect. In isoelectric focusing experiments of Bunn & Riggs (1979) all Prochilodus hemoglobin components demonstrated an alkaline Bohr effect. Thus if differences exist between the functions of the two components, they may be ones of quantity and not quality. That the components might be functionally differentiated is suggested by the complex ligand binding kinettcs earlier discussed. Perhaps the Prochilodus system represents an intermediate stage in the evolution of the functionally diverse hemoglobin systems typified by the trout. Further studies on the purified components of Prochilodus hemoglobin should reve,'d the degree of functional and structural differentiation attained by the two components. .4ekm~wledgement.s---Wc would hke to thank Ceha Bonaventura for her generous assistance m tile kinetic experiments. Maunzlo Brunorl expresses his thanks to the National Research Council (C.N R.) of Italy for financml support. Joseph Bonaventura is an Estabhshed Investigator of the American Heart Associatton and gratefully acknowledges grants from tile United States Nahonal Institute of Health and the National Science Foundation. We would hke to thank the officers and crew of tile Alpha Heh\. without whose unstmting assistance tll~s work would not have been possible, and tl~e Brazdian
government for permission to conduct research m their waters
This work was supported in part by the National Science Foundahon under grant PCM 75-06451 to the Scripps Institute of Oceanography for the support of the Alpha Helix program and by National Institutes of Health grant HL 15460. J. P. Martin is a predoctoral trainee supported by the National Institutes of Health grant GM07184 to Duke Untverslty. REFERENCES
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BONAVENTURA J.
(~¢
BRUNOR[ M. (1976) Spot hemoglobin Studies on the Root effect hemoglobin of a marine teleost d. hiol Chem. 251, 1871-1876. BRn)GI: J. W & HADOON G. C (18931 Contributions to the anatomy of fishes 11. The air bladder and Weberian ossicles in the sduroid fishes. Plnl. Trans. B, 184, 65-434. BRUNORI M. (19751 Molecular adaptation to physiological requ.rements: tile hemoglobin system of trout. In Current Topws in Cell Regulatiom Vol. 9 (Edited by HORECK~:R M. & STADTMAN E), pp. 1-39 Academic Press, New York. BUNN H. F. & RIGGS A (19791 The measurement of the Bohr effect of fish hemoglobins by gel moelectnc focusing. Comp. BuJchem. Physiol. 62A, 95 100. DAVIS B. J. (19641 Disc electrophoresis--ll. Method and application to human serum proteins Am~. N.Y Acad Sci. 121, 404-427 FARMER M., FVHN H. J., FVHN U. E H & NOI~LE R. W (19791 Occurrence of Root effect hemoglobms in Am~,zonian fishes. Comp. Biochem. Ph)'suH. 62A, II 5-124. FINK W. L. & FINK S U. (19791 Central Amazonia and its fishes. Comp. Btochem. Physml (this issue) F',HN H. J, FVHN U. E H., DAWS B. J, FINK W. L., GARUCK R. L. & POWERS D. A (19781 Hemoglobin heterogeneity of Amazonmn fishes. Comp. Bmchem Physiol. 62A, 39-66. FVHN U. E. H. & SULLIVAN B (1975) Elasmobranch hemoglobins: dmaerization and polymerizatton in various spectes. Comp. Btochem Phy.siol. 50B, 119-129 GARUCK R. L., BUrN H. F., FVHN H. J., FVHN U. E H., MAR'tIN J P., NOBLI=- R. W. & POWI-Rs D. A (19791 Functional studies on the separated hemoglobin components of an air-breathing catfish, Hoplostermu, littorale. Comp. Bioehem Physiol. (this assue). GIARDINA B., BRUNORI M., BJNOTTt J., GtOVENCO S. & AN'rONINI E. (19731 Studies on the properties or fish hemoglobins: kinetics of reaction with oxygen and carbon monoxide of the ~solated hemoglobin components from trout. Eur. J. BIochem. 39, 571-579. HASHIMOTO D., YAMAGUCHI Y. & MATSUURA U. (1960) Comparative studies on two hemoglobins of salmon--IV oxygen dissociation curves. Bull. Jap. Soe. sciem Fish. 26, 827 834. ISAACkS R. E., KIM H. D., BARTLI;rr G. R. & HARKNESS D. R (19771 Inositol pentaphosphate in erythrocytes of a freshwater fish, piraracu (Arapaima glgas). Lilt" Sci. 211, 987-990. Kaoca-t A. & LEITCH I. (19191 The respiratory function of blood m fishes, d. Phvstol., Loml. 52, 388-300 LOWI:-McCoNNFLL R. H. (1975) Ft.~h Communitws in Tropwal Fre.~h Water.s. Longman, London.
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JOSIPH P MARTIN et al
MARTIN j. P. BONAVI n rt'rA J, BRUNORI M.. F'~ ItN H. J.. F~HN U E H . GAru('K R. L. & P o w l r s D. A. (1978) IsolatIon and characterization of the hemoglobin components of Mylo~soma sp. Charactdae) Comp. Btochem. Phy.siol. (this issue) NOBLI= R W , PARKm~RST L. J. & GIBSON Q H. [1970) The effect of pH on the reactions of oxygen and carbon monoxide with the hemoglobin of the carp, Cl'prinu.s carpto. J. hiol Chem. 245, 6628 6633. ORNSTHN L (19641 D~sc electrophores~s I Background and theory. 4ms. N.E Acad. Sct. 121, 321-349. POWI-RS D. A., FVHN H. J. F~HN U. E. H, GARLICK R. L., MARTIN J. P. & WOODS S. (1979) A comparahve study of blood from forty-five speoes of A m a z o m a n fishes Comp. Btochem. Physiol. (this issue) RV.tSCl-tL E 11976)The hemoglobin of fresh water teleost
Hoplias malaharwus: heterogeneity and polymerization. Comp. B,whem. Ph)'.siol 55B, 255-257 RIGGS A & WOI_BAClt R A [1956) Sulfyhydryl groups and the structure of hemoglobin. J. yen. Ph)'mol 39, 585-605. TAn A. L., NOBLr R. W. & GmSON Q. H (1973) C o n d m o n s restricting allostenc transitions in carp hemoglobin. J. htol. Chem. 248, 2880 2888. WLBI:R R E., SULLWAN B., BONAVENVURA J. & BOYAW:NtURA C. (1976)The hemoglobm of the primitive fish .4mm cah'a: Isolation and functional characterization of the individual hemoglobin components Biochim. htophyx. 4eta 434, 18 31. WhBl:R R. E., JOHNASEN K., L'¢KKI_BOE O. & MALOIY G O (19771 The oxygen binding properties of hemoglobins from aestivating and actwe African lungfish. J. exp. Zool. 199. 85-99.
(13(10-9629 79 0101-1)I955'0200 0
THE ROOT EFFECT HEMOGLOBIN OF THE JARAQUI, A TELEOST FISH, P R O C H I L O D U S SP.* JOSEPH P. MARTIN,~'I" JOSEPH BONAVENTURA, 2 MAURIZIO BRUNORI, 3 ROBERT L. GARLICK ¢ and DENNIS A. POWERSs )Department of Zoology, Duke UmversJty Marine Laboratory, Beaufort, NC 28516, U.S.A.: "Department of Biochemistry, Duke Umversity Medical Center and Duke University Marine Laboratory, Beaufort, NC 28516, U.S.A.; ~CNR Center for Molecular Bmlogy. Institute of Chemistry and Biochemistry, Faculty of Medicine, University of Rome, Rome, Italy: 4Department of Zoology, Umverslty of Texas at Austin, Austin, TX 78712, U.S.A.: ~Department of Biology, Johns Hopkins Umverslty. Balnmorc. MD 21218. U.S.A Abstrac!
I The hemoglobin system of the jaraqui, Prochtlodu.s sp., consists of multiple hemoglobin
components.
2. The oxidized denvauve elutes as a single molecular wetght spec,es m gel filtratmn experiments. The carbox.v denvauve has an apparent molecular weight of 60,000 as determined by gel filtratmn. 3 A 44-fold change m p, occurs between pH 6.4 and 8.6 in stripped hemoglobm solutions. This change increases to 388-fold over the same pH range m the presence of I m M ATP. 4. Cooperauwty in oxygen binding, as measured by , m the Hill equation, is greater than one tit pH ~,alues abovc 6.7 but less than one below this value.
5. The hemolysate displays a Root effect, being only 44°, saturated tit pH 6 4 in the presence of I mM ATP and eqtuhbrated with air at I atm. 6 At 30 C and pH 7.6 the whole blood possesses a low p,, 4.7 mm Hg, lower than that of many other Amazonian teleosts. 7. Both the carbon monoxide combination rate and the oxygen d~ssoctation rate are pH and ATP dependent. Between pH 6.2 and 8 8 the CO,,,, rate increases 10-fold. ATP reduces the rate at intermediate pH values. The O,.,. rate increases 2.4-fold between pH 8.8 and 6.7. Add)Iron of I mM ATP causes a 4.5-fold increase over the same pH range. 8. The low , values below pH 6.7 and the heterogeneity of the CO combmat|on and O , dissociation processes suggests that the hemoglobin components may be functionally dtfferentmted and/or intramolecular dilTerences exist m the kinetic properties of ~- and fl-hke chains. INTRODUCTION T h e family P r o c h i l o d o n t i d a e is a g r o u p of characold fish widely distributed m rivers t h r o u g h o u t tropical South America. Species of this widespread g r o u p conduct yearly s p a w n i n g mlgrattons often over distances exceeding 700 k and travel m large schools (i.e. greater than I x 105 individuals). They are mud eaters, digesting the bacteria and o t h e r small o r g a n i s m s living in b o t t o m detritus, and an ~mportant food item, being one of the fish species most c o m m o n l y cons u m e d by the riparmn peoples of m a n y South American countries. A l t h o u g h Prochflodus spp. regularly frequent fast-moving river habitats, they also inhabit the lower reaches of rivers, riverside pools, channels, small depressions and o x b o w lakes, often under dense beds of floating m a c r o p h y t e s (i.e. floating m e a d o w s ) ( L o w e - M c C o n n e l l , [975) O u r studies on Prochflodus sp. h e m o g l o b i n were c a m e d out for two reasons: first, Prochilodus sp. are a cultivated food source: consequently, they are readily obtained. Second, these species inhabit a * This work will also appear as a Portuguese translation i n .'~CI~I
4III~IZOIIIC~I
"I"To whom reprint requests should be addressed ++Abbrevmtions used m this study arc the following: B~sTrJs, (2hydroxyethyl)mfino trts (hydroxymethylJmethane: Tns. tris(hydroxynlethyljammomethane: ATP. adettosinc tnphosphate: EDTA. ethylenediammctetraacet~c acid: I', second order carbon monoxide combination velocity constant; A, tirsl order oxygen dissocmtton constant: p,., the oxygen partial pressure at which half the awfilable heme sites tire occupied 195
remarkable range of aquatic environments. Thus we h o p e d that their h e m o g l o b i n s would exhibit molecular properties characteristic of fast water species (e.g. Bohr effect, Root effect) such as trout (Salmo irideus, Brunori, 1975) and salmon (O,corhy,chus keta, Hashimoto et aL, 1960) and yet display properties (e.g. high oxygen affinity) typical of the h e m o g l o b i n of species such as carp Cyprimts carpio (Krogh & Leitch, 1919; Noble et al., 1970), the bowfin, Amia cah'a (Weber et al., 1976) and Hoplias malabaricus (Powers et al., 1979), fish that periodically experience hypoxic conditions. MATERIALS
AND
METHODS
Isolation Specimens of Prochilodu~ sp. were collected m November and December, 1976 during an expedition on lhe R.V. 41pEa Heh\ on the Amazon Rwer about 50km upstream on the Rio Solim6es from the junction with the Rio Negro The fish were captured with gdl nets and transported to the Alpha Heh\ for study. Blood was obtained by cardmc puncture and drawn into cold heparinized glass syringes [1001d of sodium heparin (5000 i.u./ml)in 1.7",. NaCI/5 ml of blood]. The red blood cells were washed 3 tmtes m 10 ,,olumes of cold I mM Tris, pH 8.0,:~ 1.7", NaCI and lysed in 3 volumes of I mM Tris, pH 8.0 for I hr at 0 C. One tenth volume of I M NaCI was added to the hemolysate and the mixture was centrifuged at 28.000 y for 15 mm to remove red cell ghosts arid cellular debris The supernatant was then stripped of salts and organic phosphates by passage through a 2.5 x 50cm column of Scphadex G-25 followed by treatment on a detomzing cohntm with the following resins from top to
JOSFPII P MARTIN el al.
196
botlorn 2 cm Dowex-50(w) a m m o r n u m form. 2 c m Dowex-I acetate form, 20cm BioRad AG 501-X8 (D) mixed bed resin. The purified hemoglobin was stored at 5 C unhl reqtnred. A hemolysate sample was frozen at - 7 0 C and transported on dry ice to Beaufort, NC, and stored at - 2 0 C for 2 months. Molecular wmght determmattons by gel filtrahon of a carboxy derivattve, and all rapid kinetic studies were perforrned on this sample The thawed sample was reduced by treatment with a small amount of dnhaomte, after which the hemoglobin was purified b.,, passage through a I x 25cm Sephadex G-25 column eqmhbrated with I m M Trls, pH 8.0 and used immedmtel,, m the 02 dlssocmtJon and CO combinatzon experiments
Electt'opltoresls Vertical polyacrylamtde gel electrophorests (pH 8.9. 7 5". gels) was done at room remperature according to Davts (I 964) and Ornstem (1964). Hemoglobin samples ( I mg/ml) m upper buffer containing 0. l M fl-mercaptoethanol and a small amount of dlthlonite were bubbled with cltrbon monoxide and apphed to the gels. Bovine serum albumin ,,,,its used as a mobdlty standard The gels were stained for 3 hr nl 0 25". Coomasste Brilhant Blue R m acettc acid nlethanol water solution ( 1 : 2 : 4 ) a n d destamed by diffusion ~lole~ ltlar weLqht ~ludle.s Gel liltrat~on e \ p e n m e n t s wnh oxidized Proelnlodus hemolysates \vere carried out on a Sepharose 4B c o h m m (2 × 90cm) m 0.05 M TNs. pH 7 5, I m M EDTA according Io F.,,hn & Sulh,.art (1975). In addition, the molecular weight of Prochdodu.s carboxy hemoglobin was determined according to Martin el al. (1979).
Root effect studie~ The hemolysate was examined for the presence of a Root effect The absorbances of mr equdibrated buffered hemoglobin solutions + I mM ATP were monitored its a runetton of pH Absorbance readings lit two wavelengths (560 and 576 ran) were converted to fracttonal saturatton by the following e q u a h o n . -1,~, - A,. .....
= fract;onal saturation.
4 .... is the absorbance of the oxygenated solution at pH 8. ,-I,,t...... ~ is the absorb:moo of the oxygenated solution at the pH under study, ,4, ..... is the absorbance of the dnh~omtc treated solution ilt pH 8, The data were plotted its fi'acttonal saturahon vs pH.
O\vqen eqmhhrnmt expertnwnls Oxygen eqmhbrm of Proehdodu,s whole blood were measured at 30 C as described by Powers et al. (1979) using a Hem-O-Scan oxygen dissocmtlon analyzer (American Instruments Corporation). Oxygen equihbrm of pur~fled hemoglobins were carried out at 20 C as described b.,, R~ggs & Wolbach (1956). The hernoglobin solutions (6(.) ItM m heme)were brought to 0 05 iomc strength in Tris or Bis Tr~s buffers, p, values v, ere estnnated under various pH conditions. These expcrnnents were performed m the presence and absence of I m M ATP Rapul kllletl(' e\po'iments Oxygen dtssoc~atlon rates of the laemoglobm m buffered soltmons containing d~thtonite were determined in a Gtbson Durrum stopped flow spectrophotometer equipped wtth a pneumatic drwe and a 2 crn observauon chamber by a prewously descrtbed method (Bonaventura et a/., 1974). Atr eqtuhbrated degassed hemoglobin soluttons (7 ItM heme) were rapidly mixed w~th buffer solutions contaming excess dlthtomte and tile absorbance change of the
mixed sohltton was followed at 437.5 nrn Dissocmtton rates were measured its a function of pH v,,ith and without I mM ATP. The Jomc strength of the buffer after mixing was 0.05 I, and the concentration of ATP was I m M Carbon monoxide combnlatlorl rates of Prochdodus hemoglobn3s were examined b~ stopped-Ilow spectrophoIometry according to Bonaventura el al (1974) and the influence of pH :rod orgamc phosphates on the rates were ascertained at 437 5 nm. The heme concentration after mixmg was 3 51tM All experiments were performed at 20 C m TNs or Bis TrJs buffers The ionic strength of the buffer after mixing was 0 0 5 I . Analysis of all the kinetic data wits facilitated by use of a P D P I I,E Computer (Digital Equipment Corporation) and a data acqtnsil~on and storage device (DASAR, American Instruments Corporahon).
R ES U LTS
Characterization o l hemolysate,~ Prochdodus h e m o g l o b i n s exh~bn s t r u c t u r a l features similar to t h o s e of o t h e r teleost h e m o g l o b i n s . T h e h e m o l y s a t c s c o n t a i n e d two h e m o g l o b i n s , p h e n o t y p e II a c c o r d i n g to F y h n et al. (1979): o t h e r Prochilodus p h e n o t y p e s consist of up to three c o m p o n e n t s ( F y h n et al., 1979). T h e hemol.~sate did not p o l y m e r i z e after o x i d a t i o n by p o t a s s m m ferricyanide a l t h o u g h polymerization o f Hoplias malaharicus hemoglobin ( a n o t h e r A m a z o n teleost) h a s been reported by Reischl (1976). T h e c a r b o x y d e r w a t i v e h a s an a p p a r ent m o l e c u l a r weight of 60,000. Ligaml hireling data Prochilodus h e m o l y s a t e evinces a R o o t effect (Fig. I) w h i c h is e n h a n c e d in the presence of I m M A T P . At p H 7.0 the h e m o g l o b i n s a t u r a t i o n level is lowered to 87"o at a t m o s p h e r i c o x y g e n tension. Below p H 7.0 the s a t u r a h o n level d r o p s further, r e a c h i n g a m i n i m u m of 4 4 " . at p H 6 4 . O x y g e n equilibria of w h o l e blood (Fig. 2) s u p p o r t the f o r e g o i n g Root effect d a t a in that in b l o o d equilib r a t e d with 5.6",, CO., the 100",. s a t u r a h o n is n o t a t t a i n e d at a t m o s p h e r i c o,~ygen p a r t m l pressure. A large B o h r effect is evident in e q u i l i b r i u m expertm e n t s with blood (Fig. 2) a n d stripped h e m o l y s a t e s (Fig. 3). At p H 7.6 a n d 30 C the e r y t h r o c y t e p, is
I
I
I
,o
O8 O6
-7 04
0.2 0
i
i
70
80
!
90
pH Fig. I. Tile dependence of the fractional saturation+ Y, of air equdibrated Ilemoglobnl solutions as a funehon of pH. The experm~ent was performed m T n s and Bis-Tns buffers 005 / + I m M ATP.
Tile root effect hemoglobin of tile jaraqui I0
I
I
y 0,'5
197 . . . . . .
I
////////'//////1
////// J
~
/
/ , ~ / / / .
-/ "~" / ~
/
P 05
0
/ .~- / ~
I IO
LOG
I 15
20
P(Oz)
Fig 2 Tile fractional saturatton. Y. of Prochflodtts whole blood vs tile log of the oxygen partial pressure at various pH values and 30 C. Curve I ( -) the mean curve of the oxygen equihbria of the bloods of four mdwlduals at pH 76 with no CO2 Curve 2 ( --)--the mean curve of the ox)gcn cquihbrvl of the bloods of two indwlduals at pH 70 m the presence of 5.6% CO, Curve 3 ( - ) the mean curve of the oxygen equihbria of tile blood of two mdtviduals at pH 6.7 in the presence of 5.6". CO, 4.7 mm Hg, substantmlly lower than those reported for other Amazonian species known to possess Root effect hemoglobins (t,e. Osteoglo.ssum hwirrhosum, Hoplostermm~ httorale and M.vlo.s.soma sp., Powers et aL, 1979). The/)~ of stripped hemolysate at 20 C, pH 7.6 is 0.63 mm Hg wdhout and 0 . 7 6 m m Hg with I mm A T P present m soltmon. The p, values increase as pH is lowered and the inhlbnory effects of ATP increase, until at pH 6.4 they become 16.22 and 182ram Hg, respcctwely. Thus the p~ changes approximately 44-fold in stripped and 388-fold in stripped hemoglobin + I m M ATP between 6.4 and 8.6. At pH 5.9 the stripped hemoglobin soltmon attains a p! of 34.7 mm Hg, about a 94-fold change over the p~ at pH 86. The Bohr effect, measured between pH 7.0 and 7.5, was Alog F:/ApH = - 0 . 4 6 for stripped hemoglobin solution and Alog
E
2 I
25
0
0 0 ID
o
I
I
o •
oo
I
Q
p d A p H = - 0 . 6 0 for stripped hemoglobin + I m M ATP. At pH 7.0, A T P addition causes a 4.8-fold increase in p.: at pH 6.4 it causes an 15.7-fold increase m p.. Reference to F~gs 2 and 3 indicates that oxygen binding is cooperative (n = 1.3 at pH 7.0 in stripped hemoglobin solution). However, at pH values below 6.7 n decreases to less than one, suggesting either intra- or mtermolecular heterogeneity of oxygen binding (Antoninl & Brunori, 1971). The addition of I m M ATP does not seem to affect the n value significantly at any pH.
Rapid l, inettc resuhs Figure 4 represents the fraction of the maximal absorbance change obtained at pH 8.8, observed at zero time of the oxygen dissociation process, attained at various pH values. These data are the results of experiments performed in the presence of I mM ATP.
$
l
i
i
I00
2.0
075 AA AAmox
15
--It'll EL 1.0 E~n O
050
__.1 0,5 0
60
¢o
~Io
~;o
pH -0.5
d.o
¢o
I
8o
9'o
pH
Fig. 3 Tile dependence of p, and II of Prochilodu.s hen]oglobms on pH Stripped (©) and stripped + I mM ATP (0) henlolvsatcs Experimental condmons arc as described
In le ".,I
Fig. 4 A plot of A.4/A 4 ...... at 0 time for the oxygen dissoclation reachon of Proclulodus hemoglobins in the presence of dlthlonitc and I m M A T P at various pH values. The
heine concentration after mixing was 3.51tM. A.4 =s the absorbance change noted at the pH trader study. AA...... is tile absorbanee change measured at pH 8.8. Conditions arc as described m text
198
JOShPH P. MARTIN
As the pH is reduced the rate increases and a progressively greater percentage of the dissociation process occurs within the dead time of the apparatus, 2.3 msec. Thus the percentage of the total reaction which is observed decreases as the pH is lowered. The incrcasc in the 02 .... rate suggested in Fig. 4 parallels the reduction in per cent saturation of hemoglobin solutions at low pH recorded in Fig. I. Indeed, a large lncrcase in the O,,., rate at low pH is characteristic of Root effect hemoglobins. The hemoglobin of spot (Leio.stomus ramhuru.q exceeds 300/see below pH 6.0 as measured by stopped flow spectrophotomerry (Bonaventura et al, 1976), and that of the Root effect hemoglobin of trout (Salmo irideu.s), determined by temperature jump studies, attains a rate of l l00/sec at pH 7 0 (Glardina et aL, 1963). Of course, the reduction in the rnaximal absorbance difference at low pH may also arise as a conseqtlence of a pH influence on the O _ , rate which was not determined in this study. The oxygen dissociation data arc snmmarized in Fig. 5. The 02 .... rate was heterogeneous at all pH values tested. The plotted rates represent those obtained during the second quarter of the reaction as the initial rate could not be ewduated at low pH values. These dissociation rates are both pH and ATP sensitive. Between pH 8.8 and 6.2 the 02 .... rate increases approx 5.5-fold in stripped solutions, while in the presence of I mM ATP the change is 4.5-fold between pH 8.8 and 6.7. At pH 7.4 addition of ATP causes a 2.2-fold increase in the dissociation rate. Data for the CO combination reaction are plotted as a function of pH and ATP concentration in Fig. 6. The combination process was multiphasic at all pH values studied. The difference between the initial and final rates increased with decreasing pH The plotted rates represent the initial rates of repletion. The second order rate constant for CO binding is pH dependent,
e l tt].
9.0
,
/.-/ //
6.0 "~-0")
'0
3.O
/
0
i 6.0
r I
I
I
70
8.0
9.0
PH
Fig. 6. The second order combination veloaty constant of carbon monoxide binding to Prochtlodu,s hemoglobin (l') at various pH values. Conditions are as described in text. The concentration of berne after mixing was 3.5 HM Stripped hemolysate (O), stripped hemolysate + ImM ATP (Q) changing 10-fold between pH 6.2 and 8.8 in stripped hemoglobin solutions. At low pH, 6.2, I' equals about 5.7 × 104/M per sec as compared to 20 x 104/M per sec at pH 6.5 and 5 x 104/M per sec at pH 6.2 obtained for pure hemoglobin solutions of carp (Noble et al., 19701 and spot fish (Bonaventura et al., 1976), respectively. The maximum CO .... rate, obtained at pH 8.8, is 6.4 × 10~/M per sec. I mM ATP exerts an effect on the CO,,.. rate at intermediate pH values, reducing the combination rate by 41",, at pH 7.4. DISCUSSION
I
I
I
150
\ \ \ J
\
I00
\
50
6.0
I
I
ZO
8.0
I
9.0
pH Fig 5 The first order dissoemtion constant (k) for oxygen vs pH. The plotted rates represent the second quarter of the reacuon. The fieme concentration after mixing was 3 5 ItM. Condmons are as described m text (O) stripped Prochflodus hernoglobins, (O) stripped fiemoglobms + I mM ATP.
The genus Prochilodus consists of open river, mudeating specms (Fink & Fink, 1979). These fish spend much of their time foraging on river bottoms. In other eplbenthic Amazon species (e.g. members of various catfish famihes), which require a slight negative buoyancy to remain stationary on the river bed, the swim bladder has secondarily evolved into a sound sensing and emitting organ and is no longer employed as a hydrostatic device (Alexander, 1967). Although Prochilodus possesses a functional swim bladder and hemoglobins that display considerable Bohr and Root effects, it lacks the oxygen-secreting apparatus of the swim bladder, the fete mirabile (Bridge & Haddon, 1893). However, as pointed out by Farmer et aL (1979), it apparently does have the related apparatus of the eye, the choroid fete, and this is often associated with the occurrence of a Root effect. The large Bohr and Root effects probably reflect the diverse habitat distribution of the species. ProchiIodus sp. whole blood displays a high oxygen affinity at pH 7.6 and 3 0 C (/3, = 4 . T r a m Hg), a higher affinity than that reported for 44 out of 47 Amazonian fish species by Powers et aL (1979), especially higher than those of species whose hemo-
199
The root effect hemoglobin of the jaraqui globins display Root effects (i.e. M3'h),~,~oma sp., Martin et al., 1979; Hoplosternum littorale, Garlick et al., 19791. In fact, the affinity is comparable to those attained by certain aestivating fish species (i.e. Amia cah,a, Weber et al., 1976; Protopterus amtectans, Weber et al., 1977; LepMosiren paradoxa, Powers et td., 19791 which survive for long periods of time buried in mud. The high erythrocyte oxygen affimty may offer some physiological advantage in the hypoxic conditions found in various Prochilodus sp. habitats. The p~ of blood at pH 7.6 and 30~C is higher than that of the stripped hemoglobm solution + I m M ATP measured at 2 0 ' C under similar pH conditions. The difference undoubtedly arises in part because of the dtfferences in temperature and also because hemoglobin within fish erythrocytes may be modulated by c o m p o u n d s other than A T P (lsaacks et al., 19771. The heterogeneity of the kinetic data and the low n values, obtained in tonometric experiments below pH 6.7 indicate that the components of the ProchHodus hemoglobin system may be functionally differentiated. However, part of this heterogeneity may stem from a nonequivalence in ligand binding of ~t- and ,8-hke chains at low pH. lntramolecular functional heterogeneity is known in other fish hemoglobins having been reported for spot (Bonaventura et al., 1976), trout (Brunori, 1975), M y l o s s o m a (Martin et al., 19791 and carp (Tan et al., 19731. The results of oxygen dissociation and carbon monoxide combination experiments are in good agreement with those of oxygen equilibrium experiments in that changes induced in k and I" by pH and organic phosphates are paralleled by the influence of these modulators on the p., for oxygen. The Root effect of ProchHodus hemoglobins is chiefly mediated through the effects of pH on the Oz,,., constant, as is the case in spot, trout and carp hemoglobins, and variation in both the C O .... and 02,,, constants underlie the organic phosphate effect. In isoelectric focusing experiments of Bunn & Riggs (1979) all Prochilodus hemoglobin components demonstrated an alkaline Bohr effect. Thus if differences exist between the functions of the two components, they may be ones of quantity and not quality. That the components might be functionally differentiated is suggested by the complex ligand binding kinettcs earlier discussed. Perhaps the Prochilodus system represents an intermediate stage in the evolution of the functionally diverse hemoglobin systems typified by the trout. Further studies on the purified components of Prochilodus hemoglobin should reve,'d the degree of functional and structural differentiation attained by the two components. .4ekm~wledgement.s---Wc would hke to thank Ceha Bonaventura for her generous assistance m tile kinetic experiments. Maunzlo Brunorl expresses his thanks to the National Research Council (C.N R.) of Italy for financml support. Joseph Bonaventura is an Estabhshed Investigator of the American Heart Associatton and gratefully acknowledges grants from tile United States Nahonal Institute of Health and the National Science Foundation. We would hke to thank the officers and crew of tile Alpha Heh\. without whose unstmting assistance tll~s work would not have been possible, and tl~e Brazdian
government for permission to conduct research m their waters
This work was supported in part by the National Science Foundahon under grant PCM 75-06451 to the Scripps Institute of Oceanography for the support of the Alpha Helix program and by National Institutes of Health grant HL 15460. J. P. Martin is a predoctoral trainee supported by the National Institutes of Health grant GM07184 to Duke Untverslty. REFERENCES
ALI:'XANDER R. MCN (19671 Functlomtl Demgn m Fishe.s, 160 pp. Hutchinson Umversity Library London. ANrONINi E. & BRUNORI M. (1971) Hemoglobin and Myogh)bm in Thew Reaction.~ with Ligamls, 436pp. North-Holland, Amsterdam. BONAVLNTURA C., SULLIVAN B. • BONAV[NTURA J, (19741 The effects of pH and anions on functtonal properties of hemoglobins from Lemm" ]id~u.s.luh,u.s. J. hml. Chem. 249, 3768-3775. BONAVENTURA C., SULLIVAN B.,
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(~¢
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