Functional and molecular properties of corpuscular haemoglobin from the bloodworm glycera gigantea

Netherlands Journal of Sea Research 7 : 316-327 (1973) 7th European Symposium on Marine Biology

FUNCTIONAL AND MOLECULAR PROPERTIES OF CORPUSCULAR HAEMOGLOBIN FROM THE BLOODWORM GLYCERA GIGANTEA* by R. E. W E B E R * *

(Netherlands Institute for Sea Research, Texel, The Netherlands)

CONTENTS I. I n t r o d u c t i o n

. . . . . . . . . . . . . . . . . . . . . . . . . .

II. M a t e r i a l a n d M e t h o d s . . . . . . . . . . . . . . . . . . . . . . I I I . Results a n d Discussion . . . . . . . . . . . . . . . . . . A. W h o l e coelomic fluid . . . . . . . . . . . . . . . . . B. H b in solution . . . . . . . . . . . . . . . . . . . . I. p H effect . . . . . . . . . . . . . . . . . . . . . 2. t e m p e r a t u r e effect . . . . . . . . . . . . . . . . . 3. c o n c e n t r a t i o n effect . . . . . . . . . . . . . . . . 4. A T P effect . . . . . . . . . . . . . . . . . . . . 5. molecular differentiation of H b a n d O~-affinity . .

. . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . .

IV. G e n e r a l Discussion . . . . . . . . . . . . . . . . . . . . . . . . V. S u m m a r y

316 317 318 318 319 319 320 321 321 322 324

. . . . . . . . . . . . . . . . . . . . . . . . . . .

325

V I . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

325

I. I N T R O D U C T I O N

The ecological niches inhabited by annelid worms are characterized by a great diversity of respiratory conditions. Annelids accordingly exhibit a multiplicity of respiratory adaptations, which are important factors governing their distribution. With regard to "choice" of respiratory pigments, annelids show a similar lack of conservatism, possessing one or more types of pigments with a large range of structural and functional properties. The giant extracellular haemoglobin (Hb) and chlorocruorin molecules (molecular weight 2.7 to 3 × 10~) circulating in a closed blood vascular system are, however, most commonly found. The Hb of the burrowing

worm

Glycera is i n m a n y

ways opposite.

Glycerids lack a closed blood vascular system and the haemoglobin is * W o r k was carried out partly at Stazione Zoologica, Naples, Italy, d u r i n g tenureship of support from the N e t h e r l a n d s ' Ministry of E d u c a t i o n a n d Sciences. ** Present address: Zoophysiology L a b o r a t o r y , University of Aarhus, D e n m a r k .

HAEMOGLOBIN

FROM

GLYCERA

317

enclosed in corpuscles contained in a voluminous coelomic fluid. The pigment molecules are small: for G. rouxii SVEDBERG ~ PEDERSEN (1940) record a sedimentation constant of 3.5 suggesting a molecular weight of 34,500. The Hb of the American species G. dibranchiata has in recent years become a focal point for several molecular and physiological studies. Although PADLAN & LOVE (1968) record homogeneity in molecular weight, HOFFMAN & MANGUM (1970) reported the presence of 2 molecular species identified variously as monomers and dimers (VINoGRADOV, MACttLIK & CHAO, 1970) and as monomers and polymers (SEAMONDS, FORSTER & GOTTLEIB, 1971a; MANGUM & CARHART, 1972). From 02 uptake experiments on normal and CO-poisoned animals HOFFMAN& MANGUM(1970) have demonstrated the participation of the Hb in 02 transport at all ambient 02 tensions tested, including air saturation. The available information on functional characteristics of glycerid Hb is intriging but fragmentary and apparently limited to Glycera dibranchiata. The present report attempts the functional characterization of the Hb of the mediterranean G. gigantea with regard to the factors influencing O2-affinity, and the eco-physiological significance of the Hb to the organism. Some physico-chemical properties of the Hb are moreover reported to trace structure-function correlations.

Acknowledgements.--I am indebted to Messrs E. Pauptit and G. W. Kraay, Texel, for invaluable technical assistance. II. MATERIAL AND METHODS Specimens of Glycera gigantea Quatrefages, 1865 averaging 10 cm in length were collected subtidally from Mergellina Bay, Naples, Italy (where the tidal difference is small). The specimens collected burrowed in sediments with a median grain size of approximately 240 ~z, below about 3 metres of clear turbulent water. G. gigantea is an extremely agile predaceous polychaete. Most striking is its extremely large proboscis armed with strong jaws, which is shot out with flashing speed during preying and burrowing. Their burrows lack marked consolidation by mucous or surface oxidation of sediments and are periodically irrigated by undulatory body movements (LINDROTS, 1941; MANGUM & MIYAMOTO, 1970). For 02 binding studies of Hb solutions the coelomic fluid was collected in 0.9% NaC1 solution. The corpuscles were precipitated centrifugationally and lysed with 2 volumes of distilled water after which the red cell ghosts were separated by centrifugation. The Hb solutions were

318

R.E. WEBER

dialysed against NaC1 (ionic strength, I = 0.2) or phosphate buffer (pH = 7.2, I = 0.2) and concentrated by ultrafiltration. Where present autoxidation to methaemoglobin was countered by brief dialysis against 0.1% sodium dithionite in Tris-buffered NaC1, followed by extensive dialysis against CO saturated media. Hb-O2 equilibria were determined with the diffusion chamber method (SICK • GERSONDE, 1969) at 20°C (unless otherwise indicated). At this and lower temperatures no significant autoxidation occurred during the equilibrium registration. To investigate the influence of organic phosphates on Oz equilibrium, Hb solutions were first "stripped" chromatographically, on columns (30 X 5 cm) containing Sephadex G-25 Fine gel (BERMAN, BENESCH~Z: BENESCH, 1971) eluting the Hb with Tris buffered 0.1 M NaC1 solution (pH 7.56) at 54 ml/hour. Haemoglobins with different isoelectric point (pI) were separated by iso-electric focusing in stable pH gradiensts (VESTERBERG~: SVENSSON,1966) using 440 ml preparative columns. III. R E S U L T S

AND DISCUSSION

A. W H O L E C O E L O M I C F L U I D

The oxygen affinity of the red cells in vivo was estimated by recording 02 equilibria ofcoelomic corpuscles within 5 to 10 minutes after collection from fresh worms. The Hb in the corpuscles showed moderately %

OxyHb IO0,

80. ,/.1 f ' t ' / f ' 1 ' " "

60.

"~"

/ ..............................................................................................................; t . : ....................................................................................... / 40"

20'

/./'/" /' /./'/" .,/"/ "/"

pO2 (mm Hg)

Fig. 1. Oxygen equilibria of Glycera gigantea H b in coelomic corpuscles, coelomic fluid p H = 7.37 (continuous curve), and dissolved in Tris-HC1 buffer,/0H = 7.32, ionic strength, I = 0.2, H b concentration approximately 0.02 g/ml (broken curve).

HAEMOGLOBIN

FROM

319

GLYCERA

high oxygen affinity with ps0's of 7.2, 7.2 and 7.6 mm Hg in 3 determinations (Fig. 1). The equilibrium curves are weakly sigmoid with an n value (haem-haem interaction constant) of 1.8, indicating the presence of cooperative interaction between the haem units. The p H of coelomic fluid measured directly after extraction was 7.4 to 7.5. B.

HB

I.

IN

P H

SOLUTION

EFFECT

Haemolysed and dissolved in buffers a low Hb-O2 affinity was evidenced (Ps0 = 13 to 16 mm Hg, Figs 1 and 2a). It will also be seen that O~ affinity is essentially p H independent; the Bohr factor (A log Pso/ A pH) as represented by the drawn regression (Fig. 2a) being -0.02. The sigmoid coefficient n is similarly invariant o f p H (Fig. 2b). The absence of a p H influence on the haem-haem and proton-haem interactions show that the oxygen-affine centres in Glycera H b are independent of ionizable groups, and that there is no p H induced facilitation of oxygen delivery in organs bathed by the coelomic fluid. This seems correlated with the absence of a closed circulatory system and the related lack of a significant p H gradient between the gills and sites of oxygen delivery (cf. FLORKIN, 1949; ~V~ANGUM,1970).

~og

t~.-

i

P50

t °(ramHgl

P50

•

2

F

1.2-

0 A

•

•

A

fl 6

•

I.O-

t 2

o A

A

z,

•.

A

•

•

I

~0

810

~0

"

pH

f

SO

Fig.2. Influence ofpH on (a) the half-saturation oxygen tension (Pso) and (b) the Hill coefficient (n) of Glyceragigantea Hb solutions in phosphate buffers (solid symbols) and in Tris-HC1 buffers (open symbols). Circles and triangles refer to data obtained from separate Hb preparations, I = 0.2 and 0.1, respectively. Hb concentration approximately 0.02 g/ml.

320

R.E. WEBER

The absence of a Bohr effect in blood pigments of marine invertebrates is, however, not strictly correlated with the lack of a blood vascular system (MANWELL, 1959). In Glycera a selection pressure favouring pH insensitivity may moreover be explained by its tubiculous habitat where temporary increases in CO2 concentrations would otherwise interfere with 02 loading in the gills (cf. bullfrog tadpole Hb, MCCRUTCHEON, 1936). The pH insensitivity of the whole hemolysate of G. gigantea red cells is remarkable when compared with the finding that in G. dibranchiata the high molecular Hb component is essentially pH insensitive while the monomeric form has a BoAr factor of about--1.1 (HOFFMAN• MANGUM, 1970). 2. T E M P E R A T U R E

EFFECT

Increase in temperature results in decreased Hb-O2 affinity of Glycera Hb in accordance with the exothermic nature of oxygenation generally observed in respiratory proteins (Fig. 3). Based on these data the apparent heat of oxygenation, AH, derived from the integrated van 't Hoff equation: d logp,, AH = --2.303R 1

d7 25 12

20

15

temp. (°C) I0

?

\

log Pso

i4 P5o [mm Hg) b2

0,8 A H : -2,30

0.6

d (-~--)

"

\ z\

3:4

~'.~ 316 temp.(+ × IO')

Fig.3. Influence of temperature on the half-saturation oxygen tension (Ps0) of Glycera gigantea H b dissolved in phosphate buffer, p H = 7.17, I = 0.1. H b concentration 0.02 g/ml. Regression equation: log P~0 = (-9861.6 x ~1) + 10.820 ( N = 9; correlation coefficient, r = 0.99).

HAEMOGLOBIN

FROM GLYCERA

321

where R = gas constant, amounts to --13.1 kcal/mole. No correction was made for the heat of solution of oxygen (--3.1 kcal/mole at 25 ° C), or for effects of temperature-induced pH variation (due to the absence of a Bohr effect). The temperature sensitivity of Hb from subtidal Glyceragigantea thus conforms to that generally recorded for Hbs (AH ~- 10 to 14 kcal/ mole, ANTONINI & BRUNORI, 1970), in contrast to the intertidal Arenicola marina where the strongly reduced temperature effect found in Hb and myoglobin (WEBER, 1973, and unpublished data) appears adaptive to its greater ambient temperature variation. It would thus be of great comparative interest to determine the temperature influence on Hb of G. dibranchiata, which shows maximum population density at the low water mark (KLAwE & DICKIE, 1957). 3" C O N C E N T R A T I O N

EFFECT

Dissolved in phosphate buffer (pH 7.17, I = 0.1) solutions of Glycera Hb at 4.8, 2.4 and 1.2 g/100 ml showed Ps0 values of respectively 20.2, 18.6 and 16.5 m m Hg. This slightly positive correlation between concentration and Ps0 is in accordance with that generally found in Hbs (WYMAN, 1964), but fails to account for the high Ps0 values found after haemolysis, since the corpuscular Hb concentrations are high (HOFFMAN • MANGUM, 1970). 4" A T P E F F E C T

The intracorpuscular location of Hb in Glycera renders it of great interest for investigating the possible role of intracellular factors regulating Ps0 in invertebrates. Following BENESCH & BENESCH'S (1967) demonstration that allosteric binding of diphosphoglycerate (DPG) to deoxygenated mammalian haemoglobin depresses its oxygen affinity, analogous effects of DPG, adenosine triphosphate (ATP) and inositol hexaphosphate (IHP)--apparently correlated to environmental oxygen conditions--have become known in Hbs from all classes of vertebrates (JoHANSEN & LENFANT, 1972). As far as known no similar co-factor effect has, however, been demonstrated in invertebrate Hbs. To study the influence of organic phosphates on 02 equilibrium, ATP was chosen since it has a well-defined function in glycolysis and appears to be present in all (vertebrate) red cells investigated (RAPOPORT & GUEST, 1941). The results (Fig. 4) suggest a significant depressing influence of ATP on 02 affinity, analogous to that in vertebrates. Again, no significant co-factor binding was apparent at alkaline

322

R.E. WEBER

pH, suggesting that the negatively charged phosphate interacts electrostatically with positively charged amino acid residues of the protein. The effect on GlyceraHb was, however, small compared to vertebrates; l,l[

log

o

PSO

o .

1.08

pH

6.97 :

// t/

pH 7.'79

.......

"I"

1.00"

mole ATP/18,000g Hb

Fig.4. Effect of added ATP on oxygen aEmity of G~cera &iganteaHb, dissolved in Tris buffer, pH = 6.97 (open symbols) and 7.79 (solid symbols). Circles and triangles

refer to data from separate Hb preparations. in human Hb a DPG/Hb-tetramer ratio of 2.5 (molar concentrations) doubles theps0 value (BENESCH& BENESCH,1969). In the present experiments n was about 1.4 and showed no consistent variation with ATP concentration. Essentially the same results were obtained in 3 separate determinations. As far as known, this is the first demonstration of the influence of an organic phosphate on ps0 of invertebrate Hbs. The observations are moreover significant since--excepting the tetrameric Hb of the primitive coelacanth fish Latimeria (WooD, JOHANSEN • WEBER, 1972; WEBER, BOL, JOHANSEN & WOOD, 1972)--previous evidence for organic phosphate effects is limited to tetrameric Hbs yielding S-shaped Hb-O2 equilibrium curves (BENESCH & BENESCH, 1970). Evualation of the full significance to the biology of G. gigantea must, however, await estimation of intracorpuscular phosphates in relation to various acclimation conditions. It is moreover possible that other corpuscular molecules may exert more potent modifying effects on cellular p 5o. 5" MOLECULAR DIFFERENTIATION OF HB AND O2-AFFINITY

Molecular weight.--In Sephadex gel-filtration experiments red cell extracts revealed the presence of 4 molecular components. The elution volumes of the first ( × in Fig. 5) and last components corresponded to the limits of the fractionation range of the gel, indicating molecular weights respectively above 150,000 and below 5,000 daltons. These

H A E M O G L O B I N FROM GLYCERA

323

fractions, however, lacked the characteristic Soret absorption maxima near 410 nm, which characterize haemoproteins. Two peaks with intermediate elution characteristics showed high A410/A28o ratios and thus 0'0'280

Hb H

Blue Dextran

" i\/

~

/

~

. / I"~.~\

J HbL "\.

eluote volume (ml)

Fig.5. Chromatography of Glycera gigantea coelomic cell extract (continuous curve) and of Blue Dextran and whale myoglobin on column (1.5 × 60 cm) of Sephadex G-100 gel. Elution buffer ----0.05 M Tris buffer in 0.1 M NaCI, pH 6.8; flow rate 15 ml/hr. Optical densities at 280 nm monitored with an LKB Uvicord II absorptiometer. H b H and HbL indicate the heavy and light Hb components.

consist respectively of Heavy and Light Hb components (Fig. 5). In relation to elution rates of proteins with known molecular weights, masses of about 60,000 and 18,000 daltons were estimated for these indicating respectively oligomers and monomers. Sedimentation constants (S~0°,w) of 4.4 and 1.6 determined for these (WEBER & BOL, unpublished data) indicate tetrameric and monomeric compositions (SvEDBERG & PEDERSEN,1950). In freeze-dried material the Hb-H/HbL ratio was lower than in fresh material indicating dissociation of the oligomer into monomers. Iso-electric point.--In iso-electric focusing experiments (WEBER, unpublished data) Glyceragigantea carboxy-Hb could be resolved into at least 10 components, with iso-electric points of the main components at 15°C of about 7.3, 7.0, 6.7, 6.3, 6.1 and 5.6. These values show remarkable agreement with values of 7.4, 7.1, 6.5, 6.1 and 5.6 recorded for G. dibranchiata carboxy Hbs (SAEMONDS, FORSTER • GEORGE, 1971). Following their preparative isolation, the 4 Hb components apparently occurring in highest concentrations, showed marked differences in ps0 values (Table I). The fact that the component with pI of 7.4 has the lowest ps0 aligns with the finding that the analogous protein in G. dibranchiata is monomeric (SEAMONDS, FORSTER & GEORGE, 1971), and that the monomers appear to have higher oxygen affinity (HoFFMAN & MANGUM, 1970). Provided that intra-corpuscular conditions influence the component Hbs in the same fashion, these results suggest

324

R.E. WEBER

TABLE I Iso-electric points (pI) and half-saturation oxygen tensions (Ps0; determined in phosphate buffer, pH 7.17, I = 0.1 ; Hb conc. approximately 0.025 g/ml) of main Glyc~a gigantea haemoglobin components separated by electro-focusing. pI (COHb; 15 ° C)

P~o (ram02; 20 ° C)

7.3 6.7 6.3 6.1

3.9, 4.2,3.8 4.6, 4.7 9.0, 9.2 11.5,12.8

a marked in vivo functional differentiation of Glycera Hb which may in fact contribute to the observed functioning of the Hb over a wide range ofpO2 tensions (see page 317). IV. G E N E R A L

DISCUSSION

The Ps0 values recorded for Glycera gigantea Hb in the whole coelomic fluid (approximately 7 mm Hg) agree well with MANGUM& CARHART'S (1972) values for G. dibranchiata corpuscles in isotonic buffer solutions. The discrepancy from Hb solutions is, however, largely unexplained, but illustrates the danger of extrapolating the previously available data on Hb solutions to the situation in vivo. The Ps0 values in the animals, however, appear considerably lower than those (around 140 mm Hg) needed to half-saturate Hb in intact specimens of G. dibranchiata (MANGUM & CARHART,1972). Moreover 02 consumption experiments with G. dibranchiata (HOFFMAN• MANGUM, 1970) show that at 20 ° C the Hb remains functional at air saturation. This marked discrepancy indicates inefficient gills and a high resistence of the epidermis to Oz diffusion. In contrast to the highly sigmoid Hb-O2 equilibrium curves of some high molecular annelid Hbs (cf. WEBER, 1970), which are apparently adapted to Oz transport at low 02 activity, the strongly hyperbolic curve of Glycera Hb aligns with its functioning over a wide range of O2-tensions. This concurs with the absence of a Bohr effect (page 319), which in invertebrates is often correlated with oxyconformity (MANWELL, 1959) as has indeed been found in G. dibranchiata (HoFFMAN& MANGUM, 1970). These features show a lack of regulatory mechanisms to extend aerobiosis under annoxic conditions and imply a high tolerance of the tissues to anaerobiosis. These considerations are in accordance with the observed ability of G. dibranchiata to shut off red cell circulation through the gills at low O2-tensions (MANGUM, 1970).

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325

For an interpretation of the physiological significance of the ATP influence on GlyceraHb-O2 affinity analogy with vertebrate Hb cannot simply be assumed. The ATP effect in GlyceraHb is small and we have no knowledge as yet of corpuscular ATP concentrations and their variation in response to environmental conditions. The regulatory significance of ATP-induced variation in Ps0 moreover seems questionable in view of the large individual variation in O2-affinity generally found within the same invertebrate species suggesting a reduced need to maintain P~0 within very narrow limits. The differentiation in O2-affinities of component Glycera Hbs (recently also demonstrated for polychaete myoglobin, WEBER ~; PAUPTIT, 1972) open exciting further fields for research in view of possible differences in the effects of factors like temperature, pH and allosteric factors between the components, possible mutual interactions of these components, and possible quantitative variation in component composition as an adaptational response to environmental conditions. V. S U M M A R Y

Oxygen-binding and some molecular properties are reported for the Hb of Glyceragigantea, which contrasts to that in most annelids in being extravascular and intracellular. The 02 affinity of Hb in whole coelomic fluid is moderately high (Ps0 4- 7 mm). The Oz equilibrium curve is slightly sigmoid (n = 1.4 to 1.8). Variation in pH effects neither Ps0 (no Bohr effect) nor n, and the temperature sensitivity of O2-binding indicates an apparent heat of oxygenation (AH) of = --13 kcal/mole. At acid pH added ATP decreases the O2-affinity analogous to the influence of organic phosphates in vertebrates, but this effect is slight and its functional significance is questionable. Glycera red cells appear to contain tetrameric as well as monomeric Hb. On the basis of iso-electric point the Hb resolves into numerous components. Four main components isolated preparatively show marked differentiation in oxygen affinity. The results are discussed in relation to the burrowing habitat and compared with available data on Hb from the American G. dibranchiata and the high molecular annelid Hbs. VI. R E F E R E N C E S ANTONINI, E. & M. BRUNORI, 1971. Hemoglobin and myoglobin in their reaction with ligands. In: A. NEUBERGER & E. L. TALUM. Frontiers of Biology 21 NorthHolland Publishing Company, Amsterdam.

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BENESCH, R. & R. E. BENESCH, 1967. The effect of organic phosphates from the human erythrocyte on the allosterie properties of hemoglobin.--Biochem, biGphys. Res. Commun. 26 (2) : 162-167. , 1969. Interaction of red cell organic phosphates with hemoglobin.--F6rvarsmedicin 5: 154-158. BENESC~, R. E. & R. BENESCH, 1970. Control mechanisms for oxygen release by hemoglobin. The reaction between diphosphoglycerate and hemoglobin.-Fedn Proc. Fedn Am. Socs exp. Biol. 29 (3) : 1101-1104. BEmMA~, M., R. BENESCH & R. E. BENESCH, 1971. The removal of organic phosphates from hemoglobin.--Archs Biochem. Biophys. 145: 236-239. FLORKIN, M., 1949. Biochemical evolution. Acad. Press, New York: 1-157. HOFFMAN, R . J . & C. P. MANOUM, 1970. The function of coelomic cell hemoglobin in the polyehaete Glycera dibranchiata.--Comp. Biochem. Physiol. 36:211-228. JOHANSEN, K. & C. LENFANT, 1972. A comparative approach to the adaptability of O2-Hb affinity. In" Oxygen affinity of hemoglobin and red cell acid-base status. Munksgaard, Copenhagen: 750-780. KLAWE, W. L. & L. M. DICKIE, 1957. Biology of the bloodworm Glycera dibranchiata Ehlers, and its relation to fish of the maritime provinces.--Bull. Fish Res. Bd Can. 115" 1-37. LINDROTH, A., 1941. Atmungsventilation der Polych~iten.--Z. vergl. Physiol. 28 (5) : 485-532. MANGUM, C. P., 1970. Respiratory physiology in annelids.--Am. Scient 58 (6): 641-647. MANCUM, C. P. & J . A. CARHART, 1972. Oxygen equilibrium ofcoelomic cell hemoglobin from the bloodworm Glycera dibranchiata.--Comp. Biochem. Physiol. 43 (4A) • 949-957. MANGUM, C. P. & D. M. MrVAMOTO, 1970. The relation between spontaneous activity cycles and diurnal rhythms of metabolism in the polychaetous annelid Glycera dibranchiata.--Mar. Biol. 7" 7-10. MAXWELL, C., 1959. Oxygen equilibrium of Cucumaria miniata hemoglobin and the absence ofa Bohr effect.--J, cell. comp. Physiol. 53: 75-83. McCRUTCHEON, F. H., 1936. Hemoglobin during the life history of the bullfrog.-J. cell. comp. Physiol. 8" 63-81. PADLAN, E. A. & W. E. LOVE, 1968. Structure of the haemoglobin of the marine annelid worm Glyceradibranchiata, at 5.5 A resolution.--Nature, Lond. 220 (5165) -" 376-378. RAPOPORT, S. & G. M. GUEST, 1941. Distribution of acid-soluble phosphorus in the blood cells of various vertebrates.--J, biol. Chem. 138: 269-282. SEAMONDS,B. & R. E. FORSTER, 1972. Ligand equilibrium and kinetic characteristics of Glycera dibranchiata hemoglobins.--Am. J. Physiol. 223: 734-738. SEAMONDS,B., R. E. FORSTER • P. GEORGE, 1971. Physico-chemical properties of the hemoglobins from the common bloodworm G~cera dibranchiata.--J, biol. Chem. 246 (17) : 5391-5397. SEAMONDS,B., R. E. FORSTER• A.J. GOTTLEIB,1971. Heterogeneity of the hemoglobin from the common bloodworm Glycera dibranchiata.--J, biol. Chem. 246 (8) : 1700-1705. SICK, H. & K. GERSONDE, 1969. Method for continuous registration of Oz-binding curves of haemoproteins by means of a diffusion chamber.--Analyt. Biochem. 32 : 362-376. SVEDBERG,T. & K. 0. PEDERSEN,1940. Die Ultrazentrifuge, Theorie, Konstruction und Ergebnisse. Theodor Steinkopff Verlag, Dresden: 1-436. VESTERBERG,0. ~£ H. SVENSSON,1966. Isoelectric fractionation, analysis and charac-

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terization of ampholytes in natural pH gradients. IV. Further studies on the resolving power in connection with separation of myoglobin.--Acta chem. scand. 20: 820-834. VINOGRADOV,S. N., C. A. MACHLm & L. L. CHAO, 1970. The intracellular hemoglobins of a polychaete.--J, biol. Chem. 245 (24) : 6533-6538. WEBBR, R. E., 1970. Relations between functional and molecular properties of annelid haemoglobins. Interactions between haems in the haemoglobin of Arenicola marina L.--Comp. Biochem. Physiol. 35: 179-189. --, 1973. On the variation in oxygen binding properties of haemoglobins of lugworms (Arenicolidae, Polychaeta). Proc. Vth Eur. Mar. Biol. Symp., Venice, Italy, Oct. 1970. Piccin Publ., Padova: 231-243. WEnER, R. E., J. F. Bol, K. JOHANSEN• S. C. WOOD,1973. Physicochemical properties of the hemoglobin of the coelacanth Latimeria chalumnae.--Archs Biochem. Biophys. 154 (1) : 96-105. WEBER, R. E. & E. PAUPTtT, 1972. Molecular and functional heterogeneity in myoglobin from the polychete Arenicola marina L.--Archs Biochem. Biophys. 148: 322-324. WOOD, S. C., K. JOrIANSEN& R. E. WEBER, 1972. Haemoglobin of the Coelacanth.Nature, Lond. 239: 283-285. WX'~AN, J., 1964. Linked functions and reciprocal effects in hemoglobin: a second look.--Adv. Protein Chem. 19: 263.