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The hemoglobin ofpseudodoras, a South American catfish: Isolation, characterization and ligand binding studies
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THE H E M O G L O B I N OF PSEUDODORAS, A S O U T H A M E R I C A N CATFISH: ISOLATION, C H A R A C T E R I Z A T I O N A N D L I G A N D B I N D I N G STUDIES* JOSEPH P. MARTIN, I~ HANS J. FYHN, 2 UNNI E. H. FYHN, 2 ROBERT L. GARLICK, 3 ROBERT W. NOBLE4" and DENNIS A. POWERS5 ~Department of Zoology, Duke Umversity Marine Laboratory, Beaufort. NC 28516, U.S.A.; "Institute of Zoophyslology, UmversJty of Oslo, Bhndern, Oslo 3, Norway; 3Department of Zoology, Umverslty of Texas at Austin, Austin, TX "/8712, U S.A., '*Department of Medicine and Biochemistry, Veterans Administration Hospital, SUNY at Buffalo, Buffalo, NY 14215, USA." SDepartment of Biology. Johns Hopkins Llmverslty. Baltimore, MD 21218. U.S.A. (Recewed 5 April 1978)
Abstract 1. The hemoglobin of the Amazontan catfish Psemlodora,s sp was isolated and characterized: it comprises a smgle component. 2 The hemoglobin's subunit composmon is s~mtlar to that of other telcost hemoglobins. The apparent native molecular weight as determined by gel filtration is 66,000. The apparent subuntt molecular weight is 14,300 by sodium dodecyl sulhltc elcctrophorests. The hemoglobin does not polymerize after oxidation by potassium ferricyanide. 3. The hemoglobin lacks a Root effect. A small Bohr effect is evident m the phosphate-free hemoglobin: Alogpt/ApH ts no more than about -0.1 to -0.2 and increases to Alogp_~/ApH = -0.4 in the presence of I mM ATP. The coopcrativity, as determined by n of the Hill equation, is low, varying from 0.8 to 1.7 between pH 6.1 and 8.6. 4. The Pt values of stripped hemoglobin solutions are extrcrnely low, less than 0.5 mm Hg at all pH values examined between pH 6.1 and 9.0 The high oxygen affinity is reflected primarily in the CO combination rate which resembles that found m myoglobins and isolated subunits of human hemoglobin. 5. Both the CO combination rate and the O, dissociatton rate determined by stopped-flow spectrophotometry are pH and phosphate sensmve. Between pH 6 2 and 8 I the CO,,. rate increases about 5-fold in the phosphate-free hemoglobin. Addmon of I mM ATP causes a depression in the rate at all pH values examined. The 0_,..,, rate decreases 7-fold going from pH 6.0 to 8.2 m strtpped hemoglobin solutions. Addition of 1 mM ATP induces a 10-fold decrease over the same range At pH values below 6.0 a depression m the O, , rate occurs m the stripped hemoglobin, indicative of an acid Bohr effect.
INTRODUCTION
The blood of fish generally contains multiple hemoglobin components. Bonaventura et al. (1976) electrophoretically surveyed approximately 100 aquatic and marine fish species. Of those examined fewer than 5". contained single hemoglobins in their blood (Bonaventura, personal communication). Sharp (1973) discovered only three fish species with a single hemoglobin out of 31 in a study of California coastal fish. Riggs (1970) has suggested that hemoglobin multiplicity might be physiologically advantageot, s to fish. Considerable effort by numerous investigators (Hashimoto et al., 1960; Tan et al., 1972; Gillen & Riggs, 1973; Tan & Noble, 1973; Weber & DeWilde, 1975; Weber et al., 1977: Garlick et al., 1979) has been expended in purifying and characterizing individual fish hemoglobins from multiple component systems with the hope that the physiological roles of these molecules might be deduced. Although the three hemoglobins of carp appear to be functionally identical (Noble et al., [970; Gillen & Riggs, 19721 detailed * A Portt,gt, ese translation of this work will appear m Acta Amazonica. i'To whom reprint requests should be addressed.
kinetic stu&es on the hemoglobin of "'single component fish species" are comparatwely rare. Bonaventura et al. (1976) elucidated the functional properties of the Root effect hemoglobin of the spot (Leiostomus xanthurus), but no other such studies have been reported. Fish hemoglobins may provide valuable paradigms for the allosteric control of ligand affinity in mammalian hemoglobins. Although a two-state model has been proposed to account for the cooperativity of oxygen binding (Monod et al., 1965) it has proved difficult to observe these structt, ral states in h u m a n hemoglobin. However, investigators are able to experiment with fish hemoglobins that have been stabilized in high and low affin,ty states (Noble et al., 1970: Tan et al., 1972, 1973; Tan & Noble 1973; Bonaventura et al., 1976). This paper describes work performed on the hemoglobin isolated from an Amazonian catfish, Pseudodoras sp. The blood of this species contains one hemoglobin component. The fish was readily obtained and the hemoglobin easily purified in large quantities. Pseudodoras sp. is an epibenthic catfish species and we wished to determine if its hemoglobin ewnced a Root effect. It was also felt that the adaptations of fish hemoglobins to the exigencies of the 207
JOSEPH P. MARTIN et al.
208
e n v i r o n m e n t c o u l d be m o s t easily s t u d i e d in a s y s t e m where selection p r e s s u r e s are focused on a single hemoglobin component. MATERIALS A N D M E T H O D S
fl-mercaptoethanol. H u m a n transferrm, bowne serum albumin, ovalbumm, :~-chymotrypsmogen-A (bovine pancreas), and sperm whale myoglobin were used as molecular weight standards. The myoglobm was obtained from MilesSeravac Co. All other standards were purchased from the Sigma Chemical Co.
Isolatton
Oxygen equilihrutm .~tudie.s
Speomens of Pseudodoras sp. were collected in November and December, 1976, during an expedition on the R.V. Alpha Helix on tile Amazon River about 50 km upstream on the Rio Solim6es from the junction with the R~o Negro. The catfish were caught by beach seine and gdl nets and transported to the Alpha Helix for study Blood was obtained by cardiac puncture and drawn into cold heparmized glass syringes [100/d of sodium heparin (5000 Lu./ml) in 1.7". NaCI/5 ml of blood]. Small samples were taken up in 501d capillary tubes and spun for 3 min in a serum centrifuge for hematocrit estimations. The red blood cells were washed 3 times m 10 volumes of cold l mM Tns, pH 8.0,* 1.7",, NaCI and then lysed in 3 volumes of I m M Tris, pH 8.0 for I hr at 0"C. One tenth volume of 1 M NaCI was added to the hemolysate and the mixture was centrifuged at 280000 for 15min to remove cellular debris. The supernatant was then stripped of salt and orgamc phosphates by passage through a 2.5 x 50cm column of Sephadex G-25 resin equilibrated m 0.1 mM Tris, pH 8.5 followed by treatment on a deionizing column with the following resms from top to bottom: 2 cm Dowex-50w a m m o n i u m form, 2 cm Dowex-I acetate form. 20cm Bin. Rad AG 501-X8 (D) m~xed bed resin. Tile purified hemoglobin was stored at 5"C until reqmred. A sample of packed erythrocytes was frozen at - 70'C, transported on dry ice to Beaufort, NC, and stored at - 2 0 " C for 2 months. In Beaufort the cells were thawed, lysed and the molecular weight of the carboxy derivative of the hemoglobin was determined by gel filtration.
Whole blood oxygen equilibria wcrc performed at 30'C by the method of Powers et al (1979) using a Hem-O-Scan oxygen dissocmtmn analyzer (American Instruments Corporation). Oxygen equdibria of purified hemoglobin were carried out at 20'C as described by Riggs & Wolbach (1956). The hemoglobin solutions (601,M) were brought to 0.05 ionic strength m Tris and Bls-Tris buffers. P,. values were determined as a function of pH. The experiments were performed on stripped hemolysates in the presence and absence of 1 mM A T P
Electrophoresis Vertical polyacrylamlde gel electrophoresis (pH 8.9, 7.5°o gels) was done at 25'~C according to Davis (1964) and Ornstein (1964). Hemoglobin samples (1 mg/ml) in upper buffer containing 0.1 M fl-mercaptoethanol and a small a m o u n t of dithmmte were bubbled with carbon monoxide and apphed to the gels. Bovine serum albumin was used as an internal standard. The gels were stained for 3 hr in 0.25"° Coomassle Brilliant Blue R in an acetic acid-methanol-water solutmn (1:2:4) and destained by diffusion. The gels were scanned at 560 nm using a Gilford gel scanner attached to a Beckman DU monochromator.
Molecular wetyht studtes Gel filtration experiments of oxidized and reduced hemoglobin solutions of Pseudodoras sp. were carried out on a Sepharose 4B column (2 x 90cm) in 0.05 M Tns, pH 7.5. I m M EDTA, at 5 ' C according to Fyhn & Sullivan (1975). In addition, the molecular weight of the carboxy hemoglobin was measured w~th another resin according to Martin et aL (1979). Molecular weights of denatured hemoglobin chains were determined by sodium dodecyl sulfate (SDS) electrophoresis according to Weber & Osborn (19691 except that the incubatmn solutmn was 6 M in urea and 0.1 M in
* Abbreviations: B~s-Tris, bls(2 hydroxyethyl)lmmo Tris(hydroxymethyll methane: Tris, Tris(hydroxymethyllaminomethane, ATP, adenosine triphosphate; EDTA, ethylenediaminetetraaceuc acid; I', second order combination velocity constant for carbon monoxide binding; k, first order dlssocmtlon velooty constant for oxygen: p~, the partial pressure of oxygen at which one half of t h e total heine sites are occupied.
Liyand binding kinetics All kinetic measurements were performed with a stopped-flow apparatus of the type originally described by Gibson & Mflnes (1964). In all cases the ionic strength of the final solution after mixing was 0.05 and when A T P was used its concentrauon after mixing was I m M The kinetic constants presented are the least squares fits to the first 65°. of the observed reactions. Tile kinetics of oxygen dlssocmtion were measured by the pH j u m p procedure as described by Noble et al (1970). Oxygenated hemoglobin in 1 m M Tns, pH 8.0 was mixed with a solution of dithionite m a 0.1 ionic strength buffer of the desired pH. The final hemoglobin concentrauon was approx 30 ,uM m heme equivalents and the reaction was followed at 540 and 560 nm. The kinetics of carbon monoxide c o m b m a u o n to deoxygenated hemoglobin were measured by mixing solutions of deoxygenated hemoglobin in 0.1 ionic strength buffers of the desired pH with a solutmn containing a known concentration, approx 85 llM, of carbon monoxide dissolved in water. After mixing, the hemoglobin concentration was approx 3 ItM in heme equlwdents. The reactmn was followed at 420 and 435 nm.
Buffers The buffers employed m the kinetics and equilibrium experiments were all of untform final iomc strength (0.05/). Tris buffers were used m expermaents above pH 7 while Bls-Tris buffers were employed m experiments below pH 7.
RESLILTS A N D D I S ( ' U S S I O N
T h e h e m a t o c r i t s of two Pseudodoras sp. i n d i v i d u a l s of weight 180 a n d 6 4 0 g were 30 a n d 31",, respectively. E l e c t r o p h o r e s i s of three h e m o l y s a t e s revealed only a single h e m o g l o b i n c o m p o n e n t . T h e m i g r a t i o n of the h e m o g l o b i n on disc gels in c o m p a r i s o n with h u m a n h e m o g l o b i n is s h o w n in Fig. 1. After 3 weeks of s t o r a g e at 5~C the m o b i l i t y of the h e m o g l o b i n c o m p o n e n t r e m a i n e d c o n s t a n t a n d no c h a r g e heterogeneity w a s detected. T h e h e m o g l o b i n d o e s not p o l y m e r i z e after treatm e n t with p o t a s s i u m ferricyanide or after storage, a feature r e p o r t e d for tile h e r n o l y s a t c of the teleost Hoplias malaharicus by Reischl (19761. O x i d i z e d a n d reduced h e m o g l o b i n s a m p l e s elutc as single s y m m e t r i cal p e a k s on S e p h a r o s c 4B C h r o m a t o g r a p h y . T h e C O - h e m o g l o b i n h a s an a p p a r e n t m o l e c u l a r weight of 66,000 a c c o r d i n g to the clution posH~on in gel filtration e x p e r i m e n t s . T h e h e m o g l o b i n elutes slightly
The hemoglobin of Pseudodoras
209
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a small Bohr effect. The shape of the binding curve denotes cooperative oxygen binding. Oxygen equilibrium experiments on stripped hemolysates with and without 1 mM ATP (Fig. 3) were performed at 20°C. The p~ at pH 7.6 of stripped hemoglobin is 0.25 mm Hg while that of stripped hemoglobin + 1 mM ATP at pH 7.54 is 0.56 mm Hg. The Bohr effect of stripped hemolysates, Alog p j A p H , appears to be between -0.1 and -0.2. Bunn & Riggs (1979) using an isoelectric focusing technique, have corroborated this result. The Bohr effect, Alog p½/APH, increases to - 0 . 4 in the presence of ! mM ATP between pH 7 and 9 but the value increases to about 1.6 in the range pH 6.5-7.0. The p~ varies 2.5-fold between pH 6.5 and 9.0 in stripped hemolysates and almost 30-fold after ATP additions. As might be expected
band in both gels is bovine serum albumin. The anode is at the bottom of the figure. before the hemoglobin of the spot which is known to be tetrameric (S2o.,, = 4.46S), as determined by sedimentation velocity centrifugation, under the specified column conditions. Tile apparent molecular weight of the denatured hemoglobin chains from Pseudodoras sp. as determined by SDS gel electrophoresis, was 14,300, which is close to that obtained for human chains (14,600). The oxygen equilibrium curve of Pseudodoras blood is presented in Fig. 2. At pH 7.6 and 30°C the red blood cell Pi is 11.0 mm Hg, while under conditions of 5.6% CO2 it shifts to 13.6 mm Hg, indicating
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Fig. 2. Oxygenation curves of Pseudodora.~ blood at pH 7.6, 30 C. Curve I (- - -) and Curve 2 ( reprcsent continuous oxygenation in tile absence and presence of 5.6". CO_,, respectively.
JOSLPH P. MARTIN el al.
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pH Fig. 4. The relatmn between pH and the first order dissooallon constant (k) of Pwudodora~ oxyhemoglobm. Condihons are as described in text. Stripped hemolysate (O), stripped hemol.~sate + 1 mM ATP (O). for a polyanion, ATP exerts a greater effect at lower pH values. The mterchain cooperativity, reflected in n, is generally low and is pH dependent (Fig. 3). At pH 6.1 the n value for stripped hemolysates is less than unity. Between pH 6.7 and 8.6 n attains its maximum value of about 1.7 and therafter declines until at pH 9.0, n = 1.0. The presence of l mM ATP does not cause any consistent increase or decrease in the value of n over the pH range examined. The rate of oxygen dissociation from the hemoglobin is shown as a function of pH, with and without 1 mM ATP, in Fig. 4. The pH dependence observed is that of a hemoglobin with a normal Bohr effect. In the absence of ATP the total variation with pH is 7-fold. I mM ATP increases the rate of this reaction at all pH wdues below 8.2 and this increases the total variation to more than 10-fold. The second order rate constants for the reaction of carbon monoxide with the deoxygenated derivative of this hemoglobin are shown in Fig. 5. again m the i
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presence and abscnce of I mM ATP. This rate constant is strongly pH dependent, increasing as the pH is increased from 6.0 to 8.0 as would be expected for a normal Bohr effect. Addition of 1 mM ATP decreases this reaction rate at all pH values examined but the effect is smallest at the highest pH. Although these kinetic results indicate a normal Bollr effect between pH 6.0 and 8.0, they suggest an acid or reverse Bohr effect below pH 6.0. In the absence of ATP this is manifest primarily in the rate of oxygen dissociation. In the presence of ATP it is seen m both rate constants but the effect in each ts smaller. The rate constants for the reaction with carbon monoxide are most unusual. These are much larger than are normally found for unmodified hemoglobin molecules. The rate observed at pH 8.0 approaches that found for myoglobins and the isolated subunits of human hemoglobin. As stated previously, Pseudodoras sp. differs lrom most fish in possessing a single hemoglobin component. The simplicity of this hemoglobin system may reflect the constancy of the physical environment and the nature of the fish's physiological requirements. Fyhn et al. (1979) noted that hemolysates with a single hemoglobin were more common among fishes from the tipper Amazon River than among fishes from temperate regions. The low variance of certain physical parameters in the Amazon (e.g. water temperature fluctuates less than 2~C yearly) and the unparalleled fish species diversity and habitat specialization in the Amazon basin may partially explain their observations. Pseudodoras hemoglobm chromatographs as a stable tetramer. It neither aggregates under oxidizing conditions nor dissociates into dimers. At 20'C the stripped hemoglobin exhibits a low p~ for oxygen binding (less than 1 mm Hg at all pH values examined) and a very small Bohr effect. Addition of I mM ATP substantially increases the Bohr effect: but the p~ remains relatively low, never exceeding 4.6mm Hg. In contrast, the whole blood at pH 7.6 and 30'C has a p~ of I 1 mm Hg. The significance of the difference between the results obtained with the whole blood and those from the hemoglobin studies cannot be assessed without knowing the temperature dependence of the oxygen affinity of this hemoglobin. The differencc observed may be due entirely to variations in the parameter, or may result from the presence in the red cell of an organic phosphate which is more effective than ATP. lssaacks et al. (1977) have discovered inositol pentaphosphate in the erythrocytes of anothcr Amazon fish species (Arapaima ,qi~jas). Tan & Noble (1973) have demonstrated with inositol hexaphosphate and Torracca et al. (1977) have shown for guanosme triphosphate that these are more effective modulaters of hemoglobin oxygen binding than is ATP in some fish species. The second order rate constant for carbon monoxide association with hcmoglobin corroborates the equilibrium results. It exceeds those known for other fish hemoglobins by nearly an order of magnitude (e.g. carp, Noble et al., 1970: spot, Bonaventura et al., 1976: Myloxsoma sp. Martin et al., 1979) and resembles the values obtained for isolated hemoglobin subunlts and myoglobins.
The hemoglobin of Pseudodora.s Other notable characteristics of P.wudodoras hemoglobin are its small Bohr effect and the absence of a Root effect. Riggs (1970) has pointed out that the magnitude of the Bohr effect in fish hemoglobin is often proportional to the fishes" activity levels. The data on P.selulodora.s hemoglobin are consistent with this generalization in that Pseudodoras is an epibenthlc catfish and, as a siluriform, is generally less active than fish of other taxa. Consequently, a large Bohr effect may not be required to facilitate oxygen unloading at the tissues. Although Pseudodoras possesses a swim bladder its lacks the usually attendant rete mirabile (Bridge & Haddon, 1893), the counter-current multiplier system wherein hemoglobin releases oxygen for entry into the swim bladder. In addition, Pseudodoras lacks a choroid rete (Farmer et al., 1979), the structure responsible for oxygen delivery to the eye The absence of these anatomical features may have prechlded the development of a Root effect hemoglobin. ATP and H + affect both the COo,' and the 0 2.... rates of P.wudodol'as hemoglobin. This is similar to the effects of these agents on other fish hemoglobins. The magnitude of these kinetic changes between pH 5.1 and 8.7 adequately accounts for the nearly 30-fold change in p~ of stripped hemolysates in the presence of 1 mM ATP. Equlhbrium data suggest an acid Bohr effect at low pH similar to that noted in trout 3 hemoglobin (Sahno +jairdnel'i) (Lau et al., 1975). The behavior of the CO,,,, and 02,.,' constants below pH 6.0 in Pseudodoras hemoglobin support the equilibrium data. However, the pH at which the acid Bohr effect is first apparent is higher in the equilibrium than in the kinetic experiments, possibly a consequencc of differences in the ligand binding behavior of CO and 02 The n values, obtained from the analysis of equilibrium data, vary as a function of pH. Maximal cooperatlvity +s attained in the physiological pH range, as in carp hemoglobin (Noble et al., 1970: n decreases above pH 8.6 and below pH 6.7. ATP does not significantly alter this pattern. At pH 6.1 the cooperatlvity drops below unity, perhaps indicating differences in the binding affinities between m- and fl-like chains. In conclusion+ Pseudmloras hemoglobin shares many of the hgand binding characteristics of Root effect hemoglobins (i.e. those of carp+ trout and spot) yet itself lacks a Root effect. Detailed structural comparisons among these hemoglobins may therefore enlighten us with respect to the structural basis of the Root effect in fish hemoglobins. ,4cknowh'dgement.s We wish to thank the officers arid crew of the 41pha Heh\ for thew cooperation and camaraderm during thRs crmse We would also hke to express our thanks to the Brazdzan government for permitting us to work m Amttzon waters. Hans J. Fyhn and Unni E. H. Fyhn acknowledge travel grants from the Norwegmn Research Count21 for Scmncc and the Humanmes. Addittonal support was provided by NSF grant DEB-76-19877 (to D. A Powers). the National Geographic Socmty (to D. A Powers)+ NSF grant PCM-76-06719 and NIH grant GM 21314 {to A. R~ggs) This work was supported m part by the Natmnal Scmnce Foundahon under grant PCM 75-06451 to the Scripps Institute of Oceanography for the support of the ,-llpha Hehx Progran+t, and b~ the National Instmltes of
211
Health grant HL 15460. Joseph P. Marlin is a predoctoral trainee supported by the National Institutes of Health grant GM 07184 to Duke University. Robert W. Noble is an Established Investigator of the American Heart Association.
REFERENCES
BONAVENTURA (.', SULLIVAN B., BONAVhNTURA J BRUNORI M (1976) Spot hemoglobin, studies on the Root effect hemoglobin of a marine teleost. J. biol. Chem. 251, 1871-1876. BRIDGI_ S. W. & HADDON G. C. (1893) Contributions to the anatomy of fishes. II The air bladder and Weberian ossicles in the sdurold fishes Plul Trans. B, 184, 65 434 BRUNOR[ M., FALCIONI G., FORTUNA G & GIARI)INA B. (1975) The effects of anions on the oxygen binding properties of the hemoglobin components from trout (Sahno irideu~s). Archs Biochem. Biophys. 168, 512-519. BRUNORI M. (1975) Molecular adaptation to physiological requirements: the hemoglobin system of trout. In Current Topic's nl Cell Regulation. Vol. 9 (Edited by HORECKhR M. & STAI)TMAN E ), pp 1 39. Academm Press, New York. BUNN H. F & RIGGS A. (1979) The measurement of the Bohr effect of fish hemoglobins by gel electrofocusing. Comp. Blocht'm. Phy.~ml. 62A, 95 I00. DAVIS B. J. (1964) Disc electrophoresls--ll. Method and application to human serum proteins. Am~. N . Y Acad. Scl 121, 404-427 FARMER M., EYHN H J, FYItN U. E H. & NOBLE R. W. (1979) Occurrence of Root effect hemoglobins m Ama/onmn fishes Comp Biochem Phl'siol 62A, 115 124. F~HNU. E H &SULLIVANB (1975) Elasmobranch hemoglobins, dlmcnzaUon and polymerization m various species Comp. Bwchem. Physiol. 50B, 119 129. h n N U E H., FYIIN H. J., DAVIS B. J., FINK W L, POWERS D. A, FINK W. L. & GARLICK R. L. (1979) Hemoglobin heterogeneity of Amazonian fishes Comp. BIochem. Plu'.~ml 62A, 39 66. GARLICK R. L., BUNN H F., F~HN H. J., FVHN U. E. H., MARrIN J P., NOI~LL R. W & POWERS D A. (1979) Functmnal studms on the separated hemoglobin components of an air-breathing catfish, Hoplosternum httorah' (Hancock}. Comp. Bmchem. Phl'.~ml. 62A, 219 226 GIBSON Q H. &. MILNI'++S L [1964) Apparatus for rapid and sensitwe spectrophotometry Btochem. J 91, 16[-171 GILLI,N R. & RIGGS A. (1972l Structure and functmn of the hemoglobins of the carp, Cvprim~.s carplo. J. hiol. Chem 247, 6039 6046. GILLIN R & RIC;GS A (1973) Structure and function of the isolated hemoglobin of the American eel .4nguHla ro~trata J Inol Chem. 248+ 1961 1969. HASnlMOTO K Y. YAMAGUCHI & MATSUURA F. (1960) Comparatwe studies on two hemoglobins of salmon IV Oxygen dlssocmhon curves Bull Jap. Soc .wwnt. Fish. 26, 827 834. ISAA('KS R. E.. KIM H. D., BARTLI-TT G. R. & HARKNFSS D R. (1977) lnosltol penlaphosphatc in erythrocytes of a freshwater fish, plraracu ( 4rapatma .qujas). L(lb Scl. 20, 987 990. LAir H. K F. WALLACH D., PI:NNI'LLV R. & NOBLE R. W. ll975) Ligand binding propertLes of hemoglobin 3 of the trout, Salmo qalrdnert. J hml. Chem. 250, 1400 1404.
MARII"< J P+. BONAVINTIIRAJ. BRtrNORI M. FYHN H J, FYII"q U. E. H., GARI.IE'K R. L, POWI.RS D. A. & WILSON M. T (1979) The isolatmn and charactenzatmn of the hemoglobin components of A4ylo.s.soma sp., an Amazomun tcleost. Comp. Bmchem. Ph)'siol 62A, 155 162.
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Josl-l,l-I P. MARTIN et al.
MONOD J., W'tMAN J. & CrIANGLAUX J. P. (1965) On the nature of allostcrlc transitions: a plausible model. J. molec. Bud. 12, 88 118 NOaLI R. W., PARKrltIRSl U J. & GmSON Q. H. (1970) The effect of pH on the reactions of oxygen and carbon monoxide with the hcmoglobin of carp, Cyprmu.s carp,;. J hurl. Chem. 245, 6628 6633. ORNSTIqN L. (1964) Disc elcctrophoresls--l. Background and theory..,hm. N.Y Acad. Sol. 121, 321 349. POWERS D A, Fvltr~ H. J., FVHN U. E. H., MARTIN J P , GARLIC'S, R. L. & WOOD S. C. (1979) A comparative study of the oxvgcn equilibria of blood from 40 genera of Am,lzontan Iishcs Comp. Bwchem Plll'~ml. 62A, 67- 86. REtS('IIL E. (1976)The hemoglobin of a fresh water teleost Hopha.~ tntllahartctt,s: heterogeneity and polymerization. Comp. Btochem. Physiol. 55B, 255-247 RIGGS A. (1970) Properties of fish hemoglobms In Fish Physiolo.qy, Vol. 4 (Edited by HOAR W. S. & RANDALL D J ), pp. 209 251 Academic Press, New York. RtGGS A. & WOLI3ACH R A. (1956) Sulfllydryl groups and the structure of hemoglobin J yen. Physiol 39, 585-605 SHARe G D. (1973) An electrophoretic study of hemoglobins of some scombrid fishes and related forms. Comp. Biochem Plly,stol 44B, 381-388. TAN A, DrYOUNG A & NOBLI R W (1972) Thc pH dependence of the aflimty, kinetics, and cooperativity of hgand binding to carp hemoglobin. Cyprttnts carplo. J. htol Chem 247, 2493 2498. TAN A. L. & Nont, I R. W (1973) The effect of Inosltol hexaphosphate on the allostertc properties of carp hemoglobin J. hwl. Che,i. 248, 7412 7416.
TAN A. L., NOBLI: R. W. & GIBSON Q. H. (1973) Condltmns restricting allosteric transitions m carp hemoglobin. J biol. Chem. 248, 2880-2888. TORRACCA A. M., RASCHETTI R., SALVlOLA R., RICCIOARDI G & WmTERHAL1ER K. H. {1977) Modulation of the Root effect in goldfish by ATP and GTP. BiochmL biophys. Acta 496, 367-373. WI-:RER K. & OSaORN M. (1969)The rehability of molecular weight determinations by dodecyl sulfate-polyacrylamide electrophoresis. J. biol. Chem. 244, 4406-4412. W~I~R R. E. & Dt-:Wml)l: J. A. M. (1975) Oxygenation properties of hemoglobin from the flatfish plaice (Pleuronecte.~ platts.sa) and flot, nder (Plalichthy.s fle.~us) J comp. Physlol I01, 99-110. WI-BER R. E., JOHANSEN K.. L'¢KKI-_'I]OE G. & MALOIY G. M. O. (1977) The oxygen binding propertms of hemoglobins from cstivating and active African lungfish, d. e.xp. ZooL 199, 85 99. WEBER R. E., SULLIVAN B., BONAVENTURAJ. & BONAVENTVRA C. (1976) The hemoglobin of the primitive fish, .4mla cah:a: ~solation and functional characterization of the individual hemoglobin components. Biochim. htophys Acta 434, 18-31. YAMAGUCHI K., KOCHI',AMA Y., HASHIMOTO K. & MATSUURA F. (1962) Studies on the multiple hemoglobins of eel II Oxygen dissociation curve and the relative a m o u n t s of components F & S. Bull Jap. So('. scient Fish. 28, 192-200. YAMAGUCHt K., YAMAGUCHI K. & MATSt'URA F (1963) Studies on two hemoglobins of loach 1I. Oxygen dlssooation curve. Bull. Jap. Soc. scwnt. Fish. 29, 180.
Bto~hcm P h l ~ t o l
I,d 62,'~ pp 2117 I o 212
0lt~l-tlC~29 79UIqlI-1121ffS(I2UI)0
PI'I(IIIIIIOll Ptc~s L i d 1979 P~fttl~,d m (tic,lit IJtttalll
THE H E M O G L O B I N OF PSEUDODORAS, A S O U T H A M E R I C A N CATFISH: ISOLATION, C H A R A C T E R I Z A T I O N A N D L I G A N D B I N D I N G STUDIES* JOSEPH P. MARTIN, I~ HANS J. FYHN, 2 UNNI E. H. FYHN, 2 ROBERT L. GARLICK, 3 ROBERT W. NOBLE4" and DENNIS A. POWERS5 ~Department of Zoology, Duke Umversity Marine Laboratory, Beaufort. NC 28516, U.S.A.; "Institute of Zoophyslology, UmversJty of Oslo, Bhndern, Oslo 3, Norway; 3Department of Zoology, Umverslty of Texas at Austin, Austin, TX "/8712, U S.A., '*Department of Medicine and Biochemistry, Veterans Administration Hospital, SUNY at Buffalo, Buffalo, NY 14215, USA." SDepartment of Biology. Johns Hopkins Llmverslty. Baltimore, MD 21218. U.S.A. (Recewed 5 April 1978)
Abstract 1. The hemoglobin of the Amazontan catfish Psemlodora,s sp was isolated and characterized: it comprises a smgle component. 2 The hemoglobin's subunit composmon is s~mtlar to that of other telcost hemoglobins. The apparent native molecular weight as determined by gel filtration is 66,000. The apparent subuntt molecular weight is 14,300 by sodium dodecyl sulhltc elcctrophorests. The hemoglobin does not polymerize after oxidation by potassium ferricyanide. 3. The hemoglobin lacks a Root effect. A small Bohr effect is evident m the phosphate-free hemoglobin: Alogpt/ApH ts no more than about -0.1 to -0.2 and increases to Alogp_~/ApH = -0.4 in the presence of I mM ATP. The coopcrativity, as determined by n of the Hill equation, is low, varying from 0.8 to 1.7 between pH 6.1 and 8.6. 4. The Pt values of stripped hemoglobin solutions are extrcrnely low, less than 0.5 mm Hg at all pH values examined between pH 6.1 and 9.0 The high oxygen affinity is reflected primarily in the CO combination rate which resembles that found m myoglobins and isolated subunits of human hemoglobin. 5. Both the CO combination rate and the O, dissociatton rate determined by stopped-flow spectrophotometry are pH and phosphate sensmve. Between pH 6 2 and 8 I the CO,,. rate increases about 5-fold in the phosphate-free hemoglobin. Addmon of I mM ATP causes a depression in the rate at all pH values examined. The 0_,..,, rate decreases 7-fold going from pH 6.0 to 8.2 m strtpped hemoglobin solutions. Addition of 1 mM ATP induces a 10-fold decrease over the same range At pH values below 6.0 a depression m the O, , rate occurs m the stripped hemoglobin, indicative of an acid Bohr effect.
INTRODUCTION
The blood of fish generally contains multiple hemoglobin components. Bonaventura et al. (1976) electrophoretically surveyed approximately 100 aquatic and marine fish species. Of those examined fewer than 5". contained single hemoglobins in their blood (Bonaventura, personal communication). Sharp (1973) discovered only three fish species with a single hemoglobin out of 31 in a study of California coastal fish. Riggs (1970) has suggested that hemoglobin multiplicity might be physiologically advantageot, s to fish. Considerable effort by numerous investigators (Hashimoto et al., 1960; Tan et al., 1972; Gillen & Riggs, 1973; Tan & Noble, 1973; Weber & DeWilde, 1975; Weber et al., 1977: Garlick et al., 1979) has been expended in purifying and characterizing individual fish hemoglobins from multiple component systems with the hope that the physiological roles of these molecules might be deduced. Although the three hemoglobins of carp appear to be functionally identical (Noble et al., [970; Gillen & Riggs, 19721 detailed * A Portt,gt, ese translation of this work will appear m Acta Amazonica. i'To whom reprint requests should be addressed.
kinetic stu&es on the hemoglobin of "'single component fish species" are comparatwely rare. Bonaventura et al. (1976) elucidated the functional properties of the Root effect hemoglobin of the spot (Leiostomus xanthurus), but no other such studies have been reported. Fish hemoglobins may provide valuable paradigms for the allosteric control of ligand affinity in mammalian hemoglobins. Although a two-state model has been proposed to account for the cooperativity of oxygen binding (Monod et al., 1965) it has proved difficult to observe these structt, ral states in h u m a n hemoglobin. However, investigators are able to experiment with fish hemoglobins that have been stabilized in high and low affin,ty states (Noble et al., 1970: Tan et al., 1972, 1973; Tan & Noble 1973; Bonaventura et al., 1976). This paper describes work performed on the hemoglobin isolated from an Amazonian catfish, Pseudodoras sp. The blood of this species contains one hemoglobin component. The fish was readily obtained and the hemoglobin easily purified in large quantities. Pseudodoras sp. is an epibenthic catfish species and we wished to determine if its hemoglobin ewnced a Root effect. It was also felt that the adaptations of fish hemoglobins to the exigencies of the 207
JOSEPH P. MARTIN et al.
208
e n v i r o n m e n t c o u l d be m o s t easily s t u d i e d in a s y s t e m where selection p r e s s u r e s are focused on a single hemoglobin component. MATERIALS A N D M E T H O D S
fl-mercaptoethanol. H u m a n transferrm, bowne serum albumin, ovalbumm, :~-chymotrypsmogen-A (bovine pancreas), and sperm whale myoglobin were used as molecular weight standards. The myoglobm was obtained from MilesSeravac Co. All other standards were purchased from the Sigma Chemical Co.
Isolatton
Oxygen equilihrutm .~tudie.s
Speomens of Pseudodoras sp. were collected in November and December, 1976, during an expedition on the R.V. Alpha Helix on tile Amazon River about 50 km upstream on the Rio Solim6es from the junction with the R~o Negro. The catfish were caught by beach seine and gdl nets and transported to the Alpha Helix for study Blood was obtained by cardiac puncture and drawn into cold heparmized glass syringes [100/d of sodium heparin (5000 Lu./ml) in 1.7". NaCI/5 ml of blood]. Small samples were taken up in 501d capillary tubes and spun for 3 min in a serum centrifuge for hematocrit estimations. The red blood cells were washed 3 times m 10 volumes of cold l mM Tns, pH 8.0,* 1.7",, NaCI and then lysed in 3 volumes of I m M Tris, pH 8.0 for I hr at 0"C. One tenth volume of 1 M NaCI was added to the hemolysate and the mixture was centrifuged at 280000 for 15min to remove cellular debris. The supernatant was then stripped of salt and orgamc phosphates by passage through a 2.5 x 50cm column of Sephadex G-25 resin equilibrated m 0.1 mM Tris, pH 8.5 followed by treatment on a deionizing column with the following resms from top to bottom: 2 cm Dowex-50w a m m o n i u m form, 2 cm Dowex-I acetate form. 20cm Bin. Rad AG 501-X8 (D) m~xed bed resin. Tile purified hemoglobin was stored at 5"C until reqmred. A sample of packed erythrocytes was frozen at - 70'C, transported on dry ice to Beaufort, NC, and stored at - 2 0 " C for 2 months. In Beaufort the cells were thawed, lysed and the molecular weight of the carboxy derivative of the hemoglobin was determined by gel filtration.
Whole blood oxygen equilibria wcrc performed at 30'C by the method of Powers et al (1979) using a Hem-O-Scan oxygen dissocmtmn analyzer (American Instruments Corporation). Oxygen equdibria of purified hemoglobin were carried out at 20'C as described by Riggs & Wolbach (1956). The hemoglobin solutions (601,M) were brought to 0.05 ionic strength m Tris and Bls-Tris buffers. P,. values were determined as a function of pH. The experiments were performed on stripped hemolysates in the presence and absence of 1 mM A T P
Electrophoresis Vertical polyacrylamlde gel electrophoresis (pH 8.9, 7.5°o gels) was done at 25'~C according to Davis (1964) and Ornstein (1964). Hemoglobin samples (1 mg/ml) in upper buffer containing 0.1 M fl-mercaptoethanol and a small a m o u n t of dithmmte were bubbled with carbon monoxide and apphed to the gels. Bovine serum albumin was used as an internal standard. The gels were stained for 3 hr in 0.25"° Coomassle Brilliant Blue R in an acetic acid-methanol-water solutmn (1:2:4) and destained by diffusion. The gels were scanned at 560 nm using a Gilford gel scanner attached to a Beckman DU monochromator.
Molecular wetyht studtes Gel filtration experiments of oxidized and reduced hemoglobin solutions of Pseudodoras sp. were carried out on a Sepharose 4B column (2 x 90cm) in 0.05 M Tns, pH 7.5. I m M EDTA, at 5 ' C according to Fyhn & Sullivan (1975). In addition, the molecular weight of the carboxy hemoglobin was measured w~th another resin according to Martin et aL (1979). Molecular weights of denatured hemoglobin chains were determined by sodium dodecyl sulfate (SDS) electrophoresis according to Weber & Osborn (19691 except that the incubatmn solutmn was 6 M in urea and 0.1 M in
* Abbreviations: B~s-Tris, bls(2 hydroxyethyl)lmmo Tris(hydroxymethyll methane: Tris, Tris(hydroxymethyllaminomethane, ATP, adenosine triphosphate; EDTA, ethylenediaminetetraaceuc acid; I', second order combination velocity constant for carbon monoxide binding; k, first order dlssocmtlon velooty constant for oxygen: p~, the partial pressure of oxygen at which one half of t h e total heine sites are occupied.
Liyand binding kinetics All kinetic measurements were performed with a stopped-flow apparatus of the type originally described by Gibson & Mflnes (1964). In all cases the ionic strength of the final solution after mixing was 0.05 and when A T P was used its concentrauon after mixing was I m M The kinetic constants presented are the least squares fits to the first 65°. of the observed reactions. Tile kinetics of oxygen dlssocmtion were measured by the pH j u m p procedure as described by Noble et al (1970). Oxygenated hemoglobin in 1 m M Tns, pH 8.0 was mixed with a solution of dithionite m a 0.1 ionic strength buffer of the desired pH. The final hemoglobin concentrauon was approx 30 ,uM m heme equivalents and the reaction was followed at 540 and 560 nm. The kinetics of carbon monoxide c o m b m a u o n to deoxygenated hemoglobin were measured by mixing solutions of deoxygenated hemoglobin in 0.1 ionic strength buffers of the desired pH with a solutmn containing a known concentration, approx 85 llM, of carbon monoxide dissolved in water. After mixing, the hemoglobin concentration was approx 3 ItM in heme equlwdents. The reactmn was followed at 420 and 435 nm.
Buffers The buffers employed m the kinetics and equilibrium experiments were all of untform final iomc strength (0.05/). Tris buffers were used m expermaents above pH 7 while Bls-Tris buffers were employed m experiments below pH 7.
RESLILTS A N D D I S ( ' U S S I O N
T h e h e m a t o c r i t s of two Pseudodoras sp. i n d i v i d u a l s of weight 180 a n d 6 4 0 g were 30 a n d 31",, respectively. E l e c t r o p h o r e s i s of three h e m o l y s a t e s revealed only a single h e m o g l o b i n c o m p o n e n t . T h e m i g r a t i o n of the h e m o g l o b i n on disc gels in c o m p a r i s o n with h u m a n h e m o g l o b i n is s h o w n in Fig. 1. After 3 weeks of s t o r a g e at 5~C the m o b i l i t y of the h e m o g l o b i n c o m p o n e n t r e m a i n e d c o n s t a n t a n d no c h a r g e heterogeneity w a s detected. T h e h e m o g l o b i n d o e s not p o l y m e r i z e after treatm e n t with p o t a s s i u m ferricyanide or after storage, a feature r e p o r t e d for tile h e r n o l y s a t c of the teleost Hoplias malaharicus by Reischl (19761. O x i d i z e d a n d reduced h e m o g l o b i n s a m p l e s elutc as single s y m m e t r i cal p e a k s on S e p h a r o s c 4B C h r o m a t o g r a p h y . T h e C O - h e m o g l o b i n h a s an a p p a r e n t m o l e c u l a r weight of 66,000 a c c o r d i n g to the clution posH~on in gel filtration e x p e r i m e n t s . T h e h e m o g l o b i n elutes slightly
The hemoglobin of Pseudodoras
209
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a small Bohr effect. The shape of the binding curve denotes cooperative oxygen binding. Oxygen equilibrium experiments on stripped hemolysates with and without 1 mM ATP (Fig. 3) were performed at 20°C. The p~ at pH 7.6 of stripped hemoglobin is 0.25 mm Hg while that of stripped hemoglobin + 1 mM ATP at pH 7.54 is 0.56 mm Hg. The Bohr effect of stripped hemolysates, Alog p j A p H , appears to be between -0.1 and -0.2. Bunn & Riggs (1979) using an isoelectric focusing technique, have corroborated this result. The Bohr effect, Alog p½/APH, increases to - 0 . 4 in the presence of ! mM ATP between pH 7 and 9 but the value increases to about 1.6 in the range pH 6.5-7.0. The p~ varies 2.5-fold between pH 6.5 and 9.0 in stripped hemolysates and almost 30-fold after ATP additions. As might be expected
band in both gels is bovine serum albumin. The anode is at the bottom of the figure. before the hemoglobin of the spot which is known to be tetrameric (S2o.,, = 4.46S), as determined by sedimentation velocity centrifugation, under the specified column conditions. Tile apparent molecular weight of the denatured hemoglobin chains from Pseudodoras sp. as determined by SDS gel electrophoresis, was 14,300, which is close to that obtained for human chains (14,600). The oxygen equilibrium curve of Pseudodoras blood is presented in Fig. 2. At pH 7.6 and 30°C the red blood cell Pi is 11.0 mm Hg, while under conditions of 5.6% CO2 it shifts to 13.6 mm Hg, indicating
IO
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Fig. 2. Oxygenation curves of Pseudodora.~ blood at pH 7.6, 30 C. Curve I (- - -) and Curve 2 ( reprcsent continuous oxygenation in tile absence and presence of 5.6". CO_,, respectively.
JOSLPH P. MARTIN el al.
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pH Fig. 4. The relatmn between pH and the first order dissooallon constant (k) of Pwudodora~ oxyhemoglobm. Condihons are as described in text. Stripped hemolysate (O), stripped hemol.~sate + 1 mM ATP (O). for a polyanion, ATP exerts a greater effect at lower pH values. The mterchain cooperativity, reflected in n, is generally low and is pH dependent (Fig. 3). At pH 6.1 the n value for stripped hemolysates is less than unity. Between pH 6.7 and 8.6 n attains its maximum value of about 1.7 and therafter declines until at pH 9.0, n = 1.0. The presence of l mM ATP does not cause any consistent increase or decrease in the value of n over the pH range examined. The rate of oxygen dissociation from the hemoglobin is shown as a function of pH, with and without 1 mM ATP, in Fig. 4. The pH dependence observed is that of a hemoglobin with a normal Bohr effect. In the absence of ATP the total variation with pH is 7-fold. I mM ATP increases the rate of this reaction at all pH wdues below 8.2 and this increases the total variation to more than 10-fold. The second order rate constants for the reaction of carbon monoxide with the deoxygenated derivative of this hemoglobin are shown in Fig. 5. again m the i
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presence and abscnce of I mM ATP. This rate constant is strongly pH dependent, increasing as the pH is increased from 6.0 to 8.0 as would be expected for a normal Bohr effect. Addition of 1 mM ATP decreases this reaction rate at all pH values examined but the effect is smallest at the highest pH. Although these kinetic results indicate a normal Bollr effect between pH 6.0 and 8.0, they suggest an acid or reverse Bohr effect below pH 6.0. In the absence of ATP this is manifest primarily in the rate of oxygen dissociation. In the presence of ATP it is seen m both rate constants but the effect in each ts smaller. The rate constants for the reaction with carbon monoxide are most unusual. These are much larger than are normally found for unmodified hemoglobin molecules. The rate observed at pH 8.0 approaches that found for myoglobins and the isolated subunits of human hemoglobin. As stated previously, Pseudodoras sp. differs lrom most fish in possessing a single hemoglobin component. The simplicity of this hemoglobin system may reflect the constancy of the physical environment and the nature of the fish's physiological requirements. Fyhn et al. (1979) noted that hemolysates with a single hemoglobin were more common among fishes from the tipper Amazon River than among fishes from temperate regions. The low variance of certain physical parameters in the Amazon (e.g. water temperature fluctuates less than 2~C yearly) and the unparalleled fish species diversity and habitat specialization in the Amazon basin may partially explain their observations. Pseudodoras hemoglobm chromatographs as a stable tetramer. It neither aggregates under oxidizing conditions nor dissociates into dimers. At 20'C the stripped hemoglobin exhibits a low p~ for oxygen binding (less than 1 mm Hg at all pH values examined) and a very small Bohr effect. Addition of I mM ATP substantially increases the Bohr effect: but the p~ remains relatively low, never exceeding 4.6mm Hg. In contrast, the whole blood at pH 7.6 and 30'C has a p~ of I 1 mm Hg. The significance of the difference between the results obtained with the whole blood and those from the hemoglobin studies cannot be assessed without knowing the temperature dependence of the oxygen affinity of this hemoglobin. The differencc observed may be due entirely to variations in the parameter, or may result from the presence in the red cell of an organic phosphate which is more effective than ATP. lssaacks et al. (1977) have discovered inositol pentaphosphate in the erythrocytes of anothcr Amazon fish species (Arapaima ,qi~jas). Tan & Noble (1973) have demonstrated with inositol hexaphosphate and Torracca et al. (1977) have shown for guanosme triphosphate that these are more effective modulaters of hemoglobin oxygen binding than is ATP in some fish species. The second order rate constant for carbon monoxide association with hcmoglobin corroborates the equilibrium results. It exceeds those known for other fish hemoglobins by nearly an order of magnitude (e.g. carp, Noble et al., 1970: spot, Bonaventura et al., 1976: Myloxsoma sp. Martin et al., 1979) and resembles the values obtained for isolated hemoglobin subunlts and myoglobins.
The hemoglobin of Pseudodora.s Other notable characteristics of P.wudodoras hemoglobin are its small Bohr effect and the absence of a Root effect. Riggs (1970) has pointed out that the magnitude of the Bohr effect in fish hemoglobin is often proportional to the fishes" activity levels. The data on P.selulodora.s hemoglobin are consistent with this generalization in that Pseudodoras is an epibenthlc catfish and, as a siluriform, is generally less active than fish of other taxa. Consequently, a large Bohr effect may not be required to facilitate oxygen unloading at the tissues. Although Pseudodoras possesses a swim bladder its lacks the usually attendant rete mirabile (Bridge & Haddon, 1893), the counter-current multiplier system wherein hemoglobin releases oxygen for entry into the swim bladder. In addition, Pseudodoras lacks a choroid rete (Farmer et al., 1979), the structure responsible for oxygen delivery to the eye The absence of these anatomical features may have prechlded the development of a Root effect hemoglobin. ATP and H + affect both the COo,' and the 0 2.... rates of P.wudodol'as hemoglobin. This is similar to the effects of these agents on other fish hemoglobins. The magnitude of these kinetic changes between pH 5.1 and 8.7 adequately accounts for the nearly 30-fold change in p~ of stripped hemolysates in the presence of 1 mM ATP. Equlhbrium data suggest an acid Bohr effect at low pH similar to that noted in trout 3 hemoglobin (Sahno +jairdnel'i) (Lau et al., 1975). The behavior of the CO,,,, and 02,.,' constants below pH 6.0 in Pseudodoras hemoglobin support the equilibrium data. However, the pH at which the acid Bohr effect is first apparent is higher in the equilibrium than in the kinetic experiments, possibly a consequencc of differences in the ligand binding behavior of CO and 02 The n values, obtained from the analysis of equilibrium data, vary as a function of pH. Maximal cooperatlvity +s attained in the physiological pH range, as in carp hemoglobin (Noble et al., 1970: n decreases above pH 8.6 and below pH 6.7. ATP does not significantly alter this pattern. At pH 6.1 the cooperatlvity drops below unity, perhaps indicating differences in the binding affinities between m- and fl-like chains. In conclusion+ Pseudmloras hemoglobin shares many of the hgand binding characteristics of Root effect hemoglobins (i.e. those of carp+ trout and spot) yet itself lacks a Root effect. Detailed structural comparisons among these hemoglobins may therefore enlighten us with respect to the structural basis of the Root effect in fish hemoglobins. ,4cknowh'dgement.s We wish to thank the officers arid crew of the 41pha Heh\ for thew cooperation and camaraderm during thRs crmse We would also hke to express our thanks to the Brazdzan government for permitting us to work m Amttzon waters. Hans J. Fyhn and Unni E. H. Fyhn acknowledge travel grants from the Norwegmn Research Count21 for Scmncc and the Humanmes. Addittonal support was provided by NSF grant DEB-76-19877 (to D. A Powers). the National Geographic Socmty (to D. A Powers)+ NSF grant PCM-76-06719 and NIH grant GM 21314 {to A. R~ggs) This work was supported m part by the Natmnal Scmnce Foundahon under grant PCM 75-06451 to the Scripps Institute of Oceanography for the support of the ,-llpha Hehx Progran+t, and b~ the National Instmltes of
211
Health grant HL 15460. Joseph P. Marlin is a predoctoral trainee supported by the National Institutes of Health grant GM 07184 to Duke University. Robert W. Noble is an Established Investigator of the American Heart Association.
REFERENCES
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