Heterogeneity and oxygen equilibria of haemoglobin from the bloodworm Glycera gigantea

Comp. Biochem. Physiol., 1976, Vol. 53B, pp. 23 to 30. Pergamon Press. Printed in Great Britain

HETEROGENEITY AND OXYGEN EQUILIBRIA OF H A E M O G L O B I N FROM THE B L O O D W O R M GLYCERA GIGANTEA* RoY E. WEBERt AND JOHN F. BOL Department of Zoophysiology, University of Aarhus, Denmark, and Department of Biochemistry, University of Leiden, The Netherlands

(Received 30 September 1974) Glycera gigantea was studied with regard to molecular weight, subunit composition, heterogeneity on the basis of isoelectric point and oxygen equilibrium of the major components. 2. In gel filtration chromatography the haemoglobin resolves into a main component (HbH) and a minor component of smaller molecules (HbL), which appear to be composed of protein chain subunits of the same size, but have sedimentation values, S20.w, of 4.4 and 1.6 indicative respectively, of tetramers and monomers. 3. The haemoglobin is highly heterogeneous on the basis of isoelectric point. Six fractions studied funtionally show considerable differentiation in oxygen affinities. 4. The data are discussed comparatively with particular reference to the coelomic haemoglobin of G. dibranchiata and vascular haemoglobin of Arenicola marina.

A b s t r a c t ~ l . Haemoglobin from the coelomic cells of

INTRODUCTION

etal., 1971a; Seamondsetal., 197lb; Seamonds & Forster, 1972; Mangum & Carhart, 1972; Imamura et al., 1972; Mizukami & Vinogradov, 1973; Bonaventura & Kitto, 1973; Harrington et al., 1973; Weber et al., 1975). In a previous communication (Weber, 1973) some oxygen-binding properties of G. 9igantea haemoglobins have been reported. The present paper extends those data, and deals with aspects of the molecular and functional heterogeneity of G..qiqantea haemoglobin. These aspects are of comparative interest in view of the occurrence of a complex system of electrophoretically-distinguishablehaemoglobins in bloodworms (Seamonds et al., 1971), while the published data on the vascular annetid haemoglobins generally show little or no heterogeneity (Levin, 1963; Weber, 1971; Weber & Pauptit, 1972b).

THE ~LOODWORMS belonging to the annelid family Glyceridae are active predators, inhabiting marine intertidal and subtidal sandy fiats. During burrowing and predation a voluminous coelomic fluid forms the hydrostatic basis for thrusting out the proboscis, which may attain half the body length. The coelomic fluid contains red cells richly supplied with haemoglobin. The cellular haemoglobins in the coelome of bloodworms contrast markedly from the extracellular, vascular haemoglobins commonly found in annelids. Whereas the molecules of the latter haemoglobins --also termed erythrocruorins (Roche, 1965)--are extremely large (mol.wt 2.7-3.0 × 10°) and have distinctive quarternary structures (Svedberg & Pedersen, 1940; I_~vin, 1963; Roche, 1965; Van Bruggen & Weber, 1974), the bloodworm haemoglobins are smaller, having mol. wt estimated at 55,000 and 34,000 respectively, in G. 9igaatea and G. rouxii (Roche & Combette, 1937; Svedberg & Pedersen, 1940) and 17,700 and 125,000 for the monomeric and polymeric forms in G. dibranchiata (Seamonds et al., 1971b). These properties thus illustrate a marked dichotomy in the evolutionary development of haemoglobins in annelids. The available data on the respiratory and structural properties of the coelomic haemoglobins of bloodworms are, however, almost entirely limited to the North American G. dibranchiata (cf. Salomon, 1941; Padlan & Love, 1968; Hoffmann & Mangum, 1970; Vinogradov et aL, 1970; Seamonds, 1971 ; Seamonds

MATERIALS AND METHODS

Glycera gigantea Quatrefages, 1865, were obtained from Mergellina Bay, Naples, and haemoglobin solutions were prepared by lysing the saline-washed coelomic cells by osmotic shock. Column chromatography was carried out at 4°C on a 60 x 1.5cm column of Sephadex G-100gel. The haemoglobin and calibration proteins were eluted with 0.1 M NaCI in 0.05 M Tris buffer, pH 7.0 and monitored with an ahsorptiometer at 280 nm. The sedimentation velocity of carboxy haemoglobin dissolved in 0.05M Tris buffer, pH6.8 and 0.1 M NaCl was determined at 20°C in a double sector cell of a Spinco model E analytical ultracentrifuge, spinning at 60,000rev/ min, while the sedimentation was recorded with a splitbeam photoelectric scanner at 280 nm. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS) was run at room temperature on 10% gels, containing 0'27% methylenebisacrylamide (Weber & Osborn, 1969) and on 12.5% gels containing 1.25% methylenebisacrylamide (Swank & Munkres, 1971) (gel size, 7 x 0.6cm)and 6M urea. Electrophoresis

*The work was supported in part by the Danish Natural Science Research Council. ~"Part of this work was carried out at Stazione Zoologica, Naples, Italy, and at the Netherlands Institute for Sea Research, Texel, Holland. 23

24

RoY

E. WEBER

time for 10 and 12.5°,+, gels were respectively 7 and 16hr and 50 V and 4 mA were applied per gel, with buffer circulating between the electrode compartments (to keep the pH constant). Prior to electrophoresis SDS and 2-mercaptoethanol were added to a final concentration of 1%. The gels were stained with amido black, The heterogeneity of Glycera haemoglobins on the basis of iso-electric point was determined both using miniature electrofocusing columns (after Koch & Backx, I969), and preparative 440ml electrofocusing columns (LKB, Bromma, Sweden), containing 1% LKB carrier ampholytes in linear sucrose gradients formed by an LKB Ultragrad gradient mixer. In the miniature columns equal parts of carrier ampholytes with pH ranges of 6 8 and 8 l0 were used. In the preparative columns three parts ampholytes of pH 6-8 were mixed with one part of pH 3-10. Columns were focused for 2 days at 5'C at 700 V, and the contents were then pumped out via the upper ends through an LKB Uvicord II u.v. absorptiometer monitoring transmittance at 280nm. pH values of the fractions collected were measured at 10 or 15°C using a Radiometer PHM 72 pH meter equipped with microelectrode unit. Oxygen equilibrium curves were recorded continuously at 436 nm with an oxygen diffusion chamber (Sick & Gersonde, 1969) coupled to an Eppendorf model I I(R)M photometer and Eppendorf type L1853 lineariser, using two calibration gases in each determination. Carbon monoxy-haemoglobin was considered dissociated when no further increase in optical density difference was found during alternative washings of the haemoglobin sample in the chamber with pure oxygen and nitrogen. Where needed haemoglobin solutions were concentrated using Sartorius ultrafiltration equipment. RESULTS

I. Gel filtration O n Sephadex G-100 gel-filtration experiments the hemolysate from Glycera 9igantea resolves into two components, H b H and HbL, which respectively comprise approximately 92 and 8')~i of the total haemoglobin. Comparing their partition coefficients, K , , (Laurent & Killander, 1964) with those of standard proteins, eluting from the same column, mol. wt of about 60,500 and 18,000 were estimated for the two forms of haemoglobin (Fig. 1). It is, however, significant that I

I

--

i

--

i

0.8 L

,.e..

0.4

" "~3"

2b H bH

o.o

'

....

si2

log mo[. wt.

Fig. 1. Partition coefficient, K~, during Sephadex gel column chromatography, as a function of mol. wt of marker proteins and of the high and low mol. wt components (HbH and HbL) of Glycera 9igantea. Marker proteins and their mol, wt are: 1, aldolase (147,000); 2a and 2b, human and horse haemoglobin (68,000); 3, oval-albumin (45,000); 4, chymotrypsinogen (25,700); 5, sperm whale myoglobin (17,800) and 6, cytochrome c (12,800).

AND

JOHN

F'. B o t .

the elution of H b H corresponded with those of both horse and human haemoglobins which according to sedimentation, osmometric and light-scattering measurements have tool. wt of about 64,000 to 68,000 (Rossi-Fanelli & AntoninL 1964). The elution volume of the minor component agrees well with that of sperm whale myoglobin (mol. wt 17.816- Edmundson & Hirs. 1962). These data indicate that Glycera gi~tamea haemoglobin in solution is in the tetrameric and monomeric states. In a haemoglobin sample that had been freezedried after dialysis against distilled water the ratio of HbH to HbL had increased to about 8 : 1, indicating dissociation of HbH into a monomeric f o r m I1. Sedimentation t,elocity The sedimentation constants were determined for

Glycera H b H and HbL, separated on a Sephadex G100 column as described above. Solutions of HbH and HbL with respective initial optical densities (O.D,) at 280 of 0'65 and 0.37 were used. For HbH a sedimentation constant, Scow, of 4"4 was found. This value equals that found for haemoglobins originating from the various classes of vertebrates (Svedberg, 1933) suggesting that H b H is tetrameric. HbL yielded a S2~w value of 1'6. showing a monomeric structure. This value is lower than that (1-91 typically lound for myoglobins (Svedberg & Pedersen, 1940: Rossi-Fanelli & Antonini, t964). For the monomer of G. dihranchiata values of 1-7 and 1-8 were found R)r the deoxygenated and oxygenated forms (Seamonds et al., 197 l b). Whereas HbL sedimented homogeneously a clear "tailing" was observed in the sedimentation of HbH, indicative of partial dissociation of the tetrameric structure under these conditions. III. SDS electrophoresis Both in 10 and -e~t".',,gels, the total haemoglobin from Glycera gigantea migrates as one component, with apparent tool. wt of respectively 12,800 and 13,000 dahons (Fig. 2, 3a,b). This indicates that the mol. wt of the polypeptide chains of H b H and HbL are the same. In view of the fact that the gel-filtration and sedimentation velocity of the Glycera H b H correspond with those of vertebrate tetramers, the relatively low tool. wt prestimably reflect inaccuracies of the relationship between mobility in the SDS gels and tool. wt, as are known to occur for proteins with mol. wt of about 15,000 daltons or less (Fish et al., 1970: Williams & Gratzer, 197t). In contrast to the homogeneity of the subunits of Glycera haemoglobin on the SDS-gels, the vascular "'erythrocruorin" of Arenicola marina on 12.5~, gels resolved into two xnain components indicating tool. wt of 12,300 and 13,700 daltons~-and several minor components of lower mol. wt (Fig. 2). On 0°~> gels only one major zone was. however, found, which migrated at a rate corresponding to 13,500 daltons. In view of the preincubation of the sample with a reducing agent, it is unlikely that the occurrence of the two main components for A. mari~la haemoglobin on 12-5',!0 gels can be ascribed to mutual differences in contormation or SDS-binding (Duncker & Ruckert. 1969). These results essentially agree with Waxman's (1971) finding that A. cristata haemoglobin contains two types of polypeptide chains, for which

Heterogeneity of bloodworm haemoglobin

25

---0

.e

B

C

736

,

708

'

6.92

•

6.78

,

6.63

e

O,S

A

....

D

E

F

G "---6.32

Fig. 2. Electrophoresis of haemoglobins from Glycera gigantea, Arenicola marina and of marker proteins on 12'5~o polyacrylamide gels in the presence of 0"1~o sodium dodecyl sulphate (SDS) at room temperature. A, B Arenicola haemoglobin (0.06mg and 0.12mg respectively); C, Glycera haemoglobin (0.07 mg); D, (top to bottom), chymotrypsinogen, horse heart myoglobin and Glycera haemoglobin; E, chymotrypsinogen, horse heart myoglobin and horse haemoglobin; F, sperm whale myoglobin and cytochrome c; G, sperm whale myoglobin and ribonuclease. The disc gels are shown with the origins at the top.

• •

6.20 6.12

• 5.90 ---5.60

Fig. 4. Section of 440ml preparative isoelectric focusing column showing heterogeneity of G. yigantea haemoglobin and iso-electric points of the main components. The focusing occurred at 5°C; pH values were measured at 10°C.

he reports mol. wt of 13,000 and 14,000. In Lumbricus terrestris Shalom & Vinogradov (1973) found evidence for six different polypeptide chains with mol. wt ranging from 12,000 to 36,000 daltons.

points (pI) of the components could not be exactly determined, due to the high degree of multiplicity and mixing incurred during the pumping out of the column contents. Peak haemoglobin concentrations were, however, found at pH values of about 7.6, 7.3, 7.0, 6.9, 6-7, 6.6, 6-4, 6'3, 6.1, 6.0 and 5.9, measured at 15°C (cf. Fig. 5, Table 1). As shown by Vesterberg & Svensson (1966) the pI value measured at a given temperature, represents the pI at that temperature, irrespective of the zone focusing temperature. Although general agreement was found in the pI values in three experiments (Table 1) differences in

IV. lsoelectric focusing Focusing a CO-saturated G. yigantea hemolysate in stable ampholine pH gradients, resolves the haemoglobin into about 14 components (Fig. 4). These were all bright red indicating that (hybrid) oxidation did not contribute to the observed heterogeneity. The pH values of the eluted fractions show that the isoelectric points of the component haemoglobins varied widely--over at least 2 p H units. The isoelectric

~4 .Q 0

]E

2 .A 4.0

i

~ I

42

i

L

4.4

i

I

4.6

i

B 4.B

£.0

'

4'2

'

4'.4

log mot. wt. Fig. 3. Relationship between mol. wt and migration of marker proteins and G. #igantea and Arenicola marina haemoglobins in A, 10~ and B, 12"5~opolyacrylamide gels in the presence of SDS. The proteins used and their mol wt are 1, pyruvate kinase (57,000); 2, glutamate dehydrogenase (53,000); 3, carbonic anhydrase (29,000); 4, chymotrypsinogen (25,700); 5, horse heart apomyoglobin (17,200); 6, horse apohaemoglobin (15,500) and 7, cytochrome c protein (11,700). The mobilities of proteins from Arenicola and Glycera haemoglobins are indicated by A and G, respectively.

26

RoY E. WEBERAND JOHNF. BOL

!8 ?

~

'

-

~

"D~L"V~-W.~ ~" ~

50 ....

i

, "~"~*~.,, 150

100 Fractions collected

~ 6

Fig. 5. Isoelectric focusing of G. gigantea haemoglobin. Continuous curve, recorded transmittance of" column contents at 280 nm; dashed curve, pH values of fractions collected, measured at 10:'C; volume of fractions, 2-4 ml: hatched areas, fractions pooled for subsequent oxygen binding studies (experiment 3, of Table I). relative concentrations were found between haemoglobins from worms collected at different times (respectively on 2 June and 23 August) so that some components that form well-defined peaks in the transmittance recordings of the one batch of worms were less well-defined in the haemolysates from the other batch.

V. Oxygen q~inity of the haemoglobin components Although a complete preparative separation of the T a b l e i. Isoelectric points

numerous components of G. gigantea haemoglobin by isoelectric focusing was not achieved (Fig. 5) the oxygen affinities of some of the more abundant components were determined in order to discern possible variations in their in vitro oxygen-binding function. To this end the pooled fractions retrieved from the column were dialysed for 24 hr at 4 ' C against three or more changes of CO-saturated phosphate buffer, ionic strength 0.1, pH 7-0 or 72. Since low column loading favours resolution of the numerous corn-

(pI) and half saturation oxygen tensions

(P50) of the main component carboxyhaemoglobins of Gl~cera ~i~antea, isolated by isoelectric focusing at 5 °C. pI values were determined using miniature columns (expt. i) and 440 ml preparative columns (expts.

2 and 3). pH values of the fractions collected from the

electrofocusing columns were measured at 15 °C in experiment 1 and 2~ in experiment 3 these were measured a£ 10 °C and then corrected to 15 °C using calibration curves of the pH values of ampholyte fractions retrieved from the columns, at these two temperatures. P50 values were measured at 20 °C using 1.0-1.5 mM haem solutions in phosphate buffer, ionic strength 0.1, pH 7.17. Experiment 1

Experiment 2

pl

pI

8.02

8.03

7.76 7.59 7.26 7.03

7.62 7.31 7.04

Experiment 3

£50 (torr)

pI

3.9,4.2,3.8

7.66 7.33 7.04

6.94

£50 (tort)

5.9,5.8,5.9

6.88

6.76 6.63

6.72

6.44 6.30

6.36 6.29

6.20

6.11

5.96 5.58

5.93

4.6,4.7 ] ~

9.0,9.2 11.5,12.8

6.73 6.58

3

6.38 6.26

I0.8,11.3 12.3,12.3,12.8

6.13 6.05

~

11.4,11.6

5.82 5.61

]

16.7,17.8

Heterogeneity of bloodworm haemoglobin ponents, insufficient haemoglobin was generally available for the removal of the carrier ampholytes by column chromatographic and precipitation techniques (Vesterberg, 1966; Nilsson et al., 1970). It is unlikely, however, that the effects of incomplete removal of the carrier ampholytes have influenced the measured oxygen affinities. After the dialysis the pH values of the different fractions were the same and the pH sensitivity of G. 9igantea haemoglobin is moreover very slight (Weber, 1973). In addition, in control experiments the two main myoglobin components of the polychaete Arenicola marina separated by isoelectric focusing (Weber & Pauptit, 1972a) showed the same differentiation in oxygen affinity irrespective of whether they were purified by the column chromatographic technique or by dialysis. As is evident from Table i, the isolated haemoglobins from Glycera gigantea show considerable variation in oxygen affinity, measured under the same conditions. At 20'~C the Ps0 values (Oxygen tensions that half-saturate the haemoglobin) vary from about 4 to 17 torr and bear a roughly inverse relationship with the pI of the protein. The fact that the components with pI values of approx 7.3 and 7-0 (at 15°C) had the lowest Pso values (about 4.0-5.9 Torr--see Table 11aligns well with the findings that in Glycera dibranchiata the components with the higher pI values are monomeric (Seamonds et al., 1971a; Weber et al., 1975) and that in this species the monomeric haemoglobin has lower Pso values than the polymeric fraction (Hoffman & Mangum, 1970; Seamonds & Forster, 1972; Mizukami & Vinogradov, 1972; Weber et al., 1975). Related to the low resolution, and the resultant difficulty to quantify the various haemoglobin components of G. 9igantea it is not possible to compute the oxygen affinity of the whole hemolysate exactly on the basis of the properties of the constituent components and their respective fractional contributions. The oxygen affinities of the main components (Table 1) are, however, compatible with the Pso value of 13 Torr previously found for the whole hemolysate (Weber, 1973) under the same conditions. DISCUSSION

The fact that the haemoglobin of Glycera 9igantea in solution is mainly tetrameric with a minor monomeric fraction illustrates the plasticity of the aggregation properties of haemoglobins in bloodworms of the family Glyceridae as a whole. The haemoglobin is dimeric in Glycera rouxii (Svedberg & Pedersen, 1940) while G. dibranchiata haemoglobin consists of monomers, and a polydisperse higher order aggregate making respective fractional contributions of about 55 and 45~o (Seamonds et al., 1971b). At present the possibility can, however, not be excluded that the minor monomeric fraction represents an in vitro dissociation product of HbH. Evidence for a dissociation/association equilibrium of HbH is provided by the asymmetric sedimentation pattern observed during analytical ultracentrifugation, and the fact that the mol. wt derived from gel filtration experiments is lower than expected for a tetrameric structure, although its subunits are of the same mol. wt as the monomer (SDS-polyacrylamide electrophoresis experiments). The oxygen affinity of

27

G. gigantea haemoglobin is moreover distinctly concentration-dependent (Weber, 1973) as in the polymer of G. dibranchiata, where the concentration-dependence correlates with molecular aggregation phenomena during deoxygenation (Mizukami & Vinogradov, 1972). By rechromatography of separated polymeric and monomeric haemoglobins of G. dibranJSchiata, Seamonds et al. (1971b), however, found that there is no interconversion within 4 days. That these haemoglobins in G. dibranchiata are not in equilibrium with one another is moreover evident from the fact that they seem to have no chains in common (Vinogradov et al., 1970) and that they show differences in amino acid compositions (Vinogradov et al., 1970; Seamonds et al., 1971a) and amin sequences (Imamura et al., 1972). Although the tetrameric structure characterizes the haemoglobins of nearly all vertebrates, it is rare in invertebrates. The tetramers reported to occur in the terebellid polychaete Amphitrite ornata (Love & Lattman, 1965) presumably represent dissociation products of the "'erythrocruorin-type" vascular haemoglobin (Mangum et al., 1974. The tetrameric structure of HbH in G. gigantea, correlates with a small but distinct sensitivity of Pso to ATP (Weber, 1973). As far as is known to us an effect of organic phosphates have not previously been demonstrated in invertebrates. The Ps0 of the high mol. wt extracellular haemoglobin from Arenicola marina is insensitive to ATP (Weber, Everaarts & Pauptit, unpublished), and the dimeric myoglobin from the gastropod mollusc Buccinum undatum similarly shows no response to ATP or DPG (2,3-diphosphoglycerate) (Terwilliger & Read, 1971). It thus seems that the tetrameric structure is essential for the allosteric interaction of organic phosphates, and that the cofactor binding in G. 9igantea may be similar to that in vertebrates, where it plugs the entrance to the central cavity of the molecule and interacts with the N-terminal amino acid residues of the p chains (Bunn & Briehl, 1970; Perutz, 1970; Arnone, 1972). Significant to these considerations seem the findings that the tertiary structure of the monomeric haemoglobin of G. dibranchiata greatly resembles the "myoglobin fold" of mammalian myoglobin and the subunits of mammalian haemoglobin (Padlan & Love, 1968,) and that 66 residues in its amino acid sequence are identical with corresponding sites in either the 7 or /3 chains of human haemoglobin, sperm whale myoglobin, or Petromyzon haemoglobin (Imamura et al., 1972). Similarly, G. dibranchiata haemoglobin has a similar degree of helical folding to human haemoglobin A, which is about twice that of the extracellular giant haemoglobin of Lumbricus terrestris (Harrington et al., 1973). Assuming that haemoglobins from such remotely related animals arose from a common polypeptide ancestor (Padlan & Love, 1968; Imamura et al., 1972), it is thus possible that allosteric interaction of organic phosphates with haemoglobin, is not a distinctive vertebrate "invention", but that it is inherent in the specific association in one molecule of four polypeptide chains each possessing the "myoglobinfold'-tertiary structure, which is moreover essential to solubilize the haem and to protect it from autoxidation. Of further relevance to these considerations would be studies of the organic phosphate effects in

28

RoY E. WEBERAND JOHN F. BOL

other invertebrate haemoglobins, particularly those with tetrameric subunit compositions, as have been reported in the echiurid worm Urechis caupo (Bonaventura & Kitto, 1973) and the clam Anadara iJ!flata (Sassakawa & Walter, 1971) and correlation with tertiary structure, subunit composition and N-terminal groups of the polypeptide chains. In the absence of data on the amino acid sequence and stereochemistry of the tetrameric haemoglobins of G. #iyaHtea that show ATP effects, the exact analogy of the cofactor effect to that in vertebrates, however, remains speculative. The homogeneity in subunit composition of the cellular haemoglobin of Glycera ,qigantea contrasts with the apparent heterogeneity in tool. wt of the constituent polypeptide chains in the haemoglobin of Arenicola marina. When considered in conjunction with a similar homogeneity and heterogeneity that has been found respectively in G. dibranchiata (Vinogradov et al., 1970; Seamonds et aL 1971h) and in A. cristata (Waxman, 1971), these results, however. appear to illustrate a distinction between the cellular haemoglobins and extracellular "'erythrocruorins" of annelids. The low moL wt obtained for G. giyantea subunits in the SDS-electrophoresis experiments (12,800-13,000 daltons)is remarkable. By using SDS electrophoresis, similarly low values have been obtained for the extracellular annelid haemoglobins (12,000 and 13,000 in Arenicola cristata, and 12,0(X) and higher in Lumbricus terrestris Waxman, 1971: Shlom & Vinogradov, 1973). Using the same technique, a single value of 17,400 daltons was however found for the whole hemolysate of G. dihram'hiata (Seamonds et al., 1971b), and sequence analyses of the monomer in this species show a value of 15,590 (lmamura et al., 1972). When considering in conjunction with these results the low values here obtained for horse haemoglobin (Fig. 3), it is clear that the inaccuracy of the SDS-electrophoretic tool. wt determinations of proteins in the low molecular ranges (Fish et al., 1970; Williams & Gratzer, 1971) applies particularly to haemoglobins, raising doubts on data of haemoglobin subunit composition that rests solely on SDS-electrophoresis results. The strikingly large variation in isoelectric points of the haemoglobin components in G. gigantea contrasts with the single pl value of 5.6 previously recorded for this species (Roche & Fontaine, 1940). The available information indicates that the giant extracellular haemoglobins of annelids are more homogeneous. For Arenicola marina Svedberg & Pedersen (1940) record a single pl value of 4.56 (confirmed by miniature column isoelectric focusing experiments of H. J. Koch & R. E. Weber, unpublished). The isoelectric focusing experiments show that those G. gigantea haemoglobin components, which on the basis of their abundance and distinctive pl values will be most readily detected by methods with a lower resolving power (e,g. electrophoresis) have pl values at 15"C of approx 7.3, 7.0, 6.7, 6.6. 6.3, 6.1, 5.9 and 5.6 Table 1. These values seem to show some alignment ,aith those of 7"4, 7'l, 6'5, 6.1 and 5'6 found electrophoretically by Seamonds et al. 11971a) for oxyhaemoglobin of G. dibranchiata at room temperature. Some of the differences are likely to be the result of different experimental conditions. The pl values

obtained by isoelectric focusing are dependent on the temperature of pH measurement and are lower at higher temperatures (Vesterberg & Svensson, 1966). The pI values are moreover dependent on ionic strength, which is higher in the electrophoresis experiments, and thus produces lower pl values (Vesterberg, 1973). These comparative data indicate that significant differences between G. gigamea and G. dibranchiata are the occurrence of well-defined components with pl (15C) at 6.7 and 6'3 in the former species. The correspondence, however, indicates the existence of homologous proteins in the two species. Comparing the two species, it will be evident that the G. ,qigantea components with pi values at 7.3 and 7.0 which presumably correspond to the monomeric components with pl values of 7.4 and 7.1 and account for more than half of the total haemoglobin in G. dibranchiata ISeamonds et al., 1971a}--constitutes a much smaller fraction of the total haemoglobin in G. ,qigantea (Fig. 5). This tallies with the fact that the monomeric fraction in G. gigantea is much smaller than in f,. dibranchiata (gel-filtration datat. The considerable differentiation in Ps0 values obtained for the different G. gigantea haemoglobin fractions suggests an internal division of labour between the different components. Such differences in oxygen affinity may be expected to contribute to the functioning of the whole haemoglobin under widely varying environmental oxygen tensions. With the aid of carbon monoxide inactivation of the haemoglobin, Hoffmann & Mangum (1970) found that at 20'~C G. dihram'hiata haemoglobin functions in oxygen transport over a range in oxygen concentration of at least I 7ml 02/1. The possibility of an interaction between the haemoglobin components, which would modify the in vivo oxygenation properties, has, however, to be borne in mind. For G. dihranchiata haemoglobin at pH 7.0 and 74, mutual interactions are indicated by the findings that the oxygen affinity of the combined fractions is not intermediate, but lower than those of the monomeric and polymeric fractions (Hoffmann & Mangum, 1970: Seamonds & Forster, 1972). that the Ps0 value of the whole hemolysate of (;. ,qi,qwztea shows good correspondence to the weighted mean of those of the main components, however, indicates that the effects of such interaction are insigniticant or absent in G. ,qigantea. Moreover, we have been unable to confirm the reported functional interaction of the monomer and polymer of G. dibramq~iata at pH 7'7 (Weber et al., 1975). Also. the Pso of the two main myoglobins from Arenicola marim~ combined, is a good mean of those of the separated components (Weber & Pauptit, 1972a). The functional differentiation of the component haemoglobins raises the question of possible quantitative variation in the components as adaptations to different ambient oxygen tensions. A variation in the relative concentrations of the components of G. gi.qantea has been observed in haemoglobins obtained from the two batches of worms used in the isoelectric focusing experiments (page 26). Similarly, densitometric analysis of electrophoretic runs show that the relative amounts of five main haemoglobins in G. dihram'hiata varies from one individual to another (Seamonds et al., 197161. Also, whereas G. dibram'hiata from the Maine coast has a ratio of polymeric to

Heterogeneity of bloodworm haemoglobin monomeric components of about 0.85 (Seamonds et al., 1971b), this ratio is about 0"3 in animals from more southern North Carolinian coasts (C. P. Mangum, Unpublished data). It appears that the cellular annelid haemoglobins show greater resemblance to the vertebrate haemoglobins and myoglobins than to the extracellular annelid haemoglobins--with which they may concur in the same individual animals--with regard to their monomeric and oligomeric structures, high degree of heterogeneity, comparatively high isoelectric points, and sensitivity to organic phosphates. This is also supported by correspondence in tertiary structure, as is evidenced by the higher degree of helical folding in Glycera dibranchiata and in human haemoglobin A than in the extracellular haemoglobin of Lumbricus terrestris (Harrington et al., 1973).

29

LOVE W. E. & LATTMANE. E. (1965) Crystalline hemoglobin from the marine annelid Amphitrite ornata. Hvalrhdets Skrifter 48, 110-111. MANGUM C. P. & CARHARTJ. A. (1972) Oxygen equilibrium of coelomic cell hemoglobin from the bloodworm Glycera dibranchiata. Comp. Biochem. Physiol. 43, 949 957. MANGUMC. P., WOODINB. R., BONAVENTURAC., SULLIVAN I. 8,~ BONAVENTURA J. (1974) The role of coelomic and vascular hemoglobins in the annelid family Terrebellidae. Comp. Biochem. Physiol. 51A, 281-284. MIZUKAM1H. • VINOGRADOVS. N. (1973) Oxygen association equilibria of Glycera hemoglobins. Biochim. biophys. Acta 285, 314-319. NILSON P., WADSTROMT. 8~, VESTERBERGO. (1970) Separation of proteins from carrier ampholytes after isoelectric focusing. Biochim. biophys. Acta 221, 146-148. PADLAN E. A. 8z, LOVEW. E. 0968) Structure of the haemoglobin of the marine annelid worm Glycera dibranehiata, at 5.5,~ resolution. Nature, Lond. 220, (5165), 376-378. Acknowledyements--I am thankful to Messrs. E. Pauptit and G. W. Kraay (Texel) and Miss Winnie Heidemann PERUTZ M. F. (1970) Stereochemistry of cooperative effects in haemoglobin. Bohr effect and combination with (Aarhus) for valuable technical help. Drs. D. H. Spaarorganic phosphates. Nature, Lond. 228, 734-739. garen (Texel) and W. L. Bakhuis (Amsterdam) helped in ROCHEJ. (1965) Electron microscope studies on high molecollecting the haemoglobin. cular weight erythrocruorins (invertebrate haemoglobins) and chlorocurorins of annelids. In Studies in Comparative Biochemistry (Edited by MUNDAY, K. A.) Vol. 23, pp. 23-80. Pergamon Press, Oxford. REFERENCES ROCHE J. & COMBETTER. (1937) Recherches sur les 6rythARNONE A. (1972) X-ray diffraction study of binding of rocruorins (h6moglobines d'Invert6brds). Bull. Soc. Chem. 2,3-diphosphoglycerate to human deoxyhaemoglobin. biol. 19, 613 626. Nature, Lond. 237, 146-149. ROCHE J. & FONTAINEM. (1940) Le pigment respiratoire BONAVENTURAJ. & KITXOG. B. 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SASSAKAWAS. & WALTERH. (1971) Blood clam (Anadara EDMUNDSONA. B. & HIRS C. H. W. (1962) On the structure in[tata) hemoglobins. Partition in ageous two-polymer of sperm whale myoglobin--l. The amino acid composiphase systems and alkali denaturation. Biochim. biophys. tion and terminal groups of the chromatographically Acta 244, 461465. purified protein, d. molec. Biol. 5, 663-682. SEAMONDS B. (1971) Anomalous binding of hydroxide, FISH W. W., REYNOLDSJ. A. & TANFORD C. (1970) Gel cyanide, azide, and fluoride ions with a Glycera methechromatography of proteins in denaturing solvents. moglobin. In Probes of Structure and Function of MacroComparison between sodium dodecyl sulfate and guanimolecules and Membranes. Probes of Enzymes and Hemodine hydrochloride as denaturants. J. biol. Chem. 245, proteins. Vol. 11. pp. 317 320. Academic Press, New 5166-5168. York. HARRINGTON J. P., PANDOLFELLIE. R. & HERSKOVITST. SeAMONDSB. & FORSTERR. E. (1972) Ligand equilibrium T. 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RoY E, WEBER AND JOHN F. BOt_

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The intracellular hemoglobins or a polychaete. J. biol. Chem. 245, (24), 6533- 6538. WAXMAN L. (1971) The hemoglobin o[ qremcola cri.stata. J. biol. Chem. 246, 7318 7327. WEBER K. & OSBORN M. (19691 The reliability of molecular weight determinations by dodecyl-sulfate potyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406 4412. WEB~:R R. E. (1971) Oxygenational properties of w~scuk,r and coelomic haemoglobins from Nephtys homber~/ii (Polychaeta) and their functional significance. Ncth. J. Sea Res. 5, 240 251. W[!BER R. E. (1973) Functional and molecular properties of corpuscular haemoglobin from the bloodworm (;lycera gigantea. Neth. J. Sea Res. 7, 316 327. WE,BER R. E. &, PAUPT1T E. (1972a) Molecular and l'unc tional heterogeneity in myoglobin from the polychete .4remcola marina L. Archs Biochem. Biophys. 148. 332 324. WIiBER R. E. & PAUPTIT E. 11972b) Vergelijkende studies over haemoglobinen wm mediterrane polychaeten, lcr~/. koninkl. Ned. Acad. WetensUz. 80, 155 160. WEBER R. E., SULLIVAN B., BONAVtNIURA J. ~z BONAVIN-

TURA C. (1975) Functional and structural properties of Glycera dibranchiata haemoglobins. (Unpublished data1. WILLIAMS J. G. & GRATZER W. B. (1971) Limitations of the detergent-polyacrylamide gel electrophoresis method for molecular weight determination of proteins..I. ('l,'(,matow. 57, 121 125.