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Oxygen equilibrium studies of the radular muscle myoglobins of the gastropod molluscs, buccinum undatum L. and Bustcon canaliculatum L.
Znt.3. Biochm.,
1971,
OXYGEN MUSCLE
2,
253-261.
[Scientechnica
EQUILIBRIUM
MYOGLOBINS
BUCCIJVUM UNDATUM ROBERT
(Publishers)
STUDIES
Ltd.]
253
OF THE RADULAR
OF THE GASTROPOD
MOLLUSCS,
L. AND BUSYC0.N CAJVALICULATUM
C. TERWILLIGER*
AND
KENNETH
L.
R. H. READ
Department of Biology, Boston University, Boston, Mass. 022 I 5, and New England Aquarium, Boston, Mass. 021 IO, U.S.A. (Received 20 May, 1970) ABSTRACT I. The radular muscle myoglobin of Buccinum undutum is dimeric in the carbonmonoxy-, oxy-, and deoxy- forms and has a sedimentation coefficient of approximately 2.7. 2. The radular muscle myoglobin of Busycon canaliculatum is dimeric in the carbonmonoxy- and oxy- forms and has a sedimentation coefficient of approximately 3.1. 3. Purified Buccinum myoglobin combines with oxygen according to a pH-independent sigmoid dissociation curve with n equal to 1.4 and P,, of 13.0 mm. Hg at 20’ C. 4. Purified Bugcon myoglobin combines with oxygen according to a slightly sigmoid dissociation curve with n equal to 1.2 and P,, of approximately 0.7 mm. Hg at 20’ C. 5. The oxygen afBnity and sigmoid nature of the oxygen dissociation curve of Buccinum myoglobin are not affected by the concentration of the myoglobin or by the presence of 2,3_diphosphoglycerate, a-glycerophosphate, lactate, adenosine triphosphate, or phosphocreatine. 6. An increase in the concentration of sodium chloride in the buffer from 0.1 M to 2-o M does not affect the sigmoid nature of the dissociation curve of Buccinum myoglobin but does decrease the value of P,, at 25’ C. from 16.3 mm. Hg to 13.2 mm. Hg. 7. The overall heat of oxygenation (AH) is -8.4 Cal. per mole for Buccinum myoglobin and -8.8 Cal. Per mole for Busycon myoglobin. 8. The oxygen affmities of Buccinum and Busycon myoglobins are greater than the oxygen affinities of their respective haemocyanins. g. The oxygenation and structural properties of Buccinum and Busycon myoglobins are compared with one another and with vertebrate haemoglobins and myoglobins.
THE radular muscle myoglobins of gastropod molluscs so far investigated are either monomeric or dimeric (Rossi-Fanelli, Antonini, and Povoledo, 1958; Read, I g66a, b, 1967, 1968; Tentori, Vivaldi, Carta, Antonini, and Brunori, I 968 ; Terwilliger and Read, 1969, rg7ob; Koppenheffer and Read, 1970; Koppenheffer, Terwilliger, and Read, I 97 I). The monomeric buccal muscle myoglobins of the opisthobranch molluscs A&siu depilans and A. limacina exhibitpH-independent hyperbolic oxygen equilibrium curves characteristic of the monomeric vertebrate myoglobins l Present address: Oregon Institute of Marine Biology, University of Oregon, Charleston, Oregon 97420, U.S.A.
(Rossi-Fanelli and others, 1958). Studies of the oxygen equilibria of dimeric molluscan myoglobins are limited to those performed on the radular muscle myoglobin of Fwitriton oregoncnsis (Manwell, I g6oa; Terwilliger and Read, 1g7ob). Manwell reported that this pigment combines with oxygen according to a pH-independent, slightly sigmoid oxygen equilibrium curve. The radular muscle myoglobins of the gastropod mollusc, Buccinum undatum (Terwilliger and Read, 1g6g) and Busycon cunealtiulatum (Read, 1g66b; Johnson, Koppenheffer, and Read, 1970) are homogeneous dimeric pigments with apparently identical subunits. This paper describes the equilibria of these myoglobins with oxygen.
254
TERWILLlGER
MATERIALS AND METHODS Buccinum udatum L. was obtained from the Marine Biological and Development Corporation, Brunswick, Maine, and Busycoo canaliculatum L. from the supply department, Marine Biological Laboratory, Woods Hole, Mass. The radular muscles were removed from the animals, homogenized with a mortar, pestle, and sand in O-I M-sodium phosphate b&r, pki 7.4, 0.1 M in NaCl, and the thick homoaenate centrifuged at I 7,& r.p.m. The pellet w& re-extracted twice with the sodium c~o~de~ph~phate buffer and the resulting su~rna~n~ pooled. The myoglobin solution was brought successively to 40 and 60 per cent ammonium sulphate concentration, centrifuged, and the precipitate discarded. The myoglobin was precipitated by bringing the ammonium sulphate concentration to 85 per cent; it was then spun down and the pellet containing the myoglobin redissolved in the extraction buffer. Unless specifically stated otherwise, the myoglobin was isolated in the oxy- form. The myoglobin was then chromatographed on a column of Sephadex G-75 (Superfine) in equilibrium with the extraction buffer (Terwilliger and Read, ~$9). The fraction containing the myoglobin was used for the determination of the oxygenation curves of the pigment. If a change of buffer was to be effected, the mvosdobin was passed through a short coh&n of S&p&&x G-25 in eouihbrium with the desired buffer. The entirepreparation could be carried out in less than 24 hours and was performed in a coid room at about 4” C. 2,3Diphosphoglyceric acid (2,3-DPG), obtained from Sigma Chemical Co. as the pentacyclohexylammonium salt, was converted to the hydrogen form by passage. through a column containing
AND READ
ht.
J_ 3~oe~~.
carried out gently in an ice-bath and frothing of the myoglobin solution is avoided, there is only slight denaturation of the protein with complete deoxygenation attained in about 2o-30 minutes. The amount of oxygen combined with the myoglobin was determined spectrophotometrically with a Zeiss PMQ II spcctrophotometer equipped with a constant temperaturecell holder. Readings were taken at 540, 560, and 580 nm. for dilute solutions and at 600, 670, and 760 nm. for more concentrated solutions; the averages of the values obtained for per cent oxygenation were used for const~cting the oxygen diiation curve. The partial pressure of oxygen was calculated as stated in Benesch and others (1965). Haemocyanin was colIected from the animals by cutting the foot and allowing the blood to collect in a cooled beaker. The fluid was centrifuged at I 7,000 r.p.m. to remove suspended materials and studied immediately. The method of determining the oxygen equilibrium of the haemocyanins was similar to that used for the myoglobins except that readings were taken at 345 nm. for the dilute and 585 nm. for the more concentrated samples. RESULTS Wmxm
M~LEGuLAR
The c~bonmonoxyand oxy- forms of ~~~~n~ myoglobin chromatograph on Sephadex G-75 (Superfine) as molecules with an apparent molecular weight of 34,000. At low Table I.-SEDIMENTATIONCOEFFICXENTS Buccinum MYOGLOBIN AT 20’ C. I ANIMAL
Buccinum Bu~i~um Busycon
BZISpfl
1
I
I
No.
CONDITION
OF
DETERMlNATIONS
GxY Deoxy CXY Deoxy
OF
; zi
t
I
S,o+. (Svedberg units) 2.65 &o-o* 2.70 io-03 3*r3fo*og 3’34
Buffer o-t M sodium phosphate, pH 7.4, O-I M in NaCl. Myoglobm concentration approximately 005 per cent. * Errors expressed as standard errors of the mean. concentrations of myoglobin, the apparent molecular weight of both forms is unchanged. The sedimentation coefficients of the carbonmonoxy-, oxy-, and deoxy-myoglobins (buffer 0.1 iEf sodium phosphate, $-I 7.4, O-I M in NaCI, 20’ C.) were about 2-7 (lab& f). The oxy- and carbonmonoxyforms of Busycon myoglobin also chromatograph on
OXYGEN
1971, 2
AFFINITIES
OF MOLLUSC
Sephadex G-75 (Superfine) with an apparent molecular weight of 34,000, a value which is unaffected by dilution of the pigment. The sedimentation coefficients for the oxy- and carbonmonoxy-Busycon myoglobins, purified in the same way as Buccinam myoglobin, are about 3-1. Because Busycon myoglobin has such a high affinity for oxygen, it was difficult to keep it in the deoxy-form during the centrifusration; thus the sedimentation coefficient of- Busycwz deoxymyoglobin could not be accurately determined. Preliminary studies,
MYOGLOBINS
255
slightly sigmoid (n= I ‘4) as shown in Fig. I. The effect of hydrogen ion concentration on the oxygen equilibrium of this pigment in 0.05 M Tris buffer at 25’ C. is shown in the same figure. AtpH 7*4,&o, and 8.7, there is no significant difference in the oxygen affinity of the pigment. The slight variability in position of the curves is probably the result of manipulation of the myoglobin when the PH was being adjusted. At lower values of pH (6.0-6.5) the myoglobin is slowly denatured and accurate measure-
05 -
7 a
b
_I
o-
-IS-
I
I 05
03 I
I
’ O LogPO> 1.5
,O
FIG. I .-The effect of&H on the oxygen equilibrium of Buccinum myoglobin. Myoglobin concentration 5.0 x 10-6 M. Experiment at pH 7’4 was carried out in 0.1 M sodium phosphate buffer, 0.1 M in NaCl and 0.05 M Tris-HCI, 0.1 M in NaCI. At pH 87, 8.0 tbe buffer was 0.05 M Tris-HCl, 0.1 M in NaCl. Temperature, 26’ C. 0, PH 7.4; A, pH 8.0; 0, pH 8.7.
in which there was some detectable deoxymyoglobin in the cell after centrifugation, indicate that the deoxymyoglobin of Busycon probably also retains a dimeric form since the mixture of oxy- and deoxy-myoglobins appeared homogeneous in the ultracentrifuge with a sedimentation coefficient of 3.3. OXYGEN
EQUILIBRIUM
The oxygen dissociation curve of Buccinum myoglobin was consistently found to be
I I.0
I
LogPal
I.5
I PO
FIG. z-The effect of sodium chloride concentration on the oxygen equilibrium of Buccinum myoglobin. Myoglobin concentration 7.5 x 10-5 M. Buffer 0.1 M sodium phosphate, PH 7.4. Temperature, 25’ C. A, 2 M NaCl; 0, I M NaCl; A, 0.1 M NaCl. ments of the dissociation curves become difficult. The experiments at pH 7-4, carried out in phosphate or Tris buffers, showed no differences in the oxygen dissociation curves. Studies of the effect of ionic strength on the oxygenation properties ofBuccinum myoglobin
were performed by increasing the sodium chloride content of a myoglobin solution in 0.1 M sodium phosphate buffer, fiH 7.4. As illustrated in Fig. 2, an increase in sodium chloride concentration from 0.1 M to 2.0 M does not appear to affect the slope of the curve but does result in a slight displacement of the curve to the left representing a
TERWILLtCIER
256
change in P,, of 16.3 mm. Hg to 13.2 mm. Hg at 25’ C. The oxygen dissociation curves of Budnum myoglobin were carried out at different concentrations of pigment from 3 x IO-~ M to 1*8x IO-~ M; the concentration of myoglobin in the muscles is probably not much greater than the latter value. Fig. 3 indicates that the slope and position of the curves are not noticeably affected by the concentration of the myoglobin.
i5
0
I
I'0
I
AND READ
ht.
J.
~ioc~~.
Calculation of the overall heat of reaction (AH) by the van? Hoff equation from the slope of the line in Fig. 5 gave a value of -8.4 Cal. per mole. Identical experiments were performed on Busycon radular muscle myoglobin. Thii pigment, however, has a higher affinity for oxygen than Buccinum myoglobin, and the protein appears to be much more labile. Rep~du~ble values for the P,, of Barsycon myoglobin of about 0’7 mm. Hg at 20” C.
I
2.0 LogPO? I’S FIG. 5.-Effect of myoglobin concentration on the oxygen equilibrium of Buccinum myogiobin. A, 3 x 10-s M; 0, 7.5 x IO-~ M; 0, 1.8 x ro-’ M, Buffer o’ I M sodium phosphate, pH 7.4, o*I M in NaCl. Temperature, 25OC.
Fro. 4.--The effect of tcmnerature on the OXYgen eqiilibrium curves of B&hum u&turn my&globin. Buffer 0.1 M sodhs~ phosphatcsodium chloride, #I-Z7.4. Myoglobin 75 x so-5 M.
The effect of the molecules 2,3-DPC, ~-glyceroph~p~te, lactate, ATP, and phosphocreatine on the oxygen equilibrium of Bwinum myoglobin were studied; the concentration of these molecules was 50 times that of the haem. Hill plots of the results were all collinear with the control; there was no change in oxygen affinity or sigmoidicity in the presence of any of these molecules. The effect of temperature on the oxygenation of Buccinum myoglobin is shown in Fig. 4. The sigmoidicity of the curve is not affected by lowering the temperature whereas the oxygen affinity is increased. Fig. 5 shows that between IO and 30~ C. a linear relationship exists between log K and I IT.
were obtained but the Avalues, always slightly greater than 1.1, were quite variable. Fig. 6 shows a typical curve for Busycon myoglobin. The results of the experiments on Buptm myoglobin showed in general that the oxygenation properties of this molecule were similar to those obtained for the Buccinum pigment except for the higher oxygen affinity and usually lower sigmoidicity. Of the former molecule, however, less confidence is placed in the data. There is a linear relationship between log K and r/T for Busycon myoglobin; a value for AH of -8-8 Cal. per mole was calculated (Fig. 6). A comparison of the relative oxygen affinities of the radular muscIe myoglobin
‘97’,2
OXYGEN
AFFINITIES
OF MOLLUSC
and the circulating haemocyanin was carried out for both species. Fig. 7 indicates that the oxygen affinities of the myoglobins of both species are greater than the oxygen affinities of their respective haemocyanins.
257
MYOGLOBINS
latter, which was calculated from its aminoacid composition (Read, 1g66b; Terwilliger and Read, 1969). This may be the result of the presence of some metmyoglobin monomer in the Buhwm samples as has been found
(“K-‘)
of log. K (X= I/P,,) versus I/T for But&urn myoglobin.
FIG. S.--Graph
I/T
(‘K-‘)
FIG. 6.-Plot of log. K versus I/T for Busycon myoglobin. Buffer characteristics and myoglobin concentration same as in Fig. 4 for Bwxinum myoglobin. -1.0
DISCUSSION Values obtained for the sedimentation coefficients (S,,,) for both Buctinum and Busycon myoglobins (Table Z) are consistent with a molecular weight in the neighbourhood of 3o,ooo-35,000 for the two proteins. The values for Btucinum myoglobin are consistently lower than for the Bqvcon pigment despite the lower molecular weight of the
-1.0
0
Log PO,
I.0
2.0
FIG. 7.-The oxygen dissociation curves of Buccinum and Busycon myoglobins and haemocyanins at 20’ C. A, Busycon myoglobin; 1 Burycon haemocyanin; 0, But&urn myoglobin; 0, Buccinum haemoeyanins. previously (Terwilliger and Read, 1969) or may in some way reflect the instability of the Busycon myoglobin; we are at a loss as
258
TERWILLIGER
AND READ
ht. J. Biochem.
to how to explain this observation at the n value of the dissociation curve was greater for myoglobin which had not been present. Values of Ss,,, for the oxy-, carbonmonoxy-, manipulated during its isolation from the and deoxy-forms of Buccinum myoglobin muscle. This property which may be true foe indicate that the molecular weight in all Busycon myoglobin could explain the variathese ligand states is the same. Therefore, it tion in n value which has been observed for appears that the pigment is a dimer in the this protein in our study. The dimeric deoxy-form or when combined with carbon haemoglobins of the holothurians Cucumaria monoxide or oxygen. Buccinum myoglobin is miniata and Molpadia oiilitica show sigmoid unlike lamprey (Petromyxon marinw) haemodissociation curves with n values of I *3- I ~6 globin which aggregates upon deoxygenation (Manwell, 1959; Terwilliger and Read, (Briehl, 1963). Although Buccinum myo1970~; Terwilliger, 1970). These values globin can be cleaved with p-hydroxymeragree well with the values of n found for curibenzoate into monomeric subunits which B~c~nurn my~lobin. On the other hand, the can be recombined to form a dimer (Terdimeric haemoglobin of the insect ~~~op~~l~ williger and Read, xg6g), dissociation of shows a hyperbolic dissociation curve (n= I) dimer to monomer does not seem to occur as similar to that of the monomeric myoglobins a result of deoxygenation. In the oxy- and of vertebrates (Keilin and Wang, I 946 ; Rossicarbonmonoxy-forms, the molecular weight of Fanelli and Antonini, 1938). The sigmoid oxygen equilibrium curves of the radular Buccinum myoglobin, determined by Sephadex muscle myoglobins of the chitons Cvptochiton chromatography, is not dependent on concentration of the pigment; thus, Buccinum stelleri, Katharina tunicata, and khnochiton cons&cuus must be re-examined in light of the myoglobin is dissimilar to human haemogiobin in that it does not appear to exist in a recent observation that both monomeric and dimeric pigments occur in Amphineuran state of rapid association-dissociation equiradular muscle tissue (Manwell, I g38a, librium (Guidotti, 1964). Preliminary data rg6oa; Terwilliger and Read, rg7ob). indicate that the same conclusions are valid The oxygen dissociation curve of Buccinum for ~usycon myoglobin. myoglobin is not affected by changes of the The oxygen equilibrium curve of Buccinum hydrogen ion concentration over a physiomyoglobin is sigmoid under all conditions logical range of pH; in this respect Buccinum examined with n equal to I -4. Sigmoid dissociation curves are indicative of systems myoglobin resembles vertebrate myoglobins but differs from human haemoglobins. The composed of multiple interacting subunits. It therefore appears that the two subunits of dimeric haemoglobins of Cucumaria miniata and Fusitriton oregonensis also exhibit a PHthis dimeric myoglohin do not combine with independent oxygen dissociation curve (Manoxygen independently of one another. well, xg6oa, b). However, the dimeric and Buycon myoglobin exhibits a slightly monomeric haemoglobins of the insect Chironsigmoid dissociation curve (n > I *I). Athough emus th~rnrn~exhibit a pH-dependent oxygen there was some variability in the exact value affinity (Sick and Gersonde, 1969). of n in 0~ experiments, it was consistently At low values of pH (6.0 and lower) I-I or greater. This observation is also in accord with the dimeric nature of Busycon Bnccinum myoglobin is denatured. This is true for most haemoglobins that have been myoglobin. investigated. The reversibility of the acid Sigmoid oxygen equilibrium curves associdenaturation of Buccinum myoglobin as comated with dimeric haemoglobins have been pared with that of human haemoglobin has reported for several invertebrates. The not yet been investigated. dimeric radular muscle myoglobin of the Increasing sodium chloride concentration gastropod Fusitriton oregonensis shows a sigmoid dissociation curve with a value of R enhances the oxygen affinity of Buccimtm myoglobin yet does not appear to affect the equal to I ‘3 (Manwell, 1g6oa; Terwilliger shape of the oxygen dissociation curve. The and Read, 1g7ob). Manwelf reported that
197’,2
OXYGEN
AFFINITIES
OF MOLLUSC
oxygen dissociation curves of vertebrate haemoglobin, on the other hand, show a decrease in oxygen affinity of the pigment with an increase in salt concentration and an increase in sigmoidicity (Rossi-Fanelli, Antonini, and Caputo, x96x ; Antonini, Wyman, Brunori, Bucci, Fronticelli, and Rossi-Fanelli, 1968). The oxygen dissociation curve of human myoglobin is unaffected by an increase in sodium chloride concentration (Rossi-Fanelli and Antonini, r 958). Chlorocruorin, however, like Buccinum myoglobin, shows an increase in oxygen affinity with an increase in ionic strength of the buffer (Antonini, Rossi-Fanelli, and Caputo, I 962). An explanation for this effect is not clear to us at present. The presence of 2,8-DPG, phosphocreatine, lactate, ATP, or a-glycerophosphate in fifty-fold molar excess per haem has a negligible effect on the dissociation curve of Bwcinum myoglobin. In this respect Btucinum myoglobin differs from human haemoglobin the oxygen affinity of which is lowered by 2,8-DPG or ATP (Benesch and Benesch, I g6g). The affinity of Buccinum myoglobin for oxygen is unaffected by the concentration of the pigment over the range of concentrations examined in our study; this is also true for vertebrate myoglobin (Rossi-Fanelli and Antonini, 1958). On the other hand, the dependence of the oxygen equilibrium on the concentration of the protein is significant for human haemoglobin (Rossi-Fanelli and others, 1961) and is quite pronounced for lamprey haemoglobins (Wald and Riggs, I g5 I ; Briehl, I 968). The concentration effect is an indication of intermolecular interactions and has been attributed to the fact that human haemoglobin is in a state of association-dissociation equilibrium (Guidotti, 1964). The lack of a concentration effect for Buccinum myoglobin, as well as the fact that its apparent molecular weight does not change with dilution on Sephadex, indicates that the association constant of this molecule is probably quite high. The values obtained for the overall heat of oxygenation, AI-I, of haemoglobins are quite variable (Rossi-Fanelli, Antonini, and Caputo, 1964). The AH values for human
MYOCLOBINS
259
haemoglobin and myoglobin are - 13.0 and - 13.1 Cal. per mole respectively (Wyman, I 948 ; Rossi-Fanelli and Antonini, I 858). Snake haemoglobin has been reported to have a AH value as high as - 15’5 Cal. per mole (Sullivan, 1967)) although Roughton, Otis, and Lyster (1955) reported a value of -8.2 Cal. per mole for sheep haemoglobin and Manwell (195813) found values of -8.8 and -9.8 Cal. per mole for adult and foetal spiny dogfish haemoglobins. Rossi-Fanelli and Antonini (1960) reported a remarkably low AH for the haemoglobin of the tuna fish Thunnus of - I *8 Cal. per mole. The dimeric haemoglobin of Cucumaria miniata has a heat of oxygenation of -8.4 Cal. per mole which is similar to that found for both Buccinum and Busycon myoglobins (Manwell, r859). Both the molluscan myoglobins and Cucumaria pigments consist of homogeneous systems and molecules in which the AH values are not contributed to by linked groups such as in the Bohr effect; therefore, these AH values, which include the heat of solution of oxygen, probably represent the true value for heat of oxygenation. The consistency of the AH values of these molluscan myoglobins with those found for other haemoglobins strongly suggests that the molecules were not denatured by the extraction procedure and that the oxygenation properties which have been measured are those of the native protein. Furthermore, the similarity of the heat of oxygenation of these radular muscle myoglobins with those of vertebrate haemoglobins suggests that the molluscan myoglobins do indeed function -as pigments which combine directly with oxygen in the intact organism. There have been many hypotheses to explain the process by which haemoglobin combines with oxygen according to a sigmoid dissociation curve. Many hypotheses stress the importance of the a, 8 dimer as the functional subunit (see review by Antonini, 1967; Guidotti, 1967). Recently, the primary role of the a, f.3dimer in the associationdissociation equilibrium as well as its being the origin of co-operativity of the tetrameric molecule has received additional support (Anderson, Antonini, Brunori, and Wyman, I 870). A study of the a, /3 dimer is complicated
260
TERWILLIGERAND RWD
by the presence of the Bohr effect which is dependent on both a and j3 chains in the dimeric array. The forces that hold the subunits of Buccinum myoglobin together as a dimer seem to be relatively strong yet not covalent; in this respect Buccinum dimer is similar to the a, p dimer of vertebrate haem~lobin. The oxy~nation curve of B~ci~ myoglobin also exhibits si~oidici~ which is presumably an inherent property of its dimeric structure as is also thought to be true for the associating of a, p dimers of tetramerit vertebrate haemoglobin. A preliminary comparison of the relative oxygen affinities of the radular muscle myoglobins and the circulating haemocyanins of both Buccinum and Burycon indicates that the myoglobin should facilitate the diffusion of oxygen from the circulating haemolymph to the radular muscle tissues. However, the data in Fig. 7 should be treated with some caution as the Bohr effect for the haemocyanins was not investigated. It is, however., unlikely that the Bohr effect would be very great (Redfield, I 934). Our conclusions relative to the role of myoglobin in facilitating the transport of oxygen from circulating haemocyanin to the tissues are thus consistent with those of Manwell ( Ig58a, I g6ob).
ht. J. Btichm.
ANTONINI, E., WY.~, J., BRUNORI,M., Buccx, E., FRONTXELLI,C., and RO~~I-FANELL~, A. (x963), ’ Studies on the relations between moleCular and functional properties of Hb. IV. The Bohr effect in human Hb measured by proton binding *, 3. biol. Chem., 938, 2gp-zg57. BENESCH, R., and BENESCH, R. E. (rg6g), ‘ Intracellular organic phosphate-s as regulators of ~~g~~~2~ by hemoglobin ‘, Jvature,Land.,
BEN&H, R., MACDUFF,G,, and BENESCH, R. E. (19651, ‘ Determination of oxygen equilibria
with a versatile new tonometer ‘, Anayt. Biochem., XI, 81-87. BENESCH, R. E., BENESCN, R., and C. I. Yu (I&Q, ‘ The oxygenation of hemoglobin in the presence of z,3-~ph~phoglycerate. Effect of temperature, PH, ionic strength and hemoglobin concentration ‘, Biochcmishy, N.?“., 6, 2567-2571. BRLEHL,R. W. (1~6s), ‘ The relation between the oxygen equilib&& and aggregation of subunits in lamprey hemoglobin ‘, 3, biol. Chenz., ~9~ 2361-2366. GUIDOTTI.G. fxaftal. ‘ Studies on the dissociation of h&an higo&bm ‘, in Strwture arrd Actiuitr of Enzymes(ed. &XX&, ‘I’. TN., HARRI, J. 11, and HARTLEY. B. S.1. London and New York: Academic P&s. ’ GUI~OTII,G. ( Ig67), ‘ Studies on the chemistry of the hemoglobin ‘, J. Biool. C&m., q~, 3672-3720. JOHNSON,J. P., KOPPENHEFFER,T. L., and READ, K. R. H. (rg7o), unpubiish~ data. KEILIN, D., and WANG, Y. L. (x94.6), ‘ Haemoglobins of Gastro@kilw larvae. Purification and properties ‘, Biach. J., 40,855-866. KOPPEN~IEFFJZR, T. L., and READ,K. R. H. (rg7o), ACKNOWLEDGEMENTS ’ The myoglobin of the gastropod mollusc We wish to thank Dr. Henry A. DePhillips, Littorina iittorea L. ‘, ht. J. Biwh., I, 457-464. Trinity College! and Dr. Frank A. Belamarich, KOPPENHEFFER, ‘I’. I;., TERWILLIGER,R. C., and Boston Universrty, for assistance and the use of READ, K. R. H. (x971 ), ‘ Myoglobin of the laboratory space at the Marine Biological Laboragastropod mollusc Lunatia keros Say ‘, Int. J. tories, Woods Hole, Mass., and Mr. L. R. Hyde Biochem., 5 I I I--I 16. for help with the ultracentrifuge. Tbii work was MANWELL, C. (Ig58a), ‘ The oxygen-respiratory aided by Grant No. 943-F from the Massachusetts pigment equilibrium of the hemocyanin and Heart Association and was also supported by mvwlobin of the amohineuran mollusc, CrvbktU.S.P.H.S. Research Grant HE 10565. && sklti ‘, 3. cell. &z& P~ysiol., 9,34ILj52_ MANWELL. C. iras8b). ‘A “ fetal maternal shift *’ in the ovb&ip&ous spiny dogfish Sqwfur REFERENCES swkleyi ‘, Physiol, z&l., 3x, 93-100. MANWELL, C. ( Igsg), ‘ Oxygen equilibrium of ANDERSON, N. M.,ANTONINI,E., BRUNORI, M., and Cucumaria mini&a hemoglobin and the absence WYMAN, J, (‘g70), ‘ Equilibrium of human of the Bohr effect ‘? J. cell. corn&Pkysiol., 53, 75with ethylisocyanide: further hemoglobin evidence for cooperativity in hemoglobin 83. MANWELL,C. (xg6oa), ’ Heme-heme interactions dimers ‘, 3i rno~~~~~~., p205-2 13: in the oxygen equilibrium of some invertebrate Hemoglobm and its ANT~NINI, . myoglobins ‘, Archs Biockem. Biopkys,, 8g, 1g4reaction with ligands*‘, SC&Z, Jy.2”., 158, I 4 I 7201. MAN~ELL, C. (rg6ob), ‘ Histologicai specificity of AN%&I E ROWI-FANELLIA and CAPUTO A. respiratory pigments. I. Comparison of the Studies on Chlbr$ruorin. I. ‘?he (*o6*1.’ ‘ .’ coelom and muscle hemoglobins of the polyoxygen equilibrium of Spirogra@kiscklorocruorin‘, chaete worm Travikiapapa and the echiuroid h&S %d%7n. &O$p, 97, 336-342.
1971, 1
261
OXYGENAFFIMTIESOF MOLLUSCMYOGLOBINS
worm, Arhyhite pugetrensis ‘, Camp. B&hem. Physiol., I, 267-276. READ, K. R. H. (1g66a), ‘ Molluscan hemoglobin and myoglobin ‘, in Physiology of Mollusca (ed. WILBUR, K. M., and YONGE, C. M.), vol. II, pp. 209-232. New York: Academic Press. READ, K. R. H. (Ig66b), ‘ The characterization of radular muscle myoglobins from the gastroloci mollusc Busvcon canaliculatum L. ‘., Comb. II biochem. Physiol., ;7, 375-390. READ, K. R. H. (1g67), ‘ The radular muscle myoglobin of the gastropod mollusc Busron car&urnGmelin ‘, Camp.Bi~~rn. Physiol., 22, z-13. READ, K. R. H. (1g68), ‘ The myoglobins of the gastropod molluscs Buqcon cont?arium Conrad, Lunatia heros Say, Littorina littorea L. and Siphonaria gigas Sowerby ‘, Co@. Biochem. Physiol., ~3, 81-94. REDFIELD,A. C. (rg34), ’ The haemocyanins ‘, Biol. Rev., 9, I 75-2 I 2. ROSSI-FANELLI,A., and ANTONINI, E. (tq58), ’ Studies on. the -oxygen and carbon mo&i& equilibria of human myoglobin ‘, Archs Biochem. Biophys., 7fr 478-492. ROSSI-FANELLX, A., and ANTONINI, E. (Ig6o), ‘ Oxygen equilibrium of hemoglobin from Thunnus thunni ‘, Nature, L5nd., 796, 8g5. ROSSI-FANELLS, A., ANTONINK, E., and CAPVTO,A. (196x1, ‘Studies on the relations between molecular and functional properties of hemoglobin. 1. The effect of salts on the oxygen equilibrium of human hemoglobin ‘, -7. biol. Ch.9 034 397-400. ROSSI-FANELLI, A., ANTONINI,E., and CAPUTO,A. ‘ Hemoglobin and myoglobin ‘, Adv. (tg6& PYO&itlchnn., ‘9, 73-222. ROSSI-FANELLI. A., ANTONINI. E., and POVOLEDO. D. (~g.$), ‘ F&&er study. on myoglobin. ‘II. Chemical and biochemical urooerties of a new type of myoglobin in moll&cs’ ‘, in Sjmposium on Protein Structure (ZUPAC) (ed. NEWBERGER, A.), pp. 144-147. London: Methuen. ROUGHTON,F. J. W., Ons, A. B., and LY~TER, R. L. J. (x955), ‘ The determination of the individual equilibrium constants of the four
intermediate reactions between oxygen and sheep hemoglobin ‘, Froc. R. Sot. B, 117~29-54. SICK, H., and GERSONDE,K. (IgSg), ‘ Die O,Bindung seigenschaften einiger larval Hamoglobine von Chronomus rh. thummi ‘, Eur. 3. Biochem., 7, 273-279. SULLIVAN,B. (rg67), ‘ Oxygenation properties of snake hemoglobin ‘, Science, N.Y., 157, 13081210. TE&&u, L., VIVALDI, G., CARTA, S., ANTONINI, E., and BRUNORI,M. ( 1968), ’ Amino acid composition of Ap&wia myoglobin ‘, Nature, Land., 21% 487. * TERWILLI&,* R. (I g7o), unpublished data. TERWILLIGER.R., and READ, K. R. H. l1o6o). , ’ Quaternah s&ucture of ihe radular muscle myoglobin of Bwcinum un&tum L. ‘, Corn& B&hem. Physiol., 31, 55-64. TERWILLIGER,R., and READ, K. R. H. (Ig7oa), ‘ The radular muscle myoglobins of the amphineuran molluscs, Katharina tunicataWood, Cry$ochiton stelleri Middendorf, and Mopalii muscosa Gould ‘, ht. J. &o&m., X, 281-291, TERWILLIGER,R., and READ, K. R. H. (lg7ob), ‘ The radular muscle myoglobins of the gastropod molluscs, Acnuua tes&dinalis s&urn Eschscholtz, Haliotis karnt~~~~~ Jones, Tegda f~~alis Adams, Fusitriton wegonensis Redfield, and Tfrois~~115sa Gmelin ‘, Int. 3. Bide., I, I
_r_
TE%%?%IXR, R., and READ, K. R. H. (rg7oc) ‘ The hemoglobins of the holothuroid echinol derms, &c&aria miniada Brandt, Cucuma& pi’ah Stimpson and Molpadia intermedia Ludwig ‘, Co@. Biochem. Physioi., 36, 33g-351. WALD, G., and RIGGS, A. (rgg~), ’ The hemoglobin of the sea lamprey, Petromyson marinw ‘, J. gen. Physiol., 35, 45-53. WYMAN,J. (x9,+8), ’ Heme proteins ‘, Adv. Protin Q=m., ch 407-53 J. Key Wwd Ina%x: Myoglobins, dimers, oxygen affinity of myoglobin, Bqwon ca~l~at~, Butcinum Saturn.
1971,
OXYGEN MUSCLE
2,
253-261.
[Scientechnica
EQUILIBRIUM
MYOGLOBINS
BUCCIJVUM UNDATUM ROBERT
(Publishers)
STUDIES
Ltd.]
253
OF THE RADULAR
OF THE GASTROPOD
MOLLUSCS,
L. AND BUSYC0.N CAJVALICULATUM
C. TERWILLIGER*
AND
KENNETH
L.
R. H. READ
Department of Biology, Boston University, Boston, Mass. 022 I 5, and New England Aquarium, Boston, Mass. 021 IO, U.S.A. (Received 20 May, 1970) ABSTRACT I. The radular muscle myoglobin of Buccinum undutum is dimeric in the carbonmonoxy-, oxy-, and deoxy- forms and has a sedimentation coefficient of approximately 2.7. 2. The radular muscle myoglobin of Busycon canaliculatum is dimeric in the carbonmonoxy- and oxy- forms and has a sedimentation coefficient of approximately 3.1. 3. Purified Buccinum myoglobin combines with oxygen according to a pH-independent sigmoid dissociation curve with n equal to 1.4 and P,, of 13.0 mm. Hg at 20’ C. 4. Purified Bugcon myoglobin combines with oxygen according to a slightly sigmoid dissociation curve with n equal to 1.2 and P,, of approximately 0.7 mm. Hg at 20’ C. 5. The oxygen afBnity and sigmoid nature of the oxygen dissociation curve of Buccinum myoglobin are not affected by the concentration of the myoglobin or by the presence of 2,3_diphosphoglycerate, a-glycerophosphate, lactate, adenosine triphosphate, or phosphocreatine. 6. An increase in the concentration of sodium chloride in the buffer from 0.1 M to 2-o M does not affect the sigmoid nature of the dissociation curve of Buccinum myoglobin but does decrease the value of P,, at 25’ C. from 16.3 mm. Hg to 13.2 mm. Hg. 7. The overall heat of oxygenation (AH) is -8.4 Cal. per mole for Buccinum myoglobin and -8.8 Cal. Per mole for Busycon myoglobin. 8. The oxygen affmities of Buccinum and Busycon myoglobins are greater than the oxygen affinities of their respective haemocyanins. g. The oxygenation and structural properties of Buccinum and Busycon myoglobins are compared with one another and with vertebrate haemoglobins and myoglobins.
THE radular muscle myoglobins of gastropod molluscs so far investigated are either monomeric or dimeric (Rossi-Fanelli, Antonini, and Povoledo, 1958; Read, I g66a, b, 1967, 1968; Tentori, Vivaldi, Carta, Antonini, and Brunori, I 968 ; Terwilliger and Read, 1969, rg7ob; Koppenheffer and Read, 1970; Koppenheffer, Terwilliger, and Read, I 97 I). The monomeric buccal muscle myoglobins of the opisthobranch molluscs A&siu depilans and A. limacina exhibitpH-independent hyperbolic oxygen equilibrium curves characteristic of the monomeric vertebrate myoglobins l Present address: Oregon Institute of Marine Biology, University of Oregon, Charleston, Oregon 97420, U.S.A.
(Rossi-Fanelli and others, 1958). Studies of the oxygen equilibria of dimeric molluscan myoglobins are limited to those performed on the radular muscle myoglobin of Fwitriton oregoncnsis (Manwell, I g6oa; Terwilliger and Read, 1g7ob). Manwell reported that this pigment combines with oxygen according to a pH-independent, slightly sigmoid oxygen equilibrium curve. The radular muscle myoglobins of the gastropod mollusc, Buccinum undatum (Terwilliger and Read, 1g6g) and Busycon cunealtiulatum (Read, 1g66b; Johnson, Koppenheffer, and Read, 1970) are homogeneous dimeric pigments with apparently identical subunits. This paper describes the equilibria of these myoglobins with oxygen.
254
TERWILLlGER
MATERIALS AND METHODS Buccinum udatum L. was obtained from the Marine Biological and Development Corporation, Brunswick, Maine, and Busycoo canaliculatum L. from the supply department, Marine Biological Laboratory, Woods Hole, Mass. The radular muscles were removed from the animals, homogenized with a mortar, pestle, and sand in O-I M-sodium phosphate b&r, pki 7.4, 0.1 M in NaCl, and the thick homoaenate centrifuged at I 7,& r.p.m. The pellet w& re-extracted twice with the sodium c~o~de~ph~phate buffer and the resulting su~rna~n~ pooled. The myoglobin solution was brought successively to 40 and 60 per cent ammonium sulphate concentration, centrifuged, and the precipitate discarded. The myoglobin was precipitated by bringing the ammonium sulphate concentration to 85 per cent; it was then spun down and the pellet containing the myoglobin redissolved in the extraction buffer. Unless specifically stated otherwise, the myoglobin was isolated in the oxy- form. The myoglobin was then chromatographed on a column of Sephadex G-75 (Superfine) in equilibrium with the extraction buffer (Terwilliger and Read, ~$9). The fraction containing the myoglobin was used for the determination of the oxygenation curves of the pigment. If a change of buffer was to be effected, the mvosdobin was passed through a short coh&n of S&p&&x G-25 in eouihbrium with the desired buffer. The entirepreparation could be carried out in less than 24 hours and was performed in a coid room at about 4” C. 2,3Diphosphoglyceric acid (2,3-DPG), obtained from Sigma Chemical Co. as the pentacyclohexylammonium salt, was converted to the hydrogen form by passage. through a column containing
AND READ
ht.
J_ 3~oe~~.
carried out gently in an ice-bath and frothing of the myoglobin solution is avoided, there is only slight denaturation of the protein with complete deoxygenation attained in about 2o-30 minutes. The amount of oxygen combined with the myoglobin was determined spectrophotometrically with a Zeiss PMQ II spcctrophotometer equipped with a constant temperaturecell holder. Readings were taken at 540, 560, and 580 nm. for dilute solutions and at 600, 670, and 760 nm. for more concentrated solutions; the averages of the values obtained for per cent oxygenation were used for const~cting the oxygen diiation curve. The partial pressure of oxygen was calculated as stated in Benesch and others (1965). Haemocyanin was colIected from the animals by cutting the foot and allowing the blood to collect in a cooled beaker. The fluid was centrifuged at I 7,000 r.p.m. to remove suspended materials and studied immediately. The method of determining the oxygen equilibrium of the haemocyanins was similar to that used for the myoglobins except that readings were taken at 345 nm. for the dilute and 585 nm. for the more concentrated samples. RESULTS Wmxm
M~LEGuLAR
The c~bonmonoxyand oxy- forms of ~~~~n~ myoglobin chromatograph on Sephadex G-75 (Superfine) as molecules with an apparent molecular weight of 34,000. At low Table I.-SEDIMENTATIONCOEFFICXENTS Buccinum MYOGLOBIN AT 20’ C. I ANIMAL
Buccinum Bu~i~um Busycon
BZISpfl
1
I
I
No.
CONDITION
OF
DETERMlNATIONS
GxY Deoxy CXY Deoxy
OF
; zi
t
I
S,o+. (Svedberg units) 2.65 &o-o* 2.70 io-03 3*r3fo*og 3’34
Buffer o-t M sodium phosphate, pH 7.4, O-I M in NaCl. Myoglobm concentration approximately 005 per cent. * Errors expressed as standard errors of the mean. concentrations of myoglobin, the apparent molecular weight of both forms is unchanged. The sedimentation coefficients of the carbonmonoxy-, oxy-, and deoxy-myoglobins (buffer 0.1 iEf sodium phosphate, $-I 7.4, O-I M in NaCI, 20’ C.) were about 2-7 (lab& f). The oxy- and carbonmonoxyforms of Busycon myoglobin also chromatograph on
OXYGEN
1971, 2
AFFINITIES
OF MOLLUSC
Sephadex G-75 (Superfine) with an apparent molecular weight of 34,000, a value which is unaffected by dilution of the pigment. The sedimentation coefficients for the oxy- and carbonmonoxy-Busycon myoglobins, purified in the same way as Buccinam myoglobin, are about 3-1. Because Busycon myoglobin has such a high affinity for oxygen, it was difficult to keep it in the deoxy-form during the centrifusration; thus the sedimentation coefficient of- Busycwz deoxymyoglobin could not be accurately determined. Preliminary studies,
MYOGLOBINS
255
slightly sigmoid (n= I ‘4) as shown in Fig. I. The effect of hydrogen ion concentration on the oxygen equilibrium of this pigment in 0.05 M Tris buffer at 25’ C. is shown in the same figure. AtpH 7*4,&o, and 8.7, there is no significant difference in the oxygen affinity of the pigment. The slight variability in position of the curves is probably the result of manipulation of the myoglobin when the PH was being adjusted. At lower values of pH (6.0-6.5) the myoglobin is slowly denatured and accurate measure-
05 -
7 a
b
_I
o-
-IS-
I
I 05
03 I
I
’ O LogPO> 1.5
,O
FIG. I .-The effect of&H on the oxygen equilibrium of Buccinum myoglobin. Myoglobin concentration 5.0 x 10-6 M. Experiment at pH 7’4 was carried out in 0.1 M sodium phosphate buffer, 0.1 M in NaCl and 0.05 M Tris-HCI, 0.1 M in NaCI. At pH 87, 8.0 tbe buffer was 0.05 M Tris-HCl, 0.1 M in NaCl. Temperature, 26’ C. 0, PH 7.4; A, pH 8.0; 0, pH 8.7.
in which there was some detectable deoxymyoglobin in the cell after centrifugation, indicate that the deoxymyoglobin of Busycon probably also retains a dimeric form since the mixture of oxy- and deoxy-myoglobins appeared homogeneous in the ultracentrifuge with a sedimentation coefficient of 3.3. OXYGEN
EQUILIBRIUM
The oxygen dissociation curve of Buccinum myoglobin was consistently found to be
I I.0
I
LogPal
I.5
I PO
FIG. z-The effect of sodium chloride concentration on the oxygen equilibrium of Buccinum myoglobin. Myoglobin concentration 7.5 x 10-5 M. Buffer 0.1 M sodium phosphate, PH 7.4. Temperature, 25’ C. A, 2 M NaCl; 0, I M NaCl; A, 0.1 M NaCl. ments of the dissociation curves become difficult. The experiments at pH 7-4, carried out in phosphate or Tris buffers, showed no differences in the oxygen dissociation curves. Studies of the effect of ionic strength on the oxygenation properties ofBuccinum myoglobin
were performed by increasing the sodium chloride content of a myoglobin solution in 0.1 M sodium phosphate buffer, fiH 7.4. As illustrated in Fig. 2, an increase in sodium chloride concentration from 0.1 M to 2.0 M does not appear to affect the slope of the curve but does result in a slight displacement of the curve to the left representing a
TERWILLtCIER
256
change in P,, of 16.3 mm. Hg to 13.2 mm. Hg at 25’ C. The oxygen dissociation curves of Budnum myoglobin were carried out at different concentrations of pigment from 3 x IO-~ M to 1*8x IO-~ M; the concentration of myoglobin in the muscles is probably not much greater than the latter value. Fig. 3 indicates that the slope and position of the curves are not noticeably affected by the concentration of the myoglobin.
i5
0
I
I'0
I
AND READ
ht.
J.
~ioc~~.
Calculation of the overall heat of reaction (AH) by the van? Hoff equation from the slope of the line in Fig. 5 gave a value of -8.4 Cal. per mole. Identical experiments were performed on Busycon radular muscle myoglobin. Thii pigment, however, has a higher affinity for oxygen than Buccinum myoglobin, and the protein appears to be much more labile. Rep~du~ble values for the P,, of Barsycon myoglobin of about 0’7 mm. Hg at 20” C.
I
2.0 LogPO? I’S FIG. 5.-Effect of myoglobin concentration on the oxygen equilibrium of Buccinum myogiobin. A, 3 x 10-s M; 0, 7.5 x IO-~ M; 0, 1.8 x ro-’ M, Buffer o’ I M sodium phosphate, pH 7.4, o*I M in NaCl. Temperature, 25OC.
Fro. 4.--The effect of tcmnerature on the OXYgen eqiilibrium curves of B&hum u&turn my&globin. Buffer 0.1 M sodhs~ phosphatcsodium chloride, #I-Z7.4. Myoglobin 75 x so-5 M.
The effect of the molecules 2,3-DPC, ~-glyceroph~p~te, lactate, ATP, and phosphocreatine on the oxygen equilibrium of Bwinum myoglobin were studied; the concentration of these molecules was 50 times that of the haem. Hill plots of the results were all collinear with the control; there was no change in oxygen affinity or sigmoidicity in the presence of any of these molecules. The effect of temperature on the oxygenation of Buccinum myoglobin is shown in Fig. 4. The sigmoidicity of the curve is not affected by lowering the temperature whereas the oxygen affinity is increased. Fig. 5 shows that between IO and 30~ C. a linear relationship exists between log K and I IT.
were obtained but the Avalues, always slightly greater than 1.1, were quite variable. Fig. 6 shows a typical curve for Busycon myoglobin. The results of the experiments on Buptm myoglobin showed in general that the oxygenation properties of this molecule were similar to those obtained for the Buccinum pigment except for the higher oxygen affinity and usually lower sigmoidicity. Of the former molecule, however, less confidence is placed in the data. There is a linear relationship between log K and r/T for Busycon myoglobin; a value for AH of -8-8 Cal. per mole was calculated (Fig. 6). A comparison of the relative oxygen affinities of the radular muscIe myoglobin
‘97’,2
OXYGEN
AFFINITIES
OF MOLLUSC
and the circulating haemocyanin was carried out for both species. Fig. 7 indicates that the oxygen affinities of the myoglobins of both species are greater than the oxygen affinities of their respective haemocyanins.
257
MYOGLOBINS
latter, which was calculated from its aminoacid composition (Read, 1g66b; Terwilliger and Read, 1969). This may be the result of the presence of some metmyoglobin monomer in the Buhwm samples as has been found
(“K-‘)
of log. K (X= I/P,,) versus I/T for But&urn myoglobin.
FIG. S.--Graph
I/T
(‘K-‘)
FIG. 6.-Plot of log. K versus I/T for Busycon myoglobin. Buffer characteristics and myoglobin concentration same as in Fig. 4 for Bwxinum myoglobin. -1.0
DISCUSSION Values obtained for the sedimentation coefficients (S,,,) for both Buctinum and Busycon myoglobins (Table Z) are consistent with a molecular weight in the neighbourhood of 3o,ooo-35,000 for the two proteins. The values for Btucinum myoglobin are consistently lower than for the Bqvcon pigment despite the lower molecular weight of the
-1.0
0
Log PO,
I.0
2.0
FIG. 7.-The oxygen dissociation curves of Buccinum and Busycon myoglobins and haemocyanins at 20’ C. A, Busycon myoglobin; 1 Burycon haemocyanin; 0, But&urn myoglobin; 0, Buccinum haemoeyanins. previously (Terwilliger and Read, 1969) or may in some way reflect the instability of the Busycon myoglobin; we are at a loss as
258
TERWILLIGER
AND READ
ht. J. Biochem.
to how to explain this observation at the n value of the dissociation curve was greater for myoglobin which had not been present. Values of Ss,,, for the oxy-, carbonmonoxy-, manipulated during its isolation from the and deoxy-forms of Buccinum myoglobin muscle. This property which may be true foe indicate that the molecular weight in all Busycon myoglobin could explain the variathese ligand states is the same. Therefore, it tion in n value which has been observed for appears that the pigment is a dimer in the this protein in our study. The dimeric deoxy-form or when combined with carbon haemoglobins of the holothurians Cucumaria monoxide or oxygen. Buccinum myoglobin is miniata and Molpadia oiilitica show sigmoid unlike lamprey (Petromyxon marinw) haemodissociation curves with n values of I *3- I ~6 globin which aggregates upon deoxygenation (Manwell, 1959; Terwilliger and Read, (Briehl, 1963). Although Buccinum myo1970~; Terwilliger, 1970). These values globin can be cleaved with p-hydroxymeragree well with the values of n found for curibenzoate into monomeric subunits which B~c~nurn my~lobin. On the other hand, the can be recombined to form a dimer (Terdimeric haemoglobin of the insect ~~~op~~l~ williger and Read, xg6g), dissociation of shows a hyperbolic dissociation curve (n= I) dimer to monomer does not seem to occur as similar to that of the monomeric myoglobins a result of deoxygenation. In the oxy- and of vertebrates (Keilin and Wang, I 946 ; Rossicarbonmonoxy-forms, the molecular weight of Fanelli and Antonini, 1938). The sigmoid oxygen equilibrium curves of the radular Buccinum myoglobin, determined by Sephadex muscle myoglobins of the chitons Cvptochiton chromatography, is not dependent on concentration of the pigment; thus, Buccinum stelleri, Katharina tunicata, and khnochiton cons&cuus must be re-examined in light of the myoglobin is dissimilar to human haemogiobin in that it does not appear to exist in a recent observation that both monomeric and dimeric pigments occur in Amphineuran state of rapid association-dissociation equiradular muscle tissue (Manwell, I g38a, librium (Guidotti, 1964). Preliminary data rg6oa; Terwilliger and Read, rg7ob). indicate that the same conclusions are valid The oxygen dissociation curve of Buccinum for ~usycon myoglobin. myoglobin is not affected by changes of the The oxygen equilibrium curve of Buccinum hydrogen ion concentration over a physiomyoglobin is sigmoid under all conditions logical range of pH; in this respect Buccinum examined with n equal to I -4. Sigmoid dissociation curves are indicative of systems myoglobin resembles vertebrate myoglobins but differs from human haemoglobins. The composed of multiple interacting subunits. It therefore appears that the two subunits of dimeric haemoglobins of Cucumaria miniata and Fusitriton oregonensis also exhibit a PHthis dimeric myoglohin do not combine with independent oxygen dissociation curve (Manoxygen independently of one another. well, xg6oa, b). However, the dimeric and Buycon myoglobin exhibits a slightly monomeric haemoglobins of the insect Chironsigmoid dissociation curve (n > I *I). Athough emus th~rnrn~exhibit a pH-dependent oxygen there was some variability in the exact value affinity (Sick and Gersonde, 1969). of n in 0~ experiments, it was consistently At low values of pH (6.0 and lower) I-I or greater. This observation is also in accord with the dimeric nature of Busycon Bnccinum myoglobin is denatured. This is true for most haemoglobins that have been myoglobin. investigated. The reversibility of the acid Sigmoid oxygen equilibrium curves associdenaturation of Buccinum myoglobin as comated with dimeric haemoglobins have been pared with that of human haemoglobin has reported for several invertebrates. The not yet been investigated. dimeric radular muscle myoglobin of the Increasing sodium chloride concentration gastropod Fusitriton oregonensis shows a sigmoid dissociation curve with a value of R enhances the oxygen affinity of Buccimtm myoglobin yet does not appear to affect the equal to I ‘3 (Manwell, 1g6oa; Terwilliger shape of the oxygen dissociation curve. The and Read, 1g7ob). Manwelf reported that
197’,2
OXYGEN
AFFINITIES
OF MOLLUSC
oxygen dissociation curves of vertebrate haemoglobin, on the other hand, show a decrease in oxygen affinity of the pigment with an increase in salt concentration and an increase in sigmoidicity (Rossi-Fanelli, Antonini, and Caputo, x96x ; Antonini, Wyman, Brunori, Bucci, Fronticelli, and Rossi-Fanelli, 1968). The oxygen dissociation curve of human myoglobin is unaffected by an increase in sodium chloride concentration (Rossi-Fanelli and Antonini, r 958). Chlorocruorin, however, like Buccinum myoglobin, shows an increase in oxygen affinity with an increase in ionic strength of the buffer (Antonini, Rossi-Fanelli, and Caputo, I 962). An explanation for this effect is not clear to us at present. The presence of 2,8-DPG, phosphocreatine, lactate, ATP, or a-glycerophosphate in fifty-fold molar excess per haem has a negligible effect on the dissociation curve of Bwcinum myoglobin. In this respect Btucinum myoglobin differs from human haemoglobin the oxygen affinity of which is lowered by 2,8-DPG or ATP (Benesch and Benesch, I g6g). The affinity of Buccinum myoglobin for oxygen is unaffected by the concentration of the pigment over the range of concentrations examined in our study; this is also true for vertebrate myoglobin (Rossi-Fanelli and Antonini, 1958). On the other hand, the dependence of the oxygen equilibrium on the concentration of the protein is significant for human haemoglobin (Rossi-Fanelli and others, 1961) and is quite pronounced for lamprey haemoglobins (Wald and Riggs, I g5 I ; Briehl, I 968). The concentration effect is an indication of intermolecular interactions and has been attributed to the fact that human haemoglobin is in a state of association-dissociation equilibrium (Guidotti, 1964). The lack of a concentration effect for Buccinum myoglobin, as well as the fact that its apparent molecular weight does not change with dilution on Sephadex, indicates that the association constant of this molecule is probably quite high. The values obtained for the overall heat of oxygenation, AI-I, of haemoglobins are quite variable (Rossi-Fanelli, Antonini, and Caputo, 1964). The AH values for human
MYOCLOBINS
259
haemoglobin and myoglobin are - 13.0 and - 13.1 Cal. per mole respectively (Wyman, I 948 ; Rossi-Fanelli and Antonini, I 858). Snake haemoglobin has been reported to have a AH value as high as - 15’5 Cal. per mole (Sullivan, 1967)) although Roughton, Otis, and Lyster (1955) reported a value of -8.2 Cal. per mole for sheep haemoglobin and Manwell (195813) found values of -8.8 and -9.8 Cal. per mole for adult and foetal spiny dogfish haemoglobins. Rossi-Fanelli and Antonini (1960) reported a remarkably low AH for the haemoglobin of the tuna fish Thunnus of - I *8 Cal. per mole. The dimeric haemoglobin of Cucumaria miniata has a heat of oxygenation of -8.4 Cal. per mole which is similar to that found for both Buccinum and Busycon myoglobins (Manwell, r859). Both the molluscan myoglobins and Cucumaria pigments consist of homogeneous systems and molecules in which the AH values are not contributed to by linked groups such as in the Bohr effect; therefore, these AH values, which include the heat of solution of oxygen, probably represent the true value for heat of oxygenation. The consistency of the AH values of these molluscan myoglobins with those found for other haemoglobins strongly suggests that the molecules were not denatured by the extraction procedure and that the oxygenation properties which have been measured are those of the native protein. Furthermore, the similarity of the heat of oxygenation of these radular muscle myoglobins with those of vertebrate haemoglobins suggests that the molluscan myoglobins do indeed function -as pigments which combine directly with oxygen in the intact organism. There have been many hypotheses to explain the process by which haemoglobin combines with oxygen according to a sigmoid dissociation curve. Many hypotheses stress the importance of the a, 8 dimer as the functional subunit (see review by Antonini, 1967; Guidotti, 1967). Recently, the primary role of the a, f.3dimer in the associationdissociation equilibrium as well as its being the origin of co-operativity of the tetrameric molecule has received additional support (Anderson, Antonini, Brunori, and Wyman, I 870). A study of the a, /3 dimer is complicated
260
TERWILLIGERAND RWD
by the presence of the Bohr effect which is dependent on both a and j3 chains in the dimeric array. The forces that hold the subunits of Buccinum myoglobin together as a dimer seem to be relatively strong yet not covalent; in this respect Buccinum dimer is similar to the a, p dimer of vertebrate haem~lobin. The oxy~nation curve of B~ci~ myoglobin also exhibits si~oidici~ which is presumably an inherent property of its dimeric structure as is also thought to be true for the associating of a, p dimers of tetramerit vertebrate haemoglobin. A preliminary comparison of the relative oxygen affinities of the radular muscle myoglobins and the circulating haemocyanins of both Buccinum and Burycon indicates that the myoglobin should facilitate the diffusion of oxygen from the circulating haemolymph to the radular muscle tissues. However, the data in Fig. 7 should be treated with some caution as the Bohr effect for the haemocyanins was not investigated. It is, however., unlikely that the Bohr effect would be very great (Redfield, I 934). Our conclusions relative to the role of myoglobin in facilitating the transport of oxygen from circulating haemocyanin to the tissues are thus consistent with those of Manwell ( Ig58a, I g6ob).
ht. J. Btichm.
ANTONINI, E., WY.~, J., BRUNORI,M., Buccx, E., FRONTXELLI,C., and RO~~I-FANELL~, A. (x963), ’ Studies on the relations between moleCular and functional properties of Hb. IV. The Bohr effect in human Hb measured by proton binding *, 3. biol. Chem., 938, 2gp-zg57. BENESCH, R., and BENESCH, R. E. (rg6g), ‘ Intracellular organic phosphate-s as regulators of ~~g~~~2~ by hemoglobin ‘, Jvature,Land.,
BEN&H, R., MACDUFF,G,, and BENESCH, R. E. (19651, ‘ Determination of oxygen equilibria
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