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The coelomic haemoglobin from the bloodworm Glycera rouxii. molecular and oxygenation properties
THE COELOMIC HAEMOGLOBIN FROM THE BLOODWORM GLYCERA ROUXI1. MOLECULAR AND OXYGENATION PROPERTIES ROY E. WEHEK AND WINNIE HEIDEMANN Department of Zoophysiology. University of Aarhus, DK 8000 Aarhus, Denmark, and Kristineberg Marine Biological Station 45034 Fiskebackskil, Sweden
Abstract-l. The coelomic haemoglobin from the polychaete Glycern mu-xii is mainly tetrameric. but also contains smaller. predominantly monomeric molecules. Iso-electric focusing resolves it into 3 distinct components which, at 1.5”C,are iso-electric at pH 7.67, 7.26 and 6.72. 2. Whole coelomic fluid and haemoglobin solutions exhibit similar oxygen binding properties under the same conditions precluding the implication of intracoelomocytic factors in the regulation of oxygen afFinity. At pH 7.3, half-saturation oxygen tension (P,,). Hilt’s cooperativity coefficient for oxygen binding (tr) and the Bohr factor (4 = Alog P,,,/ApH) are about 8 mm Hg, 1.5, and -0.04, respectively. 3. The data are compared with those for other Glycrra species and illustrate a convergence of functional properties despite the distinct. interspecific divergence in molecular weight distribution and electrophoretic heterogeneity encountered in this genus.
Haemoglobins have widespread occurrence among annelids. Most commonly annelid haemoglobins consist of large molecules which are freely dissolved in the vascular fluid and have a molecular weight of about 3 million. These pigments are also termed erythrocruorins (reviewed by Weber, 1977). In contrast to the extracellular and intravascular erythrocruorins. haemoglobins with lower molecular weight are encountered in coelomocytes of five families of polychaetes: Glyceridae, Terebellidae, Ophelidae, Capitellidae and Cirratulidae (Romieu, 1923). The available data on the coelomic haemogiobins almost exclusively concern Terebellidae and Glyceridae. and suggest that whereas the haemoglobins are monomeric (mol. wt near 17,000) in the former family, there is striking species to species variability in aggregation states of the pigment in the glycerids. Thus, while haemoglobin is present both as monomers and as polydisperse polymers in Giyc~a dibr~~~/~iata (Hoffman & ~angum, 1970; Seamonds t’f ~1.. 1971n). it appears to be predominantly monomeric and tetramerit in G. gigantea (Weber, 1973; Weber & Bol, 1975). The haemoglobins of both G. dihranchiata (Seamonds rt al., 1971 h; Seamonds & Forster. 1972) and G. gigantra (Weber & Bol. 1976), moreover. exhibit marked heterogeneity in electrophoretic and oxygenation properties. Interestingly, ATP. the major modulator of oxygen affinity in red cells of lower vertcbrates, slightly, but distinctly, depresses oxygen affinity of G. ytyurrtea haemoglobin (Weber, 1973). For the coelomic haemoglobin of G. rou?tii Svedberg and Pedersen (1940) report a sedimentation constant (S&,) of 3.5, which is aligned with a mol. wt near 34,500 and thus indicates a dimeric aggregation state. With the view of gaining further insight into the inter-specific plasticity in haemoglobin structure and function among glycerids, and into its possible func-
tional impli~tions, the present study focusses on the haemoglobin of G. rouxii with regard to multiplicity in aggregation state and isoelectric point. and its oxygen binding properties.
MATERiALS AND METHODS This study is based on twelve specimens of G!~crru rouvii .Audouin L Milne Edwards 1833. about IO-15 cm long which were dredged from 45 m depth in GPsij Riinna, a sound near the mouth of the Gullmarn Fjord, Sweden. The identity of the animals used was checked individually. The coelomic fluid was collected by cutting the probosces when extroverted. The coelomocytes were washed twice in local sea water (33.40, salinity) and were then precipitated by centrifugation. The cells were subseq~lently lysed by addition of thrice their volume of distilled water. and the cell ghosts were removed by I5 min centrifugation at 25.000 rev/min. All preparative steps were carried out at &4”C. Gel filtration was carried out at 6’C using a column (height, 56.8cm; dia 2.30cm) of Sephadex GlOO gel, and eluting the proteins with 0.05 M NaCl containing 0.05 M Tris buffer, pH 7.5 and O.OZ”i,sodium azide (as antimicrobial agent). The partition coetficient, K,,. was catcuFdted as (v, - V,)/(I< - f$) (Laurent & Killander, 1964), where V, is the elution volume, v the total volume of the gel bed and V, the void volume (elution volume of blue dextran and Armicoin erythrocruorin, which are totally excluded by the gel). The multiplicity and iso-electric points of the haemoglobins were determined by electrofocusing at 6 8‘ C in 110 ml preparative LKB columns, containing equal concentrations (0.4”;) of ampholines with pH ranges of 558, and 3.5. IO, respectively. At the end of focussing the column contents were fractionated (2.15 ml fractions) for measurement of pH at 15°C and optical densities (O.D.). Haemogiobin fractions used for oxygen-binding studies were dialysed for 36 hr against three changes 0.01 M Tris buffer pH 7.5. containing 5 x 1O--4M EDTA. O.D. values were measured using a Beckman DU spectrophotometer equipped with Optilab multibank 171, and Optilab multilog 3 11 control and digital read-out units. 1.51
WINN~ Haemo~lobin concentrations were calculated using molar extinction coefficients for human haemoglohin (Antonini & Brunori, 1971). which are very similar to those found for G. ~ib~u}zc~ziu~a baemoglobins (Seamonds et (il.. 1971b). Haemoglobins in solution were concentrated hq centrifugation (12 hr at 50.000 revjmin) in a Beckman I?-65B ultracentrifuge. Concentrations of ATP in coelomic fluid and in stock sotutions of this chemical were determined with Sigma test chemicals (Missouri. U.S.A.). Hacmolysatcs were “stripped” (Treed from ions) by passage through MB-3 mixed ion exchanger. Oxygen equilibrium curves of the haemoglobin were determined with a diffusion chamber techniyuc (Sick & Gersonde, 1969), modified as previously described (Weber rt ul., 1976). pH variation was obtained either by mixing carbon dioxide into the equilibration gases with WGsthof gas mixing pumps (coelomic fluid) or by addition of appropriate Tris buffers (haemogiobi~l solutions). Values of halfsatur~~tion oxygen tension (P,,) and Hill’s coefficient. tz (reflecting cooperativity for oxygen binding). were interpolated from Hill plots [log (oxyHb)/(Hb) vs log PO>] of the equilibrium data. RESt.LTS
Two samples of pooled G. rousii coelomic fluid showed very variable values for haematocrit (9 and 141>;),Hb concentration (0.7 and 1.3 mM.----monomer basis) and ATP concentr~~tion (0.41 and 0.52 mM). The haemoglobin values predict oxygen capacities of about 1.6 and 2.9 ml 0,ilOO ml fluid. which are considerably Jower than values (3.2-7.7 ml 02/100 ml
HEIDEMANN
Fig. 1. Oxygen eyuilibrium curves of whole coelomic fluid of G. rourit at 15-C and pH 7.32 (0) and 6.90 (0).
fluid) found for G. dihrmchiata (Hoffman & Mangum. 1970). Oxygen eqliilibr~urn curves of the whole coelomic Auid (Fig. 1) indicate a moderately high oxygen afinity (P,, around 8 mm at 1YC) and a very slight Bohr effect (I#I= -0.10 between pH 7.3 and 6.9).
(i) i~l~l~~ul~~ wright. Gel filtration properties of haemolysate of G. rousii coelomocytcs in relation to those of other proteins of known molecular weight, arc given in Figs. 2 and 3. The elution profile of the haemolysate reveals the presence of two main haemogfobin fractions (Hb 1 and Hb 2 in Fig. 2) which on the basis of optical densities at 420nm constitute roughly 74 and 2?“,,, respectively, of the total haem. Their K ,j values reflect mol. wt of 49.000 and 20,000
Fig. 2. Elution of the haemolysate of G. rou.xii coelomic cells and of proteins of known molecular weight. from a Scphadex GIOO column. Marker substances (with molecular weights in parentheses) used are: B.D.. Blue dextran: Aid. aldolase (147,OOO)o): R.S.A.. bovine serum albumen (67.000): O.A.. oval albumen (45.000); Ch. tr., chymotrypsinogen (25,oof)): Hb A, human tl~lelnoglobin (64,500); Mb. myoglobin from Chinstrap penguin (17.000); cyt. t’. horse heart cytochrome c (13,500). Except for HbA (freshly prepared) and penguin Mb (earlier described. see Weher. Hemlnin~sen & Johanscn. 1974) all proteins are from Boehringer. Mannheim.
Coelomic mot
08
r t
20
10
wl
30
haemoglobin
10-j
doltons
40
60
80
100
c
06
Hb2-
\
40
50
45 log
mo,
wt
Fig. 3. Partition coefficients, K,,. on Sephadex gel (
proteins
of known molecular weight. as in legend to Fig. 2.
Other
details
daltons, respectively. The value for Hb 1 is significantly lower than 6468.000 daltons found for tetramerit vertebrate haemoglobins by alternative methods of molecular weight determination such as sedimentation. diffusion. light scattering and osmometric methods (Rossi-Fanelli. Antonini & Caputo, 1964). In the present study the elution property of human haemoglobin used as standard indicates a mol. wt of 42,000 daltons. By gel filtration techniques corresponding values of 4148,000 have been reported for haemoglobins from reptiles and elasmobranchs (Sullivan & Riggs. 1967; Fyhn & Sullivan. 1975). reflecting a compact shape and/or rapid association/dissociation properties in tetrameric haemoglobins.
from Glyceru
153
rou.uii
These considerations suggest that, as for vertebrate haemoglobin, the major fraction of G. rouxii haemoglobin is tetrameric. The apparent molecular weight of Hb 2 is lower than that expected for a dimer (f34,OOO daltons), but exceeds that of myoglobin, presumably reflecting a monomer-dimer equilibrium. Figure 2 shows that the coelomocytes contain two other major protein components. The first (A in Fig. 2) curiously shows a low haem content as evidenced from small but distinct fl and soret optical density peaks (at 540 and 420nm. respectively). The ratio of is only about 0.23 in this protein O.D.,zoiO.D.~eo compared to 2.1 in Hb 1. This fraction eluted with blue dextran indicating a mol. wt exceeding 150,000 daltons (the limit of the fractionation range of the gel). The second component (B in Fig. 2) represents small molecules which lack haem groups and form the major part of intracellular proteinaceous material. (ii) Multiplicity arrd iso-ekcfric p0ir1t.s. Iso-electric focusing resolves the haemoglobin of G. ~u.~ii into 3 distinct components (I, II and III in Fig. 4) which, at 15C, are iso-electric at pH values of 7.67. 7.26 and 6.72, respectively. Planimetric analysis shows that component haemoglobins I. II and III contain about 1 1, I5 and 74”;,, respectively. of the total coelomocyte haem. In order to identify the aggregation state of the main component (Hb III). this haemoglobin (fractions 30 and 31 in Fig. 4) was subjected to column chromatography on Sephadex GIOO. O.D. measurements at 540nm showed a resolution into two approximately equal components with K,, values of 0.27 and 0.58, allowing identification as Hb 1 and Hb 2. respectively (cf: Fig. 2). It follows that the main electrophoretic
1
-
10
/
9
8
z 4
7
6
5
4 0
20
40 FRACTION
Fig. 4. Iso-electric of 2.15 ml fractions
60
80
NUMBER
focusing of G. rousii haemoglobins. 0. optical density at 420nm; retrieved from the focusing column at the end of the experiment. fractions pooled for oxygen equilibria determinations (see Fig. 5).
l , pH at 15’C. Horizontal bar.
Rou E. WEBER
154
AND WINNIE HEIDEMANN
component either exists in two aggregation states, or that it consists of tetramers. which largely dissociates at the low concentrations eluting from the column (peak O.D. values were 0.12 at 420 nm).
P,, and II values of solutions of G. rouxii haemoglobin, and their pH dependence are shown in Fig. 5. At pH 7.3 and 15 C. P,,, approximates 7.8 mm Hg, and shows only slight pH sensitivity (4 = Alog P,,/ApH 2 -0.04). The 11values (1.5) reflect distinct, pH independent cooperativity in oxygen binding. The values for stripped haemolysates are approximately the same as for haemoglobin in coelomocytes, cxcluding the possibility that intracellular ions significantly modify oxygen affinity in G. wusii as is known to occur in vertebrate red cells. This accords with the absence of marked depressing influences of added ATP, DPG and IHP on oxygen affinity (Fig. 5). In isolation, the functional properties of the main haemoglobin component (Hb III in Fig. 5) are very similar to those of the haemolysatc. At pH 7.3, Pj,, II and 4 values approximate 6.8 mm Hg. 1.5 and -0.035, rcspectivcly. The data on Hb III should be interpreted with care. since this material had, as an exception. been frozen prior to the oxygenation studies. The lower P,,, compared to the haemolysate (Fig. 5). however, accords with a concentration effect found in the haemolysate of G. gigmtcu, which has a similar hacmoglobin molecular weight distribution as in G. wu\-ii (Weber, 1973). The small amount of material available of the smaller haemoglobin components (I and II in Fig. 4) precluded their functional analysis. Fignrc 5 shows that additions of the polyanionic organophosphatcs ATP. 2.3-diphosphoglycerate and inositol hexaphosphate. which potently depress oxygen affinity of vertebrate haemoglobins, have no significant cffcct on P,, in G. ~~sii haemoglobin. (The small enhancing effect of ATP on P,,, here seen in this figure could not be confirmed by repeat measurements or haemolysates that had been frozenstored for I week).
8
c
_
n
a.0 .
‘t
.
-1 c
Fig. 5. Pj,, and II values and their pH depcndcnce of stripped haemolysate (0) and of stripped haemolysates in the presence of the organic phosphates ATP (m). DPG (diphosphoglyccrate) (A) and IHP (inositol hexaphosphate) (V). at phosphate to haem ratios of about 6. IO and 10, respectively, and of the main electrophoretic haemoglobin (Hh 111 in Fig. 4) (A). Solvent. Tris buffer. ionic strength 0.05; temperature 15 ‘C; Hb concentrations, 0.68 mM haem (haemolysate) and 0.29 mM haem (Hb III). 0 = whole coelomic fluid (cj: Fig. I).
---!
80
Glycora roux,,
c
20
I 20
10
PO,(mmHg)
Fig. 6. Effect of temperature on oxygen equilibria of G. rouvii haemolysate. Solvent. Tris buffer. ionic strength 0.05; pH at 15 C, 7.35.
The effect of temperature on oxygen affinity of G. r-ouxii haemoglobin (Fig. 6) indicates an over-all heat of oxygenation [AH = 2.303. R (Alog P,,/A’,T) Wyman, 19481 of 13.3 kcal~mole~ I, which compares with similar values of - 11 to - 13.1 kcal.moleI for G. gigarztru. G. dihrunchiutu and G. urnericur~u haemolysates (Weber. 1973; Weber et (I/., 1977). DISCI’SSION
The results of the gel filtration studies indicate that G. rousii haemoglobin is heterogeneous with regard to molecular weight, but consists mainly of tetramers and contains only a small amount in the form of smaller aggregation states. The existence of haemoglobins with different molecular weights thus appear to be common in glycerid coelomocytes. A similar molecular weight distribution is evident in G. giguntcla and G. urncricarm (Weber & Bol, 1976; Weber ct (I/., 1977). while G. dihranchiutu red cells contain polydisperse polymeric as well as monomeric haemoglobins (Seamonds ct LI/., 19710). It is not known whether or not the different forms concur in the same red cells. The present findings dispel1 the notion that G. rousii haemoglobin is dimeric. as suggested by its sedimentation constant of 3.5 S (Svedberg & Pedersen. 1940). With regard to electrophoretic heterogeneity, G. romii haemoglobin. consisting of only three distinct components, differs significantly from those in G. gigunteu and G. rlihrmchiatu. where the haemoglobin resolves into 8-10 components by iso-electric focusing (Wcber. 1973; Weber ~‘r ul.. 1977). The relative abundancies of the tetrameric haemoglobin and the main electrophoretic component (Hb III). tentatively suggest that these two are identical. This would accord with the fact that in G. dihrurdziutu the monomeric haemoglobins have higher iso-electric points (Seamonds ef (II.. 1971h: Weber rt u/.. 1977). However. viewed in conjunction with the differences in electrophoretic patterns in G. ro~sii, G. giyctrltru and G. uwricmu. the correspondcncc in molecular weight distribution of haemoglobins in these spccics argues against a consistent relation between molecular weights and iso-electric points of component haemoglobins. The absence of such a relation is. moreover.
IS5
Coelomic haemoglobin from G/JY~YUrouzii suggested by the finding that the main electrophoretic component (Hb III) elutes as two peaks in molecular sieve gel filtration (page 153), and by the demonstration of reversible (oxygenation-linked) changes in aggregation state, as occurs in G. dihvanchiata polymers (Mizukami & Vinogradov. 1972; Bonaventura & Kitto. 1973). The occurrence of significant amounts of ATP in the coelomocytes of G. rousii is reminiscent of red cells of lower vertebrates, where this organophosphate plays an important role in modulating blood PsO. In G. yigantra haemolysates ATP moreover exerts a distinct. albeit small, depressing effect on oxygen affinity (Weber, 1973). The agreement in PsO values of the whole coelomic fluid compared to those of stripped haemoglobin solutions (cf1 Fig. 5). however, precludes the implication of intracoelomocyte factors in G. rourii. This is substantiated by the absence of physiologically-significant effects of ATP. DPG and IHP on oxygenation properties of G. rouxii haemoglobin. In view of the conspicuous interspecific divergence of glycerid haemoglobins with regard to molecular weight distribution and electrophoretic heterogeneity, it is striking that the comparative data demonstrate a remarkable convergence of functional properties. Haemolysates from G. dihrar&~iata, G. arnericana, G. rousii and coelomocyte suspensions of these species and of G. gigunteu all show similar oxygen affinities (P,,, = 7-8 mm Hg near pH 7.3). low cooperativity (n = l-1.8). small Bohr effects (4 = -0.04 to -0.1) and similar temperature sensitivities (AH = - 11 to - 13 kcal.mole‘) (Hoffman & Mangum, 1970; Mangum & Carhart. 1972; Seamonds & Foster, 1972; Weber. 1973; Webcr. 1977; Weber et ul., 1977). This suggests that the interspecific variation in molecular properties of G1~‘ceru haemoglobins may relate to functions other than those concerned with oxygen transport and storage. Ackno~lrd~~rttolfs~We thank the director, Dr. J.-O. Striimberg. and staff of Kristineberg Marine Biology Station, Fiskebsckskil. Sweden, for hospitality and help. and Ole Nielsen. Aarhus. for skilled electrotechnical assistance. The work was supported by the Danish Natural Science Research Council.
REFERENCES
ANTONINI E. & BRUNORI M. (1971) Hemoglobin and myoglobin in their reactions with ligands. In Fronrirrs of Biology (Edited by N~UHERC;ER A. & TATUM E. L.). Vol. 2 I, pp. I-1 2. North-Holland. Amsterdam. BONAVENTURAJ. & KITTO G. 9. (1973) Ligand-linked dissociation of some invertebrate hemoglobins. In Cornparutioe Ph$ology (Edited by BOLIS L., SCHMIDTNIELSEN K. & MADDR~LL S. H. P.). pp. 493-507. NorthHolland. Amsterdam. FYHN U. E. H. & SrjLLlVAN B. (1975) Elasmobranch hemoglobins. Dimerization and oolvmerization in variotys species. Cornp. Biochr~l. P/I v:yioi 5OB. I 19-l 29. HOIFMANN R. J. & MANGC~M C. P. i19701 The function of coelomic cell hemoglobin in the polychaete Gl~cera dihrtmchiata
Comp. Biochcm.
Physiol.
36. 2 I I-228.
LAURENT T. C. & KILLANDER T. (1964) A theory of gel filtration and its experimental verification. J. C~TOI~LU. 14. 3 17-330. MANGUM C. P. & CARHART J. A. (1972) Oxygen equilibrium of coelomic cell hemoglobin from the bloodworm Glycrru dihranchiuta. Comp. Biochrm. Physiol. 43. 949.-957.
MIZUMAKI H. & VINOCRA~~V S. N. (1973) Oxygen association equilibria of GIycrru hemoglobins. Biwhim. hiophys. Acra 285. 314-319. ROMI~U M. (1923) Recherches histophysiologiques sur le sang et sur le corps cordiaque des Anntlides polychetcs. Arch.
Morphol.
yin.
r.\-p. 17. l-339.
S~AMONDS B. & FORSTER R. E. (1973) Ligand equilibrium and kinetic characteristics of GIycrru dihrawhiurc~ hemoglobins. Afn. J. Physiol. 223. 734- 738. SEAMONDSB.. FORSTER R. E. & GEORC;I: P. (1971~) Physicochemical properties of the hemoglobins from the common bloodworm GIycrru dihruwhiutu. J. hiol. Chon. 246(17). 5391-5397. SEAMONDS B., FORS~~R R. E. & G~~TL~II~ A. J. (1971 h) Heterogeneity of the hemoglobin from the common bloodworm Glycrrir dihrarwhiatcl. J. hiol. Clam. 246 (8). I700~1705. SICK H. & G~RSONDI: K. (1969) Method for continuous registration of O,-binding curves of haemoproteins by means of a diffusion chamber. il~1trl~t. Biochcw. 32. 362-376.
SULLIVAN B. & RIGGS A. (1967) Structure function evolution of turtle hemoglobins--I. Distribution heavy hemoglobins. Cotnp. Biocho~. Physiol.
and of 23.
437447.
SVEDRERGT. & PEDERS~N K. 0. (1940) Die, C’ltrcl-critr.!fIcyc,. Theorir, Konstruktion und Erqeh~~isw. pp. I 436. Thcodor Steinkopff Verlag. Dresden. TIKWILLIGFR R. C.. GARLICK R. L. & Tf-KWILLI~XR N. 9. (1976) Hemoglobins of Gl!,cc~ rohwro: Structures of coelomic cell hemoglobin and hod) wall myoglohln. Camp. Biochcnl. Ph!~io/. 54B. 149 153. WEBER R. E. (1973) Functional and molecular properties of corpuscular haemoglobin from the bloodworm G/ycera gigantra.
Nrth.
.I. Sea Rcs. 7. 3 l&327.
WEBER R. E. (1977) Respiratory Pigments. In Physioloq~ ofilrmelids (Edited by MILL P. J.). Academic Press. New York (In press). WEBER R. E. & BOL J. F. (1976) Heterogeneity and oxygen equilibria of haemoglobin from the bloodworm Glyccru giguntea.
Camp. Biochem.
Physiol.
53B. 23 30.
WERER R. E.. HEMMINGSEN E. A. & JoHAhskN K. (1974) Functional and biochemical studies of penguin myoglobin. Camp. Biochcm. Physiol. 49B. 197?214. WEBER R. E., LY~KEBOE G. & JOHANS~N K. (1976) Physiological properties of eel haemoglobin. Hypoxic acclimation. phosphate effects and multiplicity. J. ~x-p. Biol. 64. 75-88. WEBER R. E., SULLIVAN B.. BONAVENTIIRA J. & BONAVLNTLIRA C. (1977). Functional and structural properties of haemoglobins of G/.ycrra dihrarlchiutu and G. wwricuw (In preparation). WYMAN J. (1948) Heme proteins. In Adww.s Protri,? Chemistry (Edited by ANWN M. L. & EDSALL J. T.), Vol. 4. pp. 407-531. Academic Press. New York.
NOTE
ADDED
IN PROOF
A recent paper (Terwilliger ct ~1.. 1976) reports an electrophoretically-homogeneous haemoglobin with an apparent mol. wt of 48.000 50.000 from coelomocytes of Glrcrru robusta.
Abstract-l. The coelomic haemoglobin from the polychaete Glycern mu-xii is mainly tetrameric. but also contains smaller. predominantly monomeric molecules. Iso-electric focusing resolves it into 3 distinct components which, at 1.5”C,are iso-electric at pH 7.67, 7.26 and 6.72. 2. Whole coelomic fluid and haemoglobin solutions exhibit similar oxygen binding properties under the same conditions precluding the implication of intracoelomocytic factors in the regulation of oxygen afFinity. At pH 7.3, half-saturation oxygen tension (P,,). Hilt’s cooperativity coefficient for oxygen binding (tr) and the Bohr factor (4 = Alog P,,,/ApH) are about 8 mm Hg, 1.5, and -0.04, respectively. 3. The data are compared with those for other Glycrra species and illustrate a convergence of functional properties despite the distinct. interspecific divergence in molecular weight distribution and electrophoretic heterogeneity encountered in this genus.
Haemoglobins have widespread occurrence among annelids. Most commonly annelid haemoglobins consist of large molecules which are freely dissolved in the vascular fluid and have a molecular weight of about 3 million. These pigments are also termed erythrocruorins (reviewed by Weber, 1977). In contrast to the extracellular and intravascular erythrocruorins. haemoglobins with lower molecular weight are encountered in coelomocytes of five families of polychaetes: Glyceridae, Terebellidae, Ophelidae, Capitellidae and Cirratulidae (Romieu, 1923). The available data on the coelomic haemogiobins almost exclusively concern Terebellidae and Glyceridae. and suggest that whereas the haemoglobins are monomeric (mol. wt near 17,000) in the former family, there is striking species to species variability in aggregation states of the pigment in the glycerids. Thus, while haemoglobin is present both as monomers and as polydisperse polymers in Giyc~a dibr~~~/~iata (Hoffman & ~angum, 1970; Seamonds t’f ~1.. 1971n). it appears to be predominantly monomeric and tetramerit in G. gigantea (Weber, 1973; Weber & Bol, 1975). The haemoglobins of both G. dihranchiata (Seamonds rt al., 1971 h; Seamonds & Forster. 1972) and G. gigantra (Weber & Bol. 1976), moreover. exhibit marked heterogeneity in electrophoretic and oxygenation properties. Interestingly, ATP. the major modulator of oxygen affinity in red cells of lower vertcbrates, slightly, but distinctly, depresses oxygen affinity of G. ytyurrtea haemoglobin (Weber, 1973). For the coelomic haemoglobin of G. rou?tii Svedberg and Pedersen (1940) report a sedimentation constant (S&,) of 3.5, which is aligned with a mol. wt near 34,500 and thus indicates a dimeric aggregation state. With the view of gaining further insight into the inter-specific plasticity in haemoglobin structure and function among glycerids, and into its possible func-
tional impli~tions, the present study focusses on the haemoglobin of G. rouxii with regard to multiplicity in aggregation state and isoelectric point. and its oxygen binding properties.
MATERiALS AND METHODS This study is based on twelve specimens of G!~crru rouvii .Audouin L Milne Edwards 1833. about IO-15 cm long which were dredged from 45 m depth in GPsij Riinna, a sound near the mouth of the Gullmarn Fjord, Sweden. The identity of the animals used was checked individually. The coelomic fluid was collected by cutting the probosces when extroverted. The coelomocytes were washed twice in local sea water (33.40, salinity) and were then precipitated by centrifugation. The cells were subseq~lently lysed by addition of thrice their volume of distilled water. and the cell ghosts were removed by I5 min centrifugation at 25.000 rev/min. All preparative steps were carried out at &4”C. Gel filtration was carried out at 6’C using a column (height, 56.8cm; dia 2.30cm) of Sephadex GlOO gel, and eluting the proteins with 0.05 M NaCl containing 0.05 M Tris buffer, pH 7.5 and O.OZ”i,sodium azide (as antimicrobial agent). The partition coetficient, K,,. was catcuFdted as (v, - V,)/(I< - f$) (Laurent & Killander, 1964), where V, is the elution volume, v the total volume of the gel bed and V, the void volume (elution volume of blue dextran and Armicoin erythrocruorin, which are totally excluded by the gel). The multiplicity and iso-electric points of the haemoglobins were determined by electrofocusing at 6 8‘ C in 110 ml preparative LKB columns, containing equal concentrations (0.4”;) of ampholines with pH ranges of 558, and 3.5. IO, respectively. At the end of focussing the column contents were fractionated (2.15 ml fractions) for measurement of pH at 15°C and optical densities (O.D.). Haemogiobin fractions used for oxygen-binding studies were dialysed for 36 hr against three changes 0.01 M Tris buffer pH 7.5. containing 5 x 1O--4M EDTA. O.D. values were measured using a Beckman DU spectrophotometer equipped with Optilab multibank 171, and Optilab multilog 3 11 control and digital read-out units. 1.51
WINN~ Haemo~lobin concentrations were calculated using molar extinction coefficients for human haemoglohin (Antonini & Brunori, 1971). which are very similar to those found for G. ~ib~u}zc~ziu~a baemoglobins (Seamonds et (il.. 1971b). Haemoglobins in solution were concentrated hq centrifugation (12 hr at 50.000 revjmin) in a Beckman I?-65B ultracentrifuge. Concentrations of ATP in coelomic fluid and in stock sotutions of this chemical were determined with Sigma test chemicals (Missouri. U.S.A.). Hacmolysatcs were “stripped” (Treed from ions) by passage through MB-3 mixed ion exchanger. Oxygen equilibrium curves of the haemoglobin were determined with a diffusion chamber techniyuc (Sick & Gersonde, 1969), modified as previously described (Weber rt ul., 1976). pH variation was obtained either by mixing carbon dioxide into the equilibration gases with WGsthof gas mixing pumps (coelomic fluid) or by addition of appropriate Tris buffers (haemogiobi~l solutions). Values of halfsatur~~tion oxygen tension (P,,) and Hill’s coefficient. tz (reflecting cooperativity for oxygen binding). were interpolated from Hill plots [log (oxyHb)/(Hb) vs log PO>] of the equilibrium data. RESt.LTS
Two samples of pooled G. rousii coelomic fluid showed very variable values for haematocrit (9 and 141>;),Hb concentration (0.7 and 1.3 mM.----monomer basis) and ATP concentr~~tion (0.41 and 0.52 mM). The haemoglobin values predict oxygen capacities of about 1.6 and 2.9 ml 0,ilOO ml fluid. which are considerably Jower than values (3.2-7.7 ml 02/100 ml
HEIDEMANN
Fig. 1. Oxygen eyuilibrium curves of whole coelomic fluid of G. rourit at 15-C and pH 7.32 (0) and 6.90 (0).
fluid) found for G. dihrmchiata (Hoffman & Mangum. 1970). Oxygen eqliilibr~urn curves of the whole coelomic Auid (Fig. 1) indicate a moderately high oxygen afinity (P,, around 8 mm at 1YC) and a very slight Bohr effect (I#I= -0.10 between pH 7.3 and 6.9).
(i) i~l~l~~ul~~ wright. Gel filtration properties of haemolysate of G. rousii coelomocytcs in relation to those of other proteins of known molecular weight, arc given in Figs. 2 and 3. The elution profile of the haemolysate reveals the presence of two main haemogfobin fractions (Hb 1 and Hb 2 in Fig. 2) which on the basis of optical densities at 420nm constitute roughly 74 and 2?“,,, respectively, of the total haem. Their K ,j values reflect mol. wt of 49.000 and 20,000
Fig. 2. Elution of the haemolysate of G. rou.xii coelomic cells and of proteins of known molecular weight. from a Scphadex GIOO column. Marker substances (with molecular weights in parentheses) used are: B.D.. Blue dextran: Aid. aldolase (147,OOO)o): R.S.A.. bovine serum albumen (67.000): O.A.. oval albumen (45.000); Ch. tr., chymotrypsinogen (25,oof)): Hb A, human tl~lelnoglobin (64,500); Mb. myoglobin from Chinstrap penguin (17.000); cyt. t’. horse heart cytochrome c (13,500). Except for HbA (freshly prepared) and penguin Mb (earlier described. see Weher. Hemlnin~sen & Johanscn. 1974) all proteins are from Boehringer. Mannheim.
Coelomic mot
08
r t
20
10
wl
30
haemoglobin
10-j
doltons
40
60
80
100
c
06
Hb2-
\
40
50
45 log
mo,
wt
Fig. 3. Partition coefficients, K,,. on Sephadex gel (
proteins
of known molecular weight. as in legend to Fig. 2.
Other
details
daltons, respectively. The value for Hb 1 is significantly lower than 6468.000 daltons found for tetramerit vertebrate haemoglobins by alternative methods of molecular weight determination such as sedimentation. diffusion. light scattering and osmometric methods (Rossi-Fanelli. Antonini & Caputo, 1964). In the present study the elution property of human haemoglobin used as standard indicates a mol. wt of 42,000 daltons. By gel filtration techniques corresponding values of 4148,000 have been reported for haemoglobins from reptiles and elasmobranchs (Sullivan & Riggs. 1967; Fyhn & Sullivan. 1975). reflecting a compact shape and/or rapid association/dissociation properties in tetrameric haemoglobins.
from Glyceru
153
rou.uii
These considerations suggest that, as for vertebrate haemoglobin, the major fraction of G. rouxii haemoglobin is tetrameric. The apparent molecular weight of Hb 2 is lower than that expected for a dimer (f34,OOO daltons), but exceeds that of myoglobin, presumably reflecting a monomer-dimer equilibrium. Figure 2 shows that the coelomocytes contain two other major protein components. The first (A in Fig. 2) curiously shows a low haem content as evidenced from small but distinct fl and soret optical density peaks (at 540 and 420nm. respectively). The ratio of is only about 0.23 in this protein O.D.,zoiO.D.~eo compared to 2.1 in Hb 1. This fraction eluted with blue dextran indicating a mol. wt exceeding 150,000 daltons (the limit of the fractionation range of the gel). The second component (B in Fig. 2) represents small molecules which lack haem groups and form the major part of intracellular proteinaceous material. (ii) Multiplicity arrd iso-ekcfric p0ir1t.s. Iso-electric focusing resolves the haemoglobin of G. ~u.~ii into 3 distinct components (I, II and III in Fig. 4) which, at 15C, are iso-electric at pH values of 7.67. 7.26 and 6.72, respectively. Planimetric analysis shows that component haemoglobins I. II and III contain about 1 1, I5 and 74”;,, respectively. of the total coelomocyte haem. In order to identify the aggregation state of the main component (Hb III). this haemoglobin (fractions 30 and 31 in Fig. 4) was subjected to column chromatography on Sephadex GIOO. O.D. measurements at 540nm showed a resolution into two approximately equal components with K,, values of 0.27 and 0.58, allowing identification as Hb 1 and Hb 2. respectively (cf: Fig. 2). It follows that the main electrophoretic
1
-
10
/
9
8
z 4
7
6
5
4 0
20
40 FRACTION
Fig. 4. Iso-electric of 2.15 ml fractions
60
80
NUMBER
focusing of G. rousii haemoglobins. 0. optical density at 420nm; retrieved from the focusing column at the end of the experiment. fractions pooled for oxygen equilibria determinations (see Fig. 5).
l , pH at 15’C. Horizontal bar.
Rou E. WEBER
154
AND WINNIE HEIDEMANN
component either exists in two aggregation states, or that it consists of tetramers. which largely dissociates at the low concentrations eluting from the column (peak O.D. values were 0.12 at 420 nm).
P,, and II values of solutions of G. rouxii haemoglobin, and their pH dependence are shown in Fig. 5. At pH 7.3 and 15 C. P,,, approximates 7.8 mm Hg, and shows only slight pH sensitivity (4 = Alog P,,/ApH 2 -0.04). The 11values (1.5) reflect distinct, pH independent cooperativity in oxygen binding. The values for stripped haemolysates are approximately the same as for haemoglobin in coelomocytes, cxcluding the possibility that intracellular ions significantly modify oxygen affinity in G. wusii as is known to occur in vertebrate red cells. This accords with the absence of marked depressing influences of added ATP, DPG and IHP on oxygen affinity (Fig. 5). In isolation, the functional properties of the main haemoglobin component (Hb III in Fig. 5) are very similar to those of the haemolysatc. At pH 7.3, Pj,, II and 4 values approximate 6.8 mm Hg. 1.5 and -0.035, rcspectivcly. The data on Hb III should be interpreted with care. since this material had, as an exception. been frozen prior to the oxygenation studies. The lower P,,, compared to the haemolysate (Fig. 5). however, accords with a concentration effect found in the haemolysate of G. gigmtcu, which has a similar hacmoglobin molecular weight distribution as in G. wu\-ii (Weber, 1973). The small amount of material available of the smaller haemoglobin components (I and II in Fig. 4) precluded their functional analysis. Fignrc 5 shows that additions of the polyanionic organophosphatcs ATP. 2.3-diphosphoglycerate and inositol hexaphosphate. which potently depress oxygen affinity of vertebrate haemoglobins, have no significant cffcct on P,, in G. ~~sii haemoglobin. (The small enhancing effect of ATP on P,,, here seen in this figure could not be confirmed by repeat measurements or haemolysates that had been frozenstored for I week).
8
c
_
n
a.0 .
‘t
.
-1 c
Fig. 5. Pj,, and II values and their pH depcndcnce of stripped haemolysate (0) and of stripped haemolysates in the presence of the organic phosphates ATP (m). DPG (diphosphoglyccrate) (A) and IHP (inositol hexaphosphate) (V). at phosphate to haem ratios of about 6. IO and 10, respectively, and of the main electrophoretic haemoglobin (Hh 111 in Fig. 4) (A). Solvent. Tris buffer. ionic strength 0.05; temperature 15 ‘C; Hb concentrations, 0.68 mM haem (haemolysate) and 0.29 mM haem (Hb III). 0 = whole coelomic fluid (cj: Fig. I).
---!
80
Glycora roux,,
c
20
I 20
10
PO,(mmHg)
Fig. 6. Effect of temperature on oxygen equilibria of G. rouvii haemolysate. Solvent. Tris buffer. ionic strength 0.05; pH at 15 C, 7.35.
The effect of temperature on oxygen affinity of G. r-ouxii haemoglobin (Fig. 6) indicates an over-all heat of oxygenation [AH = 2.303. R (Alog P,,/A’,T) Wyman, 19481 of 13.3 kcal~mole~ I, which compares with similar values of - 11 to - 13.1 kcal.moleI for G. gigarztru. G. dihrunchiutu and G. urnericur~u haemolysates (Weber. 1973; Weber et (I/., 1977). DISCI’SSION
The results of the gel filtration studies indicate that G. rousii haemoglobin is heterogeneous with regard to molecular weight, but consists mainly of tetramers and contains only a small amount in the form of smaller aggregation states. The existence of haemoglobins with different molecular weights thus appear to be common in glycerid coelomocytes. A similar molecular weight distribution is evident in G. giguntcla and G. urncricarm (Weber & Bol, 1976; Weber ct (I/., 1977). while G. dihranchiutu red cells contain polydisperse polymeric as well as monomeric haemoglobins (Seamonds ct LI/., 19710). It is not known whether or not the different forms concur in the same red cells. The present findings dispel1 the notion that G. rousii haemoglobin is dimeric. as suggested by its sedimentation constant of 3.5 S (Svedberg & Pedersen. 1940). With regard to electrophoretic heterogeneity, G. romii haemoglobin. consisting of only three distinct components, differs significantly from those in G. gigunteu and G. rlihrmchiatu. where the haemoglobin resolves into 8-10 components by iso-electric focusing (Wcber. 1973; Weber ~‘r ul.. 1977). The relative abundancies of the tetrameric haemoglobin and the main electrophoretic component (Hb III). tentatively suggest that these two are identical. This would accord with the fact that in G. dihrurdziutu the monomeric haemoglobins have higher iso-electric points (Seamonds ef (II.. 1971h: Weber rt u/.. 1977). However. viewed in conjunction with the differences in electrophoretic patterns in G. ro~sii, G. giyctrltru and G. uwricmu. the correspondcncc in molecular weight distribution of haemoglobins in these spccics argues against a consistent relation between molecular weights and iso-electric points of component haemoglobins. The absence of such a relation is. moreover.
IS5
Coelomic haemoglobin from G/JY~YUrouzii suggested by the finding that the main electrophoretic component (Hb III) elutes as two peaks in molecular sieve gel filtration (page 153), and by the demonstration of reversible (oxygenation-linked) changes in aggregation state, as occurs in G. dihvanchiata polymers (Mizukami & Vinogradov. 1972; Bonaventura & Kitto. 1973). The occurrence of significant amounts of ATP in the coelomocytes of G. rousii is reminiscent of red cells of lower vertebrates, where this organophosphate plays an important role in modulating blood PsO. In G. yigantra haemolysates ATP moreover exerts a distinct. albeit small, depressing effect on oxygen affinity (Weber, 1973). The agreement in PsO values of the whole coelomic fluid compared to those of stripped haemoglobin solutions (cf1 Fig. 5). however, precludes the implication of intracoelomocyte factors in G. rourii. This is substantiated by the absence of physiologically-significant effects of ATP. DPG and IHP on oxygenation properties of G. rouxii haemoglobin. In view of the conspicuous interspecific divergence of glycerid haemoglobins with regard to molecular weight distribution and electrophoretic heterogeneity, it is striking that the comparative data demonstrate a remarkable convergence of functional properties. Haemolysates from G. dihrar&~iata, G. arnericana, G. rousii and coelomocyte suspensions of these species and of G. gigunteu all show similar oxygen affinities (P,,, = 7-8 mm Hg near pH 7.3). low cooperativity (n = l-1.8). small Bohr effects (4 = -0.04 to -0.1) and similar temperature sensitivities (AH = - 11 to - 13 kcal.mole‘) (Hoffman & Mangum, 1970; Mangum & Carhart. 1972; Seamonds & Foster, 1972; Weber. 1973; Webcr. 1977; Weber et ul., 1977). This suggests that the interspecific variation in molecular properties of G1~‘ceru haemoglobins may relate to functions other than those concerned with oxygen transport and storage. Ackno~lrd~~rttolfs~We thank the director, Dr. J.-O. Striimberg. and staff of Kristineberg Marine Biology Station, Fiskebsckskil. Sweden, for hospitality and help. and Ole Nielsen. Aarhus. for skilled electrotechnical assistance. The work was supported by the Danish Natural Science Research Council.
REFERENCES
ANTONINI E. & BRUNORI M. (1971) Hemoglobin and myoglobin in their reactions with ligands. In Fronrirrs of Biology (Edited by N~UHERC;ER A. & TATUM E. L.). Vol. 2 I, pp. I-1 2. North-Holland. Amsterdam. BONAVENTURAJ. & KITTO G. 9. (1973) Ligand-linked dissociation of some invertebrate hemoglobins. In Cornparutioe Ph$ology (Edited by BOLIS L., SCHMIDTNIELSEN K. & MADDR~LL S. H. P.). pp. 493-507. NorthHolland. Amsterdam. FYHN U. E. H. & SrjLLlVAN B. (1975) Elasmobranch hemoglobins. Dimerization and oolvmerization in variotys species. Cornp. Biochr~l. P/I v:yioi 5OB. I 19-l 29. HOIFMANN R. J. & MANGC~M C. P. i19701 The function of coelomic cell hemoglobin in the polychaete Gl~cera dihrtmchiata
Comp. Biochcm.
Physiol.
36. 2 I I-228.
LAURENT T. C. & KILLANDER T. (1964) A theory of gel filtration and its experimental verification. J. C~TOI~LU. 14. 3 17-330. MANGUM C. P. & CARHART J. A. (1972) Oxygen equilibrium of coelomic cell hemoglobin from the bloodworm Glycrru dihranchiuta. Comp. Biochrm. Physiol. 43. 949.-957.
MIZUMAKI H. & VINOCRA~~V S. N. (1973) Oxygen association equilibria of GIycrru hemoglobins. Biwhim. hiophys. Acra 285. 314-319. ROMI~U M. (1923) Recherches histophysiologiques sur le sang et sur le corps cordiaque des Anntlides polychetcs. Arch.
Morphol.
yin.
r.\-p. 17. l-339.
S~AMONDS B. & FORSTER R. E. (1973) Ligand equilibrium and kinetic characteristics of GIycrru dihrawhiurc~ hemoglobins. Afn. J. Physiol. 223. 734- 738. SEAMONDSB.. FORSTER R. E. & GEORC;I: P. (1971~) Physicochemical properties of the hemoglobins from the common bloodworm GIycrru dihruwhiutu. J. hiol. Chon. 246(17). 5391-5397. SEAMONDS B., FORS~~R R. E. & G~~TL~II~ A. J. (1971 h) Heterogeneity of the hemoglobin from the common bloodworm Glycrrir dihrarwhiatcl. J. hiol. Clam. 246 (8). I700~1705. SICK H. & G~RSONDI: K. (1969) Method for continuous registration of O,-binding curves of haemoproteins by means of a diffusion chamber. il~1trl~t. Biochcw. 32. 362-376.
SULLIVAN B. & RIGGS A. (1967) Structure function evolution of turtle hemoglobins--I. Distribution heavy hemoglobins. Cotnp. Biocho~. Physiol.
and of 23.
437447.
SVEDRERGT. & PEDERS~N K. 0. (1940) Die, C’ltrcl-critr.!fIcyc,. Theorir, Konstruktion und Erqeh~~isw. pp. I 436. Thcodor Steinkopff Verlag. Dresden. TIKWILLIGFR R. C.. GARLICK R. L. & Tf-KWILLI~XR N. 9. (1976) Hemoglobins of Gl!,cc~ rohwro: Structures of coelomic cell hemoglobin and hod) wall myoglohln. Camp. Biochcnl. Ph!~io/. 54B. 149 153. WEBER R. E. (1973) Functional and molecular properties of corpuscular haemoglobin from the bloodworm G/ycera gigantra.
Nrth.
.I. Sea Rcs. 7. 3 l&327.
WEBER R. E. (1977) Respiratory Pigments. In Physioloq~ ofilrmelids (Edited by MILL P. J.). Academic Press. New York (In press). WEBER R. E. & BOL J. F. (1976) Heterogeneity and oxygen equilibria of haemoglobin from the bloodworm Glyccru giguntea.
Camp. Biochem.
Physiol.
53B. 23 30.
WERER R. E.. HEMMINGSEN E. A. & JoHAhskN K. (1974) Functional and biochemical studies of penguin myoglobin. Camp. Biochcm. Physiol. 49B. 197?214. WEBER R. E., LY~KEBOE G. & JOHANS~N K. (1976) Physiological properties of eel haemoglobin. Hypoxic acclimation. phosphate effects and multiplicity. J. ~x-p. Biol. 64. 75-88. WEBER R. E., SULLIVAN B.. BONAVENTIIRA J. & BONAVLNTLIRA C. (1977). Functional and structural properties of haemoglobins of G/.ycrra dihrarlchiutu and G. wwricuw (In preparation). WYMAN J. (1948) Heme proteins. In Adww.s Protri,? Chemistry (Edited by ANWN M. L. & EDSALL J. T.), Vol. 4. pp. 407-531. Academic Press. New York.
NOTE
ADDED
IN PROOF
A recent paper (Terwilliger ct ~1.. 1976) reports an electrophoretically-homogeneous haemoglobin with an apparent mol. wt of 48.000 50.000 from coelomocytes of Glrcrru robusta.