Oxygen binding domains of a clam (Cardita Borealis) extracellular hemoglobin

77

Biochimica et Biophysica Acta, 537 (1978) 77--85 © Elsevier/North-Holland Biomedical Press

BBA 38033

OXYGEN BINDING DOMAINS OF A CLAM (CARDITA BOREALIS) E X T R A C E L L U L A R HEMOGLOBIN

NORA

B. T E R W I L L I G E R

and R O B E R T

C. T E R W I L L I G E R

Department of Biology, University of Oregon, Oregon Instituteof Marine Biology, Charleston, Oreg. 97420 (U.S.A.) (Received March 20th, 1978)

Summary The extracellular hemoglobin of the heterodont clam, Cardita borealis, is an unusual heme protein. The structure of the pigment as it occurs in the clam consists of a long rod-shaped p o l y m e r with widths of 210--365 /~ and lengths of 365--1200 .~ Chromatography on Sepharose 4 B at neutral pH in the presence of divalent cations suggests that the pigment has an apparent molecular weight of 12 • 106 and is heterogeneous. The subunit of Cardita hemoglobin has a molecular weight of 300 000 daltons in striking contrast to the 15--17 000 dalton subunits of most hemoglobins. However, Cardita hemoglobin, like other hemoglobins, contains one heme per 17--20 000 g protein. Cardita hemoglobin can be dissociated to a heme-containing submultiple in low ionic strength buffer (pH 9.5). Gentle proteolysis of the pigment under these conditions with subtilisin cleaves the protein into heme-containing fragments (domains} with molecular weights of 15--17 000 and integral multiples thereof. These domains, isolated b y gel chromatography, have one heme per approximately 17 000 g protein and bind oxygen reversibly. The amino acid compositions of the isolated domain fractions are very similar to one another and to the intact pigment. The oxygen binding properties of the intact pigment show neither a Bohr effect nor cooperativity between pH 7.0 and 8.0. Oxygen binding properties of the 15--17 000 dalton domains are essentially the same as those of the intact pigment. These observations support the hypothesis that the subunits of Cardita hemoglobin consist of a series of covalently linked oxygen binding domains. It appears that the unique 1 2 . 1 0 6 dalton quaternary structure of this pigment as well as its peculia 300 000 dalton subunit does n o t confer any functional properties on this protein that are n o t present in its smallest oxygen binding domain.

78 Introduction The extracellular hemoglobin of the heterodont clam, Cardita borealis, has a very unusual molecular structure. In electron micrographs, the pigment appears as rod-shaped particles with approximate dimensions of 210--365 • in width and ranging in length from 365--1200 .~ [1]. The hemoglobin chromatographs on Sepharose 4~ at neutral pH as a broad peak reflecting the heterogeneity in length seen in the electron microscope and has an apparent molecular weight of 12 • 106. In contrast to most hemoglobins which have been studied, Cardita hemoglobin can n o t be dissociated by conventional means of protein denaturation into low molecular weight (15--17 000 dalton) polypeptide chains [1,2]. Rather, denaturation of the pigment with sodium dodecyl sulfate (SDS) in reducing agent produces a subunit with a molecular weight of approximately 300 000. The pigment, however, does contain one heme per 17--22 000 g protein, a value similar to heme contents of other hemoglobins [1,2]. Recent studies of another unusual molluscan hemoglobin, that of the planorbid snail Helisoma trivolvis, have shown that this 1.75 • 106 dalton, ring shaped pigment also has a high molecular weight subunit (175 000 daltons) [3]. The Helisoma subunit can be cleaved by gentle digestion with subtilisin into heme containing polypeptides with molecular weights of 15 000 or integral multiples thereof [4,5]. Other planorbid species have also been shown to contain a hemoglobin with a high molecular weight subunit [2,6]. The subtilisin generated fractions of Helisoma hemoglobin are capable of binding oxygen reversibly, and the amino acid composition of the various fractions suggest that the heme-containing unit of this pigment may belong to the globin family of proteins [4,5]. In this paper, we report that Cardita hemoglobin can be cleaved by gentle digestion with subtilisin into 15--17 000 molecular weight oxygen binding domains. Thus both Cardita and Helisoma hemoglobin subunits may share a similar linear arrangement of covalently linked domains, even though the molecular weight o f the Cardita hemoglobin subunit as well as its association into a huge rod shaped p o l y m e r are remarkably different from those of Helisoma hemoglobin. Some functional relationships of the intact hemoglobin and its isolated domains are also presented. Materials and Methods

Cardita borealis (Conrad) was obtained from the Woods Hole supply department, Woods Hole, Mass. The clams were stored in running sea water until use. Hemoglobin was removed with a pipette from opened clams into a beaker of ice cold 0.05 M Tris-HCl buffer (pH 8.0), 0.1 M in NaC1, 0.01 M in MgC12, 1 mM in phenylmethylsulfonylfluoride. After centrifugation at 10 000 X g for 10 min, the supernatant was purified on a 1.8 X 70 cm column of Sepharose 4B at 4°C in equilibrium with the extraction buffer w i t h o u t phenylmethylsulfonylfluoride. The broad hemoglobin peak was concentrated in the cold on solid sucrose and chromatographed on a 1.8 X 27 cm column of Sephadex G-25 in equilibrium with 0.01 M sodium glycinate buffer (pH 9.5). This treatment dissociates the p i g m e n t into a 1.4 X 106 dalton or lower molecular weight submultiple as determined by Sepharose 4B chromatography [ 1 ].

79 Digestion of the dissociated hemoglobin was carried out in the pH 9.5, 0.01 M sodium glycinate buffer at 22°C. The pigment was digested for one hr with subtilisin (Carlsburg Type VIII, Sigma Chemical Co.) and the reaction stopped by addition of phenylmethylsulfonylfluoride. The concentration of enzyme used relative to the hemoglobin was 1 mg enzyme per 50 mg protein. A control aliquot of the pH 9.5 sample was treated in the same way without the addition of subtilisin. The digestion products were separated on a Sephadex G-100-120 column (1.8 X 80 cm) in equilibrium with 0.05 M Tris-HCl buffer (pH 8.0), 0.1 M NaC1, 0.01 M in MgC12 at 4°C. Sodium dodecyl sulfate gel electrophoresis was carried out on 1.5 mm slab gels [7] with a discontinuous buffer system [8]. Gel concentrations of 7.5%, 10% and 12.5% were used with a constant ratio of acrylamide to bisacrylamide of 30 : 0.8. The samples were first denatured in boiling incubation buffer containing 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol and 1 mM phenylmethylsulfonylfluoride for 1.5 min at 100°C [9]. Calibrants included phosphorylase A, bovine serum albumin, ovalbumin, ~-chymotrypsinogen A and sperm whale metmyoglobin {Sigma Chemical Co.). Amounts of protein applied to the gels were varied between 2--50 pg. The gels were stained in Coomassie Blue according to Fairbanks et al. [10]. Regular disc gel electrophoresis of the digested samples was performed on the carbonmonoxy derivatives [11]. Amino acid analyses of the domain fractions were carried out as described by Spackman et al. [12]. Oxygen binding of both the intact hemoglobin and the fractions separated by gel chromatography were studied spectrophotometricaUy [13] with a Zeiss PMQ II spectrophotometer equipped with a temperature controlled cell holder. Samples were dialysed versus the appropriate buffer before analysis. The pigment appeared to be very unstable with a tendency to form methemoglobin during the binding experiments. The oxygen binding experiments, therefore, were carried out in the presence of the reductase system described by Hayashi et al. [14]. Results

Hemoglobin structure Digestion of the oxyhemoglobin by subtilisin at pH 9.5 in 0.01 M sodium glycinate buffer results in the production of a heme-contalning polypeptide with a molecular weight of about 16 000 plus fragments with molecular weights that are near integral multiples of this value. Enzyme to protein ratios (w/w) of 1/1000, 1/500, 1/250 and 1/50 were used in digestion experiments and the digestion products analysed by sodium dodecyl sulfate slab gel electrophoresis. In all cases, heterogeneity with respect to molecular weight was observed. The 1/1000 enzyme to protein ratio yielded mostly high molecular weight fragments, about 60 000 and above, and only small amounts of material lower than 60 000. With increasing enzyme concentration, a corresponding increase occurred in bands representing material with molecular weights smaller than 60 000. Concomitantly there was a decrease in the higher molecular weight material. When the 1/50 digest is chromatographed on Sephadex G-100, two heme containing fractions are resolved {Fig. 1}. The

80

24

b J I I

I I

c I I I

g

<

os

.~.,!

04

40

80

120 ml

160

effluent

Fig. 1. C h r o m a t o g r a p h y o n S e p h a d e x G - 1 0 0 of a o n e h o u r digest of C a r d i t a h e m o g l o b i n ( 0 . 0 1 M s o d i u m glycinate buffer, pH 9 . 5 , mg subtilisin to m g h e m o g l o b i n 1 / 5 0 , 22°C). C o l u m n v o l u m e 1.8 X 8 0 cm. C o l u m n b u f f e r 0 . 0 5 M Tris-HCl0 0.1 M in NaCI, 0 . 0 1 M MgC] 2, pH 8.0. Calibrants: A, b o v i n e serum albumin; B, ~ - c h y m o t r y p s i n o g e n A; C, sperm w h a l e m y o g l o b i n .

larger peak has a leading shoulder corresponding to an apparent molecular weight of about 94 000 and a main c o m p o n e n t of molecular weight about 78 000. The minor peak corresponds to an apparent molecular weight of about 20--25 000. Analysis of the fractions under the bars in Fig. 1 on a 12.5% sodium dodecyl sulfate slab gel are presented in Fig. 2. The molecular weight values were collated from 7.5% and 12.5% gels. The control sample (Fig. 2A) consists of a band corresponding to 300 000 molecular weight material (precise value obtained from a calibrated 4% sodium dodecyl sulfate gel) as well as some maA

B

C

D

E

F

G

--100000 Im

m e

m

--

70000

--

57000

--

47000

--

37000

--

31000

--

22000

--

16500

Fig. 2. S o d i u m d o d e c y l sulfate gel e l e c t r o p h o r e s i s patterns of p r o d u c t s f o r m e d during subtilisin digestion of Cardita h e m o g l o b i n . Gel c o n c e n t r a t i o n 12.5% acrylamide. A , control; B, F r a c t i o n I; C, F r a c t i o n II; D, Fraction III; E, Fraction IV; F, Fraction V; G, w h o l e digest.

81

terial which is retarded at the top of the spacer gel. Fraction I (Fig. 2B) consists almost entirely of material with a molecular weight of 16 500 plus a trace of 30--31 000 dalton material. Some lower molecular weight material (13--14 500) is also present in small amounts. No prominent bands are evident in Fraction II (Fig. 2C). There are small amounts of material with molecular weights of 16 500, 22 000 and 31 000. An additional component corresponding to 34-37 000 daltons, not found in Fraction I, is present in Fraction II. The absence of any dominant band as well as the molecular weights of the components present is consistent with the elution position of Fraction II between the two major peaks in Fig. 1. Fraction III (Fig. 2D) analysed by sodium dodecyl sulfate electrophoresis shows prominent bands of 34--37 000, 47--52 000 and 57--60 500 with the most strongly staining bands at the 57--60 500 molecular weight region. There is a trace of some material with a molecular weight of about 100 000. The 100 000 molecular weight value is only approximate, since the highest calibrant on the 7.5% gel, phosphorylase A, has a molecular weight of only 94 000. However, the extrapolation is reasonably accurate. Fraction IV (Fig. 2E) consists of strongly staining bands with molecular weights of 49 500-52 000 and 57--60 500. There is a small amount of 100 000 molecular weight material as well as a series of bands between 64 000--71 000. There are also traces of lower molecular weight molecules which correspond to the bands seen in Fraction III. Fraction V (Fig. 2F) contains 70 000 and 100 000 molecular weight components. Most of the material is in the 100 000 dalton category. In all the fractions there is some evidence of heterogeneity with respect to molecular weight. However, the prominent bands in the gel analysis of each fraction correspond to its elution position on Sephadex G-100.

TABLEI AMINO ACID C O M P O S I T I O N OF CARDITA H E M O G L O B I N AND D I G E S T I O N P R O D U C T S

Amino acid

Intact *

Fraction I **

Fraction I I I **

Fraction I V **

Fraction V **

Lys Us ~g ~P ~r Set Gin ~o GIy ~a 1/2Cys Val Met He ~u Tyr Phe

6.9*** 6.3 3.7 12.2 4.9 4.9 11.0 2.5 5.4 6.0

7.4 6.5 2.9 11.1 5.2 5.8 12.7 2.5 6.3 9.2

7.5 8.5 2.4 13.8 4.8 4.3 11.5 2.4 4.7 6.9

7.5 7.3 2.9 14.4 4.9 4.2 10.7 2.0 4.7 6.8

7.4 7.3 3.0 14.5 5.2 4.2 11.0 1.9 5.0 8.6

5.7

5.8

5.3 10.3 3.2 7.6

5.4 9.7 3.1 7.7

.

.

6.5 1.9 6.1 9.9 2.8 7.4

.

.

6.0 .

.

5.5 .

5.2 8.6 2.7 7.6

* Terwflliger et al. ( 1 9 7 7 ) Cys and Trp e x c l u d e d . ** C o m p o s i t i o n f r o m single 2 4 h h y d r o l y s i s . *** E x p r e s s e d as t o o l per c e n t ,

. 5.1 10.1 3.1 7.0

.

82 08

0.4

-04

-Q2

/ /

02

06 Log

10

P02

Fig, 3. O x y g e n e q u i l i b r i u m c u r v e s o f i n t a c t Cardita h e m o g l o b i n • . . . . . . • and the m o n o m e r i c d o m a i n , Fraction I • e . B u f f e r 0 . 0 5 M Tris-HC1, 0.1 M in NaCl, 0 . 0 1 M in M g C I 2 , p H 8.0. T e m p e r a t u r e 20°C.

Aliquots of the fractions from Fig. 1 which contained prominent bands on sodium dodecyl sulfate gel electrophoresis were analyzed for their amino acid compositions. When the amino acid compositions of Fractions I, III, IV and V (Table I) are compared with the intact pigment, it is evident that within the error of a single amino acid analysis, the various fractions are remarkably similar. The ratio of absorbance at 415 nm (oxyheme) to 280 nm (protein) of the 16 500 molecular weight domain as well as all the other fractions is similar to that of the intact pigment.

Oxygen equilibrium Oxygen equilibrium studies of the intact pigment (heterogeneous 1 2 . 1 0 6 dalton aggregates) were carried out between pH 7 and 8. There is no effect of pH on the oxygen equilibrium of the pigment, and the hemoglobin lacks T A B L E II OXYGEN BINDING PROPERTIES OF CARDITA

HEMOGLOBIN AND PROTEOLYTIC

Sample

Log P1/2 **

N ½ **

Intact pigment Fraction V * Fraction IV * Fraction I *

0.630 0.270 0.300 0.630

0.90 1.05 1.08 0.97

• F r a c t i o n s as s h o w n in F i g . 1. • * R e p r e s e n t average values f r o m at least t w o sepaxate e x p e r i m e n t s .

FRAGMENTS

83 TABLE III ABSORPTION SPECTRUM MAXIMA OF C A R D I T A

HEMOGLOBIN

Pigment

Soret ~max

~ ~max

a ~max

Oxyhemoglobin Carbonmonoxyhemoglobin Deoxyhemoglobin

415 421 432

540 538

575 56 S 556

cooperativity with N l n = 0.90 (Fig. 3). The relatively high oxygen affinity of the intact pigment (Pl/2 = 4.5 mmHg at 20°C) is also seen in the domain fractions as shown in Table II. The domain fractions do bind oxygen reversibly but like the intact pigment they show no cooperative oxygen binding properties. The oxygen affinities of fractions V and IV (P1/2 = 2.0 mmHg) are slightly higher than those of either the intact pigment or fraction I (PIn = 4.3 mmHg). The presence of some methemoglobin in all fractions during the binding experiments, even with the reductase system present, make one cautious of attributing any significance to these variations. Relatively good isosbestic points were recorded during the oxygen equilibrium experiments. Absorption spectra data for Cardita hemoglobin are shown in Table III. Discussion

In a previous study [1], we proposed that the high molecular weight subunit of Cardita hemoglobin consists of a linear series of oxygen binding domains. The inability of the pigment to be dissociated to a polypeptide subunit with a molecular weight less than 300 000 by conventional means of protein denaturation together with the observation that the pigment contains one heme group per 17--20 000 g protein [1,2] were consistent with this hypothesis. The results of our present study support the hypothesis that this pigment is composed of a linear series of domains with molecular weights of about 16 000 and integral multiples thereof. At high pH in low ionic strength buffer, the large polymeric rod-like structures dissociate into lower molecular weight assemblages of approximately 300 000 daltons. When this smaller component is digested with subtilisin, the digestion mixture chromatographs on Sephadex G-100 (Fig. 1) into fractions which when analysed on sodium dodecyl sulfate gel electrophoresis appear to have molecular weights of 16 000 or integral multiples of this value (Fig. 2). Although there are bands present which represent molecular weights intermediate in value such as 22 000 daltons, the major bands correspond to 16 500, 34--37 000, 47--52 000, 60 000 and about 100 000 daltons. A number of different digestion conditions were attempted without any striking variations in the sodium dodecyl sulfate banding patterns, which lends further support to an ordered repetitive structure in the native polypeptide chain. Based on the molecular weight of the intact subunit (290--300 000), it should contain about 20 domains. We do not know whether the various 16 000 dalton domains are identical. The heme-containing domains appear to be held together by covalent bonds

84 or bonds resistent to the usual means of protein denaturation. This structure may be the result of a tandem duplication of the globin gene with the myoglobin-like domains linked covalently via a polypeptide bond. On the other hand, the linked myoglobin-like subunits may be the result of a post transcriptional modification, which could occur at any one of a number of stages of protein synthesis from the level of free aminoacetyl-tRNA to the level of the completed protein released from the messenger R N A - r i b o s o m e complex [15]. We have noticed some unidentified residues in the amino acid analyses which suggest that some derivatization of amino acids or carbohydrate might be possible. The presence of carbohydrate in this protein is n o t unlikely. The extracellular hemoglobin of the gastropod mollusc, Biomphalaria, contains some carbohydrate [16] and we have shown that Helisoma, another planorbid closely related to Biomphalaria, has a hemoglobin subunit like Cardita whose structure seems to be a linear series of myoglobin-like domains. The amino acid compositions of the subtilisin generated fragments (Fig. 1) which contain the major bands on sodium dodecyl sulfate gel electrophoresis (Fig. 2) are similar to one another as well as to the intact pigment. Thus the cleavage of the protein b y subtilisin, a relatively nonspecific protease, is quite specific. This implies that the domains produced by the digestion are probably very similar in sequence. Furthermore, the domains must form rather compact tertiary structures which are resistant to the proteolysis. If this is true, one would expect that the enzyme would be more likely to cleave the polypeptide in the region between the tight domain structures rather than the domains themselves. The results support this interpretation. The presence of some minor bands whose molecular weights are n o t multiples of 16 000 can be explained by limited digestion of regions of the c o m p a c t domain fragment which are more accessible to the enzyme. The oxygen binding of the intact Cardita hemoglobin shows no cooperativity nor pH dependency under the conditions investigated. Studies by Manwell [17] on the extracellular hemoglobin of a related species, Cardita floridana, indicate that the oxygen affinity of this pigment is also insensitive to pH and does not possess cooperativity since N,/2 is slight less than one. Manwell mentions that the hemoglobin of C. floridana is also sensitive to oxidation and apparently is unstable as we found for C. borealis hemoglobin. The apparent lack of homotropic and heterotropic interactions in a molecule with such a complex quaternary structure ( 1 2 . 1 0 6 dalton aggregate) is indeed perplexing. The results of oxygen equilibrium studies on the isolated subtilisin-digested products show that the 16 000 dalton heme-containin~, domains are able to bind oxygen reversibly; they are n o t only structural b u t functional entities. The oxygen binding properties of the 16 000 dalton domains, which show no evidence of cooperativity nor a Bohr effect, are consistent with those found for the intact pigment. The higher oxygen affinities of both Fractions IV and V (Table II) are n o t easily explained. The fact that the isosbestic points were constant for all fractions argues against the possibility of methemoglobin formation during oxygen binding causing the differences. One possible explanation is that the intermediate-sized polydomain mixtures may assume unusual aggregated states which in turn may affect their oxygen affinities. The intact hemoglobin as well as Fraction I (Table II) show N,/2 values of less than one. This could be

85

attributed to functional heterogeneity of the domains. However, the presence of some methemoglobin in the intact pigment as well as in the digested samples prior to the oxygen binding experiments suggests that such a conclusion from this data is n o t appropriate. One can conclude, however, that there is probably little, if any, cooperativity in either the intact 12 • 106 dalton aggregates or the domain fractions. Cardita hemoglobin, an unusual heme protein, appears to be composed of sununits which consist of a linear series of oxygen binding domains. Although the size of the subunit as well as the quaternary structure of the intact (12 • 106 dalton) molecule are strikingly different from those of Helisoma hemoglobin, the subunits of these two heme proteins do share structural similarities. Furthermore, a subunit consisting of a linear series of oxygen binding domains is a prevalent molecular pattern seen in the molluscan hemocyanins [ 18,19]. Acknowledgements We are grateful to Dr. Kobert Becker, Department of Biochemistry and Biophysics, Oregon State University, for his help with the amino acid analysis. This work was supported by NSF grant PCM76-20948. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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