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Some larval properties of Pipa carvalhoi adult hemoglobins
0200.962979 0401-OX59SO2.000
SOME PlPA
LARVAL PROPERTIES OF CARVALHOI ADULT HEMOGLOBINS NILCE C. MEIRELLES,MARIA LAISE C. VIEIRA, LUCIA P. S. AIROLDI and A. FOCESI JR
Departamento
de Bioquimica, Instituto de Biologia, Universidade Estadual P.O. Box 1170, 13.100 Campinas, SP, Brazil (Receioed
de Campinas
26 May 1978)
Pipa carcalhoi. an anuran from northeast Brazil that lives in an aquatic environment even as an adult. has four hemoglobin components which have been identified by starch-gel electrophoresis and separated by CM-cellulose chromatography. 2. Some structural and functional properties of its hemoglobins were determined and compared with those of terrestrial anurans. The oxygen affinity, Bohr effect and rate of methemoglobin formation in the presence of alkali, urea and sodium benzoate have features similar to those of tadpole hemoglobins already described. 3. The number of reactive thiol groups are 1.552 per tetramer of hemoglobin, in contrast to the hemoglobins of tadpoles. which lack thiol groups.
Abstract-l.
INTRODUCTION Interest in amphibian hemoglobin arose, in part, from the finding by McCutcheon (1936) that tadpole hemoglobin differs from that of the adult frog in its oxygen binding properties. The larval and adult bullfrog (Ram catesbeiana) hemoglobins as well as the mechanism involved in their ontogenetic switch have been extensively studied (Maclean & Jurd, 1972). The Bohr effect behavior for instance, has been considered one of the best defined biochemical differences between the two phases of the animal’s development, although other parameters such as the alkaline denaturation and the thiol groups of the hemoglobin seem also to be different (Hamada et al., 1964). Pipa carvalhoi, an amphibian that lives in northeastern Brazil has an aquatic habitat in its adult phase. In this way, the habitat change between the larval and adult animal, observed in the majority of frogs, does not occur in this species. A study of the structural and functional properties of Pipa carvalhoi hemoglobin may offer the opportunity to investigate the molecular basis of physiological adaptation to the environment. In this paper we report the presence of four hemoglobin components in the Pipa carvalhoi hemolysate and analyse some functional and structural properties of the major component (HbI). These studies, besides their physiological implications, are of interest for the information they may yield on the general problem of structure-function relationships in hemoproteins. MATERIAL AND METHODS Pipa carualhoi of 10-15 g were captured of Bahia, in the northeast of Brazil, and
in the south kept in tanks
* Abbreviations: EDTA, ethylenediamine tetracetic acid: CM-cellulose, carboxy methyl-cellulose; PMB, p-hydroxy mercuribenzoate; ATP, adenosine triphosphate; IHP, inositol-hexaphosphate; DPG, 2,3-diphosphoglycerate; N,N-bis(2-hydroxyethyl)-iminotris (hydroxyBis-Tris, methyl) methane.
containing achlorinated water at 25’C and were fed with common vegetables and chopped beef liver. The bleeding of the animals previously anesthetized by immersion in ice-water at 0°C. was carred out after heparinization (about 0.2 ml of 2 mg/ml heparin by animal). The blood was collected by cardiac puncture and the erythrocytes were separated by centrifugation at 5OOOrev/min in an Eppendorf centrifuge and washed 34 times with lo/, NaCl containing 10e4 M EDTA,* pH 7.2. The packed cells were lysed with 2 vol of 5 x 10m4 M EDTA solution and centrifuged for 10 min at 15.000 rev/min to remove the cell residues. Vertical starch-gel electrophoresis in a discontinuous buffer system (Poulik & Smithies, 1968) was performed in 10-j M EDTA, 10m3 M Tris, 0.3 M borate buffer pH 8.3 for the gel and the same buffer at pH 8.6 for the buffer vessels (Smithies, 1959) and the separated bands of hemoglobin were determined by using the peroxidase properties of the protein. CM-cellulose chromatography (0.64 ml/g) was carried out in a refrigerated glass column (30 x 1 cm). The hemoglobin components were eluted in a linear pH gradient (Maclean & Jurd. 1971) with 0.01 M ohosnhate buffer from . . pH 6.4 to 9. The oxygen binding equilibrium curves at different pHs were obtained by measuring the change in the optical density when deoxygenated hemoglobin was converted to oxyhemoglobin by addition of small increments of air at 25°C according to Rossi-Fanelli & Antonini (1958). The hemoglobin in solution containing 10m3 M EDTA was stripped by passage through the following chromatographic columns: Sephadex G25, Dowex 1 x 4, Dowex 50W x 4 and Amberlite MB 3. (Garlick et al., 1978). Sullhydryl group determination was done by spectrophotometric titration with p-hydroxymercuribenzoate (PMB) according to the method of Boyer (1954). The buffer used was 0.05 M phosphate of pH 7 and the concentration of PMB was 10-s M in both reference and sample cuvettes. The increase of absorbance at 255 nm was determined after each addition of 3.4 x 10-&M hemoglobin solution to the sample cuvette. To study the stability in alkali solution, 0.1 M NaOH solution was added to hemoglobin in 0.1 M phosphate buffer pH 7.0, up to pH 11, at 30°C. The treatment with denaturating agents was performed with 7 M urea or 1 M sodium benzoate solution, at pH 7.0 and 30°C. 859
Tube number Fig. 1. CM-cellulose chromatography and starch-gel electrophoresis (A) of Pip carralhoi The numbers of the peaks correspond to those obtained from the starch-gel electrophoresis to the analysis of individual eluted peaks.
The rate of methemoglobin formation either in alkaline solution. or by using denaturating agents was followed by the decrease in absorbance at 577 nm recorded at intervals of 5 min. The percentage of oxyhemoglobin remaining was then calculated. The extinction coefficient was determined by the method of de Duve (1948). Some denaturation experiments were performed with Rana catesheiana hemoglobin. The frogs were purchased from local commercial supplier and the total hemoglobin solution was obtained as described. RESULTS
Electrophoretic Pipa
and chromatoyraphic
analysis
carcalhoi
starch-gel
hemoglobins were separated by electrophoresis into four components
hemolysate. according
named Hbl, HbII, HbIII and HbIV in order of their increasing anodic mobility. No indication of polymorphism was detected when samples from different individual specimens were submitted to electrophoresis. Figure 1A shows the electrophoretic pattern of the Pipa carcalhoi hemolysate. Ion exchange chromatography on a CM-cellulose column gave the pattern shown in Fig. 1. As in gel electrophoresis, the hemoglobin was separated into four components which were eluted in the following order: HbIII, corresponding to 1”; of the total hemolysate, eluted at pH 6.6; HbIV, lOoi of the hemolysate, eluted at pH 7.0; HbI, 68;,, the major fraction, eluted at pH 7.6 and HbII, representing 20”/,, eluted at pH 8.2. Each purified component was identified by starch-gel electrophoresis and showed a single band corresponding to one of the four bands of the hemolysate. Bohr Effect
Studies of the oxygen equilibria of Pipa caroalhoi hemoglobin were carried out with the total hemolysate and the purified major fraction HbI. The results are shown in Fig. 2, for the total hemolysate and purified HbI fraction, both in the presence and absence of ATP 10m3 M. The Bohr effect for both total and HbI hemoglobins expressed as AlogP,,/ApH was -0.03, whereas for the unfractionated stripped hemoglobin this value was -0.14. The Hill plot shows an increase in n (Hill coefficient) value for all fractions studied when the pH increases. 0 6
I
/
7
8
PH
Fig. 2. The pH dependence of the oxygen affinity of Pip caroalhoi hemoglobins. Unfractionated hemolysate in 0.1 M phosphate buffer (0). stripped in 0.05 M BissTris buffer (a), and in the presence of lo-’ M ATP (A). Component HbI dialysed against 0.05 M Bis-Tris buffer in the absence (0) and presence of 1O-3 M ATP (0).
Sulfnydryl
groups determination
The sulfhydryl content, determined with PMB, was 1.5-2 mol titratable groups/m01 HbI. Rate .of denaturation
and auto.uidation
of Hhl
from
Pipa Figure 3 shows the results of subjecting hemolysate to alkali, urea and sodium
the total benzoate
Some larval
\ 1.0 0
properties
‘.\
\fl\T’ , \I&
I 80
40
Time (mid
Fig. 3. Denaturation of Pipa carualhoi hemoglobins in alkali solution (0), in 7 M urea (0) and 1 M sodium benzoate (A). The data obtained for Rana catesbeiana adult (R) or larval (T) hemoglobins are represented in dashed lines: R, the adult hemoglobin treated by alkaline solution, Rz by 7 M urea and R, by sodium benzoate. For Rana catesbeiana tadpole, the curve T, represents the hemoglobin treated by alkaline solution, T, by 7 M urea and T3 by I M sodium benzoate.
action; stable
Pipa
carvalhoi
hemoglobins
are
relatively
in the presence either of alkali or denaturing agents such as urea or sodium benzoate. DISCUSSION
Pipa carwlhoi, an aquatic amphibian of the Pipidae family, shows four hemoglobin fractions both by electrophoresis and chromatographic analysis. One of them, named HbIII, represents less than 1% of the total hemolysate. Although the hemolysate had been usually treated by either 2-mercaptoethanol or EDTA, the polymerization that seems to be high in other forms like Xenopus leuuis (Maclean & Jurd, 1971) might be responsible for the presence of the minor component. However, the same analysis performed in individual specimens of Pipa carualhoi showed essentially the same result both for the number and proportions of the components. In any case, the functional properties of the total hemolysate must reflect primarily those of the major component since it comprises at least 60% of the total hemoglobin. The high oxygen affinity of tadpole hemoglobin from Rana catesbeiana is quite similar (Ps,, z 4-5 mm Hg) at pH 7.3 and 20°C whereas for adults the oxygen affinity is much lower (P,, z 13mm Hg) under the same conditions (Riggs, 1951). The value of 6.0mm Hg at pH 7.4, found for Pipa carvalhoi hemoglobin is quite close to values reported for tadpole hemoglobin. The Bohr effect of the HbI in the presence of inorganic phosphate or after stripping away the allosteric effecters and using BissTris as buffer seems to be of same degree and close to zero (-0.03). It is interesting to note that from the stand-
of adult
hemoglobins
861
point of the Bohr effect, the adult Pipa hemoglobin behaves like the tadpole hemoglobin of amphibian species studied (McCarthy, 1936; Riggs, 1951). The finding by Riggs (1951) of a Bohr coefficient of -0.28 for the adult Rana catesbeiana and -0.10 for the tadpole are consistent with this assertion. The Hill coefficient, commonly interpreted as reflecting hemeeheme interaction or cooperativity of oxygen binding (Antonini & Brunori, 1971) was found to be 2.5 and increases with pH. Concerning the effect of organic phosphate on bullfrog and tadpole hemoglobins, Araki et al. (1971) have found that the tadpole hemolysate in the presence of ATP, IHP or DPG has a greatly decreased oxygen affinity. Recently, Watt & Riggs (1975) reported that tadpole hemoglobin from Rana catesbeina has a reversed Bohr effect between pH 6 and 9, which disappears completely in the presence of 0.05 M phosphate, 0.1 M NaCl and 1 mM EDTA. For the Pipa carualhoi HbI we found in the stripped protein a small increase in the Bohr effect ( -0.14). However, the treatment of HbI with 1 mM ATP caused a normal Bohr effect (-0.46) as occurs with the bullfrog tadpole hemoglobin. Another difference between larval and adult frog hemoglobins seems to be the resistance to denaturating agents (Hamada & Shukuya, 1966). Our results compared to those found with Rana catesbeiana adult hemoglobin (Hamada et al., 1964) indicate a low rate of methemoglobin formation when the protein is subjected to denaturation agents. Nevertheless, such a resistance to oxidation is not so remarkable as that found for Rana catesbeiana tadpole hemoglobin. Finally, our findings suggest a similarity between the hemoglobins of adult Pipu carrulhoi and those of the tadpole of Rum catesbeiuna. Considering that the different hemoglobin properties described for several animal species may be related to the habitat, it is possible to suggest that the larval properties presented by Pipu carvalhoi adult hemoglobins may be due to the aquatic habitat of the frog even after metamorphosis. Acknowledgemenrs-- The authors gratefully acknowledge the help of Professor Austen Riggs in stimulating discussion during an Alpha He/ix expedition on the Amazon River (Phase IV, 1976). This study was supported in part by grant from the FAPESP (Proc, 7611338). M.L.C.V. was supported by postgraduate fellowship from CAPES/U. F. Ceara.
REFERENCES
ANTONINIE. & BRUNORIM. (1971) Hemoylohin und Myoglobin in their Reactions with Ligands, p. 164. North-Holland, Amsterdam. ARAKI T.. KAJITA A. & SHUKUYA R. (1971) The effect of organic phosphates on the allosteric property of Rana catesheiana hemoglobins. Biochem. hioph?s. Res. Comman. 43, 117991185. BOYERP. D. (1954) Spectrophotometric study of the reaction of protein sulfhydryl groups with organic mercurials. J. Am. them. Sot. 16, 4331-4337. DE DUVE C. (1948) A spectrophotometric method for the simultaneous determination of myoglobin and hemoglobin in extracts of human muscle. Actu them. wand. 2, 264-289.
GARLICK L. G., BI’NN H. F.. FYHN H. J.. FYI1N u. E. H.. MARTIN J. P.. NOBLE R. W. & POWERS D. A. (1978) Functional studies on the separated hemoglobm components of an air breathing catfish. Hoplostcwuv~ littorrrlr (Hancock). C‘omp, Biochern. P/z~..si~~l.III press. HAMADA K.. SAKAI J.. SHIIKI’YA R. & KAZIRO K. (1964) Biochemical metamorphosis of hemoglobin in ROWI c~c~trshriuncr-I. Purification procedures and properties of hemoglobins from bullfrog and tadpole erythrocytes. .I Biochrm. 536). 636 642. HAMADA K. & SHLIK~NYA R. (1966) Biochemical metamorphosis of hemoglobin in Rcrmr c,trrc.sheiuntr II. Further studies on the structure and properties of tadpole and frog hemoglobins. .I. Biochrm. 59. 397~~403. MAUIAN N. & JL~RI) R. D. (19711 The haemoglobins of healthy and anaemic Xvnop~\ /trt,r~\. J. Cell. SC,;. 9, 509m 52x. MACLEAN N. & JURU R. D. (1972) The control of haemoglobin synthesis. Biol. RN. 47(3). 393 437
M(CI.TCHFON F. H. (1936) Hemoglobin function durlnp the life story of the bullfrog. .I. Ct,//. corny. P/ty\io/ 8. 63-X1. POULIK M. D. & SMITHIES. 0. (195X1 Comparison and cornblnation of the starch-gel and filter-paper electrophoretlc methods applied to human sera: two dtmensional electrophoresis. Biochrvn. J. 68, 636 643 RIGGS A. (lY511 The metamorphosis of hcmoglobln in the bullfrog. J. yen. Phv\io/. 3S( I). 3 JO. ROSSI-FANFLLI A. & ACTONINI b. (19581 Studies on the oxygen and carbon monoxide equilibria of human myoglobin. Arc,hs Bioc~hon. Bi~~ph~~.\.77. 47X-49?. S~II~HIES 0. (1959) An improved procedure for ctarch-gel clcctrophoresis: further \artation\ in the serum proteins of normal individuals. B~oclrcrv. .1. 71, 5X5 5X7 WATT K. W. K. & RIG(.? A. (19751 Hemoglobms of the tadpole of the bullfrog. Rlrmr curr.d~citrmr. Structure and function of isolated components. ./. hiol. C/wm. 250(151, 5934 5944.
SOME PlPA
LARVAL PROPERTIES OF CARVALHOI ADULT HEMOGLOBINS NILCE C. MEIRELLES,MARIA LAISE C. VIEIRA, LUCIA P. S. AIROLDI and A. FOCESI JR
Departamento
de Bioquimica, Instituto de Biologia, Universidade Estadual P.O. Box 1170, 13.100 Campinas, SP, Brazil (Receioed
de Campinas
26 May 1978)
Pipa carcalhoi. an anuran from northeast Brazil that lives in an aquatic environment even as an adult. has four hemoglobin components which have been identified by starch-gel electrophoresis and separated by CM-cellulose chromatography. 2. Some structural and functional properties of its hemoglobins were determined and compared with those of terrestrial anurans. The oxygen affinity, Bohr effect and rate of methemoglobin formation in the presence of alkali, urea and sodium benzoate have features similar to those of tadpole hemoglobins already described. 3. The number of reactive thiol groups are 1.552 per tetramer of hemoglobin, in contrast to the hemoglobins of tadpoles. which lack thiol groups.
Abstract-l.
INTRODUCTION Interest in amphibian hemoglobin arose, in part, from the finding by McCutcheon (1936) that tadpole hemoglobin differs from that of the adult frog in its oxygen binding properties. The larval and adult bullfrog (Ram catesbeiana) hemoglobins as well as the mechanism involved in their ontogenetic switch have been extensively studied (Maclean & Jurd, 1972). The Bohr effect behavior for instance, has been considered one of the best defined biochemical differences between the two phases of the animal’s development, although other parameters such as the alkaline denaturation and the thiol groups of the hemoglobin seem also to be different (Hamada et al., 1964). Pipa carvalhoi, an amphibian that lives in northeastern Brazil has an aquatic habitat in its adult phase. In this way, the habitat change between the larval and adult animal, observed in the majority of frogs, does not occur in this species. A study of the structural and functional properties of Pipa carvalhoi hemoglobin may offer the opportunity to investigate the molecular basis of physiological adaptation to the environment. In this paper we report the presence of four hemoglobin components in the Pipa carvalhoi hemolysate and analyse some functional and structural properties of the major component (HbI). These studies, besides their physiological implications, are of interest for the information they may yield on the general problem of structure-function relationships in hemoproteins. MATERIAL AND METHODS Pipa carualhoi of 10-15 g were captured of Bahia, in the northeast of Brazil, and
in the south kept in tanks
* Abbreviations: EDTA, ethylenediamine tetracetic acid: CM-cellulose, carboxy methyl-cellulose; PMB, p-hydroxy mercuribenzoate; ATP, adenosine triphosphate; IHP, inositol-hexaphosphate; DPG, 2,3-diphosphoglycerate; N,N-bis(2-hydroxyethyl)-iminotris (hydroxyBis-Tris, methyl) methane.
containing achlorinated water at 25’C and were fed with common vegetables and chopped beef liver. The bleeding of the animals previously anesthetized by immersion in ice-water at 0°C. was carred out after heparinization (about 0.2 ml of 2 mg/ml heparin by animal). The blood was collected by cardiac puncture and the erythrocytes were separated by centrifugation at 5OOOrev/min in an Eppendorf centrifuge and washed 34 times with lo/, NaCl containing 10e4 M EDTA,* pH 7.2. The packed cells were lysed with 2 vol of 5 x 10m4 M EDTA solution and centrifuged for 10 min at 15.000 rev/min to remove the cell residues. Vertical starch-gel electrophoresis in a discontinuous buffer system (Poulik & Smithies, 1968) was performed in 10-j M EDTA, 10m3 M Tris, 0.3 M borate buffer pH 8.3 for the gel and the same buffer at pH 8.6 for the buffer vessels (Smithies, 1959) and the separated bands of hemoglobin were determined by using the peroxidase properties of the protein. CM-cellulose chromatography (0.64 ml/g) was carried out in a refrigerated glass column (30 x 1 cm). The hemoglobin components were eluted in a linear pH gradient (Maclean & Jurd. 1971) with 0.01 M ohosnhate buffer from . . pH 6.4 to 9. The oxygen binding equilibrium curves at different pHs were obtained by measuring the change in the optical density when deoxygenated hemoglobin was converted to oxyhemoglobin by addition of small increments of air at 25°C according to Rossi-Fanelli & Antonini (1958). The hemoglobin in solution containing 10m3 M EDTA was stripped by passage through the following chromatographic columns: Sephadex G25, Dowex 1 x 4, Dowex 50W x 4 and Amberlite MB 3. (Garlick et al., 1978). Sullhydryl group determination was done by spectrophotometric titration with p-hydroxymercuribenzoate (PMB) according to the method of Boyer (1954). The buffer used was 0.05 M phosphate of pH 7 and the concentration of PMB was 10-s M in both reference and sample cuvettes. The increase of absorbance at 255 nm was determined after each addition of 3.4 x 10-&M hemoglobin solution to the sample cuvette. To study the stability in alkali solution, 0.1 M NaOH solution was added to hemoglobin in 0.1 M phosphate buffer pH 7.0, up to pH 11, at 30°C. The treatment with denaturating agents was performed with 7 M urea or 1 M sodium benzoate solution, at pH 7.0 and 30°C. 859
Tube number Fig. 1. CM-cellulose chromatography and starch-gel electrophoresis (A) of Pip carralhoi The numbers of the peaks correspond to those obtained from the starch-gel electrophoresis to the analysis of individual eluted peaks.
The rate of methemoglobin formation either in alkaline solution. or by using denaturating agents was followed by the decrease in absorbance at 577 nm recorded at intervals of 5 min. The percentage of oxyhemoglobin remaining was then calculated. The extinction coefficient was determined by the method of de Duve (1948). Some denaturation experiments were performed with Rana catesheiana hemoglobin. The frogs were purchased from local commercial supplier and the total hemoglobin solution was obtained as described. RESULTS
Electrophoretic Pipa
and chromatoyraphic
analysis
carcalhoi
starch-gel
hemoglobins were separated by electrophoresis into four components
hemolysate. according
named Hbl, HbII, HbIII and HbIV in order of their increasing anodic mobility. No indication of polymorphism was detected when samples from different individual specimens were submitted to electrophoresis. Figure 1A shows the electrophoretic pattern of the Pipa carcalhoi hemolysate. Ion exchange chromatography on a CM-cellulose column gave the pattern shown in Fig. 1. As in gel electrophoresis, the hemoglobin was separated into four components which were eluted in the following order: HbIII, corresponding to 1”; of the total hemolysate, eluted at pH 6.6; HbIV, lOoi of the hemolysate, eluted at pH 7.0; HbI, 68;,, the major fraction, eluted at pH 7.6 and HbII, representing 20”/,, eluted at pH 8.2. Each purified component was identified by starch-gel electrophoresis and showed a single band corresponding to one of the four bands of the hemolysate. Bohr Effect
Studies of the oxygen equilibria of Pipa caroalhoi hemoglobin were carried out with the total hemolysate and the purified major fraction HbI. The results are shown in Fig. 2, for the total hemolysate and purified HbI fraction, both in the presence and absence of ATP 10m3 M. The Bohr effect for both total and HbI hemoglobins expressed as AlogP,,/ApH was -0.03, whereas for the unfractionated stripped hemoglobin this value was -0.14. The Hill plot shows an increase in n (Hill coefficient) value for all fractions studied when the pH increases. 0 6
I
/
7
8
PH
Fig. 2. The pH dependence of the oxygen affinity of Pip caroalhoi hemoglobins. Unfractionated hemolysate in 0.1 M phosphate buffer (0). stripped in 0.05 M BissTris buffer (a), and in the presence of lo-’ M ATP (A). Component HbI dialysed against 0.05 M Bis-Tris buffer in the absence (0) and presence of 1O-3 M ATP (0).
Sulfnydryl
groups determination
The sulfhydryl content, determined with PMB, was 1.5-2 mol titratable groups/m01 HbI. Rate .of denaturation
and auto.uidation
of Hhl
from
Pipa Figure 3 shows the results of subjecting hemolysate to alkali, urea and sodium
the total benzoate
Some larval
\ 1.0 0
properties
‘.\
\fl\T’ , \I&
I 80
40
Time (mid
Fig. 3. Denaturation of Pipa carualhoi hemoglobins in alkali solution (0), in 7 M urea (0) and 1 M sodium benzoate (A). The data obtained for Rana catesbeiana adult (R) or larval (T) hemoglobins are represented in dashed lines: R, the adult hemoglobin treated by alkaline solution, Rz by 7 M urea and R, by sodium benzoate. For Rana catesbeiana tadpole, the curve T, represents the hemoglobin treated by alkaline solution, T, by 7 M urea and T3 by I M sodium benzoate.
action; stable
Pipa
carvalhoi
hemoglobins
are
relatively
in the presence either of alkali or denaturing agents such as urea or sodium benzoate. DISCUSSION
Pipa carwlhoi, an aquatic amphibian of the Pipidae family, shows four hemoglobin fractions both by electrophoresis and chromatographic analysis. One of them, named HbIII, represents less than 1% of the total hemolysate. Although the hemolysate had been usually treated by either 2-mercaptoethanol or EDTA, the polymerization that seems to be high in other forms like Xenopus leuuis (Maclean & Jurd, 1971) might be responsible for the presence of the minor component. However, the same analysis performed in individual specimens of Pipa carualhoi showed essentially the same result both for the number and proportions of the components. In any case, the functional properties of the total hemolysate must reflect primarily those of the major component since it comprises at least 60% of the total hemoglobin. The high oxygen affinity of tadpole hemoglobin from Rana catesbeiana is quite similar (Ps,, z 4-5 mm Hg) at pH 7.3 and 20°C whereas for adults the oxygen affinity is much lower (P,, z 13mm Hg) under the same conditions (Riggs, 1951). The value of 6.0mm Hg at pH 7.4, found for Pipa carvalhoi hemoglobin is quite close to values reported for tadpole hemoglobin. The Bohr effect of the HbI in the presence of inorganic phosphate or after stripping away the allosteric effecters and using BissTris as buffer seems to be of same degree and close to zero (-0.03). It is interesting to note that from the stand-
of adult
hemoglobins
861
point of the Bohr effect, the adult Pipa hemoglobin behaves like the tadpole hemoglobin of amphibian species studied (McCarthy, 1936; Riggs, 1951). The finding by Riggs (1951) of a Bohr coefficient of -0.28 for the adult Rana catesbeiana and -0.10 for the tadpole are consistent with this assertion. The Hill coefficient, commonly interpreted as reflecting hemeeheme interaction or cooperativity of oxygen binding (Antonini & Brunori, 1971) was found to be 2.5 and increases with pH. Concerning the effect of organic phosphate on bullfrog and tadpole hemoglobins, Araki et al. (1971) have found that the tadpole hemolysate in the presence of ATP, IHP or DPG has a greatly decreased oxygen affinity. Recently, Watt & Riggs (1975) reported that tadpole hemoglobin from Rana catesbeina has a reversed Bohr effect between pH 6 and 9, which disappears completely in the presence of 0.05 M phosphate, 0.1 M NaCl and 1 mM EDTA. For the Pipa carualhoi HbI we found in the stripped protein a small increase in the Bohr effect ( -0.14). However, the treatment of HbI with 1 mM ATP caused a normal Bohr effect (-0.46) as occurs with the bullfrog tadpole hemoglobin. Another difference between larval and adult frog hemoglobins seems to be the resistance to denaturating agents (Hamada & Shukuya, 1966). Our results compared to those found with Rana catesbeiana adult hemoglobin (Hamada et al., 1964) indicate a low rate of methemoglobin formation when the protein is subjected to denaturation agents. Nevertheless, such a resistance to oxidation is not so remarkable as that found for Rana catesbeiana tadpole hemoglobin. Finally, our findings suggest a similarity between the hemoglobins of adult Pipu carrulhoi and those of the tadpole of Rum catesbeiuna. Considering that the different hemoglobin properties described for several animal species may be related to the habitat, it is possible to suggest that the larval properties presented by Pipu carvalhoi adult hemoglobins may be due to the aquatic habitat of the frog even after metamorphosis. Acknowledgemenrs-- The authors gratefully acknowledge the help of Professor Austen Riggs in stimulating discussion during an Alpha He/ix expedition on the Amazon River (Phase IV, 1976). This study was supported in part by grant from the FAPESP (Proc, 7611338). M.L.C.V. was supported by postgraduate fellowship from CAPES/U. F. Ceara.
REFERENCES
ANTONINIE. & BRUNORIM. (1971) Hemoylohin und Myoglobin in their Reactions with Ligands, p. 164. North-Holland, Amsterdam. ARAKI T.. KAJITA A. & SHUKUYA R. (1971) The effect of organic phosphates on the allosteric property of Rana catesheiana hemoglobins. Biochem. hioph?s. Res. Comman. 43, 117991185. BOYERP. D. (1954) Spectrophotometric study of the reaction of protein sulfhydryl groups with organic mercurials. J. Am. them. Sot. 16, 4331-4337. DE DUVE C. (1948) A spectrophotometric method for the simultaneous determination of myoglobin and hemoglobin in extracts of human muscle. Actu them. wand. 2, 264-289.
GARLICK L. G., BI’NN H. F.. FYHN H. J.. FYI1N u. E. H.. MARTIN J. P.. NOBLE R. W. & POWERS D. A. (1978) Functional studies on the separated hemoglobm components of an air breathing catfish. Hoplostcwuv~ littorrrlr (Hancock). C‘omp, Biochern. P/z~..si~~l.III press. HAMADA K.. SAKAI J.. SHIIKI’YA R. & KAZIRO K. (1964) Biochemical metamorphosis of hemoglobin in ROWI c~c~trshriuncr-I. Purification procedures and properties of hemoglobins from bullfrog and tadpole erythrocytes. .I Biochrm. 536). 636 642. HAMADA K. & SHLIK~NYA R. (1966) Biochemical metamorphosis of hemoglobin in Rcrmr c,trrc.sheiuntr II. Further studies on the structure and properties of tadpole and frog hemoglobins. .I. Biochrm. 59. 397~~403. MAUIAN N. & JL~RI) R. D. (19711 The haemoglobins of healthy and anaemic Xvnop~\ /trt,r~\. J. Cell. SC,;. 9, 509m 52x. MACLEAN N. & JURU R. D. (1972) The control of haemoglobin synthesis. Biol. RN. 47(3). 393 437
M(CI.TCHFON F. H. (1936) Hemoglobin function durlnp the life story of the bullfrog. .I. Ct,//. corny. P/ty\io/ 8. 63-X1. POULIK M. D. & SMITHIES. 0. (195X1 Comparison and cornblnation of the starch-gel and filter-paper electrophoretlc methods applied to human sera: two dtmensional electrophoresis. Biochrvn. J. 68, 636 643 RIGGS A. (lY511 The metamorphosis of hcmoglobln in the bullfrog. J. yen. Phv\io/. 3S( I). 3 JO. ROSSI-FANFLLI A. & ACTONINI b. (19581 Studies on the oxygen and carbon monoxide equilibria of human myoglobin. Arc,hs Bioc~hon. Bi~~ph~~.\.77. 47X-49?. S~II~HIES 0. (1959) An improved procedure for ctarch-gel clcctrophoresis: further \artation\ in the serum proteins of normal individuals. B~oclrcrv. .1. 71, 5X5 5X7 WATT K. W. K. & RIG(.? A. (19751 Hemoglobms of the tadpole of the bullfrog. Rlrmr curr.d~citrmr. Structure and function of isolated components. ./. hiol. C/wm. 250(151, 5934 5944.