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Crystallographic data for haemoglobin from the lanceolate fluke Dicrocoelium dendriticum
./. Mol.
Rid.
(1981) 146, 641-647
Crystallographic Data for Haemoglobin from the Lanceolate Fluke Dicrocoelium dendriticum Two crystal modifications of the monomeric haemoglobin from the flatworm I)icrocoelium drvrdriticum have been obtained by vapour diffusion against buffered polyethylene glycol solutions. Both the triclinic and hexagonal crystals contain c~yanomethaemoglobin. The triclinic modification, space group PI, a = 37.1 A. h = 39.9 A. c = 49.0 A, I = 88%‘, /3 = 76%“, y = 646”. with two molecules. M, = IS.750 each. per unit, cell, has been selected for a detailed crystallographic st,lldy.
Structural information on the globin fold is now available to near-atomic resolution for three different animal phyla. The chordates are represented with mammalian haemoglobins from man, Homo sapiens (Baldwin, 1980: Fermi, 1976 : Frier & I’erutz, 1977). and horse, Equus caballus (Ladner et al., 1977), and myoglobins from Physeter catodon (Takano. 1977a,6) and seal. Phoca Gtulivxcr sperm whale, (Scouloudi & Baker, 1978), as well as haemoglobin from the agnathic sea lamprey. I’etromypott marinus (Hendrickson et al., 1973). Other phyla for which structural information on the globin fold has been presented are the annelids, with GZ~cyc~ dihranchiatn haemoglobin (Padlan & Love, 1974), and the arthropods, with larval haemoglobin 111 from Chirovzomus thummi thu,mmi (Steigemann $ Weber. 1979). Moreover. the three-dimensional structure of leghaemoglobin from the root nodules of the yellow lupin, Lupinus Euteus L., has been elucidated (Vainshtein et al.. 1977). Additiona,] information is likely to become available shortly for the chordates deer. Orlo~oil~rts virgiavvrts (Girling et al.. 198(I), and trout, Salmo irideus (L. (‘. Andrews. W. E:. Love. (:. MacNichol, (1. A. Reed & M. Brunori, Abstr. Nwv~mrr Meet. -4mrr. (‘rystallograph. .+Iss. p. Tl, 1977), for the mollusc Aplysia limacina (Ungaretti et al., 197X). and for the echinoderm Molpadia arenicolu. a sea cucumber (W. M. (“arson. RI. Prrntrup, I,. L. Reed & M. L. Hack&, .A bstr. Bummw Meet. =I mrr. ( ‘rysfnllogmph .-Iss. p. 71, 1977). Despite a growing interest in invertebrat)(l respiratory proteins (Wood. 1979), structural information on the globin fold from animals more primitive than annelids is still lacking. However. several of thtJsf> lower animals are known to possess haemoglobin (Lee & Smith. 1965). Amongst them is the lanceolate fluke Dicrocoelirrm devvdriticum. a flatworm that lives in the bile duct’s of certain mammals (Wilford Olsen. 1974). The monomeric haemoglobin f’rom the body fluid of this parasite is perhaps the most primitive animal haemoglobin functionally characterized to date (Tuchschmid it al.. 197X). It, rrpresents the phylum Platyhelminthes and has low sequence homology with other globins (K. J. Wilson, personal communication). This predestinates it for studies on strwctural prot>ein evolution. A recent, study on globin evolution by Lesk & (‘hothia
ti41
.I. D.G. SMIT AND K. H. WINTEKHALTEK
(1980) states that only those amino acid replacements that preserve the geometry of the haem pocket, even at the expense of the relative positions of the helices in the globin fold, are compatible with a functional molecule and thus with the survival of the individual. In Z). dendriticuwz haemoglobin. some unusual amino acid replacements are observed (K. J. Wilson, personal communication). Phenylalanine CD4, whose side-chain packs between helices B and (’ in all known globin structures, is replaced by leucine: and leucine BlO, a residue involved in the packing of helix B to E, is replaced by tyrosine. The structure of the evolutionary ancient haemoglobin from Zj. deudriticum could also shed more light on the convergent or divergent evolution of the haem pocket for different classes of haem proteins such as globins, cytochromes, catalases and peroxidases (Argos & Rossmann, 1979). The orientation of the haem is similar in all known chordate globin structures. Of the two non-chordate structures, the haem orientation in (:. haemoglobin III a dibranchiatu is similar to those for chordates but in Phirortomus 180” rotation around the ‘1, y-methylene axis of the haem group is observed predominantly (Steigemann Xr Weber. 1979; La Mar et al.. 1980). The haem orientation in D. de,/driticum haemoglobin should be revealed by our structure determination and thus will provide more information about the generality of haem orientation in non-chordates and its functional significance. The distal histidine has been observed in all chordate globin structures (Dayhoff, 1976), with the exception of some rare human mutants (for review, see Winslow & Anderson, 1978 ; Tucker et al., 1978). Non-chordates often have the distal histidine replaced, as in (:. dihranchiata by leucine and in -4. bimacin.a by valine (Dayhoff, 1976). Chironomws represents a special case: the distal histidine is present in the sequence but is forced out of the haem pocket by isoleucine El 1 (Steigemann &. Weber. 1979). Removal of this distal histidine from the haem pocket leads to a high partition coefficient between CO and 0, : M = (~02. [HbCO I)/(pCO. [HbO, I) (Tucker et al.. 1978). n. dendriticum haemoglobin represents an extreme example of it. since its distal histidine is replaced by a glycine but position El 1 is still occupied by the usual small valine. The oxygen affinity of haemoglobin depends on steric fact,ors: a restrained iron movement and hindrance of ligand binding in t,he haem pocket (Perutz, 1979; Baldwin & Chothia, 1979). For mammalian myoglobins and isolated haemoglobin chains, the p,, value is around @6 mmHg but invertebrates generally have higher p,, values (Antonini Rr.Brunori. 197 I ). I). dendriticuw~ haemoglobin has rt al.. 1978). which is still the much lower p,,, value of 0.085 mmHg (Tuchschmid considerably higher than the 0.004 mmHg observed for the perienteric fluid erythrocruorin from the nematode Ascaris Iumbricoidrs (Davenport, 1949). However, the oxygen affinity of ll. dendriticuwc haemoglobin is higher than that for any globin structure reported to date. In addition. the oxyhaemoglobin absorption spectrum shows an ad540 nm to A 570 nm ratio greater than unity (Tuchschmid ct al.. 1978), in marked contrast to the spectra of mammalian oxyhaemoglobins. It seems that this ligand can bind at different angles in different globin folds (Steigemann & Weber, 1979: Phillips, 1978) but more structural data are needed. We are presently determining the structure of haemoglobin from the lanceolate fluke Il. drntriticum in view of its remarkable position in the evolutionary scale and the properties mentioned above.
LETTER TO THE EDITOR
GKl
Purified /I. dent&cum haemoglobin, isolated from flukes obtained from infested livers of sheep. was kindly provided by Dr K. Wilson. University of Zurich and Dr P. Tuchschmid. Kinderspital, Zurich. This material represents haemoglobin II rechromatographed over diethylaminoethane-Sephadex as characterized 1)~ et al. (1978). Although this material splits into two components with Tuchschmid isoelectric points of 451 and 4.53 upon isoelectric focusing (Tuchschmid it nl.. 1!)7X). no separation attempt was made before crystallization. The haemoglobin was crystallized in the cyanomet form, using polyethylene glycol as precipitant. with a hanging drop vapour diffusion procedure (Reid et al., 1973) at room temperature. (‘rystals grow from 30 to 3S0/, (w/v) polyethylene glycol solutions. containing (Pl M-potassium acetate (pH 4 to 5) and 5 mM-KC%. The success of thr method depends to some extent on the molecular weight fraction of the polyethylene glycol used, and the best resultIs are obtained with polyethylene glycol 20(H). The nat,ure of the buffer is also important (Smit. 1979): use of potassium acetate instead of succinate consistently yields about tenfold thicker crystals. Init,ial crystallization is slow. often taking months, and mostly yields small rrystals. Therefore, we used small de no00 grown crystals as macroscopic seeds at concentrations of polyethylene glycol below those needed for initial crystallization grow larger well-shaped t,o to crystals. Large, crystals (up 1.7 mm x 0.5 mm x 0.4 mm) were then obtained by repeatedly exchanging (ovtar a period of one week) the polyethylene glycol solution in the equilibration reservoir against solutions of increasing concentration of polyethylene glycol approaching the concentration for dr no~o crystallization. Two crystal modifications. an hexagonal and a triclinic form, were obtained under apparently identical crystallization conditions. Since the low pH maintained during crystallization might) lead to loss of cyanide from the haemoglobin, we compared the absorption spectra. from 500 to 650 nm, of single. triclinic crystals at pH 4.5 in 400/, (w/v) polyethylene glycol 2000, and of cyanomethaemoglobin in solution at pH 7%. These spect,ra (Fig. 1) are identical, demonstrating the presence of cya”ometharmoglol)iIl in the crystalline state even at pH 45. Spectra of crystals were recorded wit,h a Zeiss lYVlSF’ I spectrophotometer as described (Smit et al.. 1977). (‘rystallographic data for the two crystal modifications are given in Table 1. We have chosen the triclinic crystals for a high-resolution study because they grow preferentiaIl>,. become larger, and diffract better. Moreover, their X-ray deterioration is slow : 21. precession photographs show a strong diffraction pattern out to the edge of t,ht> phot,ographs. even after a previous 100 hours exposure to CuKn radiation (1.5 kW. N-tittered, fine-focus tube). After 300 hours exposure on a diffractometer. the mean int’eljsitjy dropped by less than lOO/o. The hk0 zone of the triclinic crystals shows I)s”“dohrxagotlal symmetry (Fig. 2) but a relationship with the h/c0 zone of the hexagonal modification seems not apparent (Table 1). The density of the triclinica cryst,als is more than 1.20 g ml- ’ as determined from Ficoll-400 solutions (Westbrook, 1976). In a bromobenzene/orthoxylene gradient column (Lo\{, & Richards. I!I52). a density of 1.24 g ml-’ was observed, consistent with t’hr presence of two monomers per unit cell. An anomalous difference Patterson (Rossmann. 1961) for t)he native protein at 3.3 A nominal resolution confirmed the prest~nc~~ of t,\ro iron atoms (i.e. two monomers) per unit cell. The smallest iron--iroll
644
J. D. G. SMIT
AND
K. H. WINTERHALTER
0.1 I I I I 5x)540560580600 Wavelength
I 65 (nm)
Fro. 1. Comparison of absorption spectra (566 to 656 nm) of Hb’CNin solution and from a single triclinic crystal. () In solution; conditions: 5 mr+bis-Tris (pH 7.8) 1 mM-KCN. (------) Crystalline; conditions: 469, (w/v) polyethylene glycol 2606, 61 M-potassium acetate (pH 45), 5 mMKCN. The absorption spectrum of hemoglobin in solution has been raised by 062 O.D. unit for ease of comparison. Spectra of crystals were recorded on the universal microspectrophotometer of the Wissenschaftlicher Dienst/Kriminalpolizei Zurich. The wavelength scale is given by the dispersion of the quartz monochromator (MI&III).
FIG. 2. Precession photograph (3.4 A nominal resolution) of the hk6 zone of a triclinic cysnomethyaemoglobin crystal showing pseudohexagonal symmetry. Ni-filtered CuKo radiation, precession angle p = 13”.
I). dendriticum
37.1 91.5
39.9 91.5
49.0 28.3
88.8 90
TABLE
1
768 90
B (7 64.6 120
data for Dicrocoelium
63,600 205,206
dendriticum
‘33
2
z
1.90 2.04
v,t (A/dalton)
haemoglobin
t Calculated after Matthews (1968). -W, (U. dent&cum haemoglobin) = 16,750. calculated from Kunz (1975). 1 The V, value (Matthews. 1968) suggests that the asymmetric unit contains one molecule.
PI P62 or P64
Space group
Crystallographic
035 0.40
Solvent content (v/v)
12.15 12.5
Resolution (4 (h/2 sin 0)
ti4ti
.l. L). G. SMIT AND K. H. \I’INTERHALTEK
distance observed is about 28.5 A (Smit, 1980), a value within the range of intramolecular iron-iron distances in tetrameric haemoglobin (Yerutz. 1969). The occurrence of two monomers per asymmetric unit provides an opportunity to assess the significance of structural features observed for different liganded states with respect to differences due solely to crystal packing. As a first step to this goal. data collection on the cyanomethaemoglobin to higher resolution is in progress. We thank Professor J. Dunitz for providing technical equipment, Professor J. Drenth for the opportunity to collect 3-dimensional data, Dr R. Halonbrenner for recording spectra of crystalline material, and Miss U. Neininger for technical assistance. This work was supported by grant no. 0.330.077.98/3 from the EidgenGssische Technische Hochschule, Zurich.
Laboratorium fiir Biochemie I Eidgeniissische Technische Hochschule Ziirich, Switzerland
.JAN DEKK G. Sm-t KASPAR H. WINTERHALTER
Received 9 October 1980 i A4uthor to whom correspondence should be addressed.
REFERENCES and Myoglobin in Their Reaction with Antonini, E. &, Brunori, M. (1971). Hemoglobin Ligunds, North-Holland Publishing Co., Amsterdam, New York and Oxford. Argos, P. & Rossmann, M. G. (1979). Biochemistry, 18, 49514960. Baldwin, J. & Chothia, C. (1979). J. Mol. Biol. 129, 17&220. Baldwin, J. M. (1980). J. Mol. Biol. 136, 103-128. Davenport, H. E. (1949). Proc. Roy. Sot. ser. B, 136, 25Tt270. Dayhoff, M. 0. (1976). Editor of Atlas of Protein Sequence and Structure, vol. 5, suppl. 2, pp. 191-224, National Biomedical Research Foundation, Washington. Fermi, G. (1976). J. Mol. BioZ. 97, 237-256. Frier, J. A. & Perutz, M. F. (1977). J. Mol. Biol. 112, 97-l 12. Girling, R. L., Houston, T. E., Schmidt, W. C. Jr & Amma, E. L. (1980). Acta Crystullogr. sect. A, 36, 43-50.
Hendrickson, W. A., Love, W. E. & Karle, J. (1973). J. Mol. Biol. 74, 331-361. Kunz, P.A. (1975). Ph.D. thesis, University of Zurich. Ladner, R. C., Heidner, E. J. & Peru@ M. F. (1977). J. MoZ. Biol. 114, 38,5414. La Mar, G. N., Smith, K. M., Gersonde, K., Sick, H. & Overkamp, M. (1980). J. Biol. Chem. 255, 6&70. Lee, D. L. & Smith, M. H. (1965). Exp. Parasitol. 16, 392-424. Lesk, A. M. & Chothia, C. (1980). J. Mol. Biol. 136, 225-270. Low, B. W. & Richards, F. M. (1952). J. =Imer. Chrm. Rot. 74, 166&1666. Matthews, B. W. (1968). J. Mol. Biol. 33, 491497. Padlan, E. A. & Love, W. E. (1974). J. Biol. Chem. 249, 4067-4078. Peru@ M. F. (1969). Harvey Lect. 63, 213-261. Perutz, M. F. (1979). Annu. Rev. B&hem. 48, 327-386. Phillips, S. E. V. (1978). LVuture (London), 273, 247-248. Reid, B. R., Koch, G. L. E., Boulanger, Y ., Hartley, B. S. & Blow, D.M. (1973). J. Mol. Biol. 89, 199-201. Rossmann, M. G. (1961). Acta Crystallogr. 14, 383-388. Scouloudi, H. & Baker, E. N. (1978). J. Mol. Biol. 126, 637460.
LETTER
TO THE
EDITOR
Smit, J. D. G. (1979). J. Chim. Phys.-Chim. Biol. 76, 805-810. Smit, .J. D. G. (1980). Eqxrientia, 36, 733. Smit. J. D. G.. Grandjean, O., Guggenheim, R. & Winter-halter, (London),
lidi
K. H. (1977). ;Vafrtw
266, 53G-538.
Strigemann. W. 8: Weber, E. (1979). J. Mol. Biol. 127, 309-338. Takano, T. (1977n). J. Mol. Biol. 110, 537-568. Takano, T. (1977b). J. Mol. Biol. 110, 569-584. Tuchschmid. I’. E., Kunz, P. A. & Wilson, K. J. (1978). Eur. J. B&hem. 88, 387-394. Tuckrr. I’. W.. Phillips, S. E. V., Perutz, M. F., Houtchens. R. & Caughey, W. S. (1978). I’m-. Sat. .-lead. Sci., /J.S.d 75, 1076-1080. I’ngarctti. L.. Bolognesi, M.. Cannillo, E., Oberti, R. & Rossi, (:. (1978). .4&a (‘rystnlloqr. srrt., B , 34 . 365K-3662. t \.ainshtein. B. K.. Arutyunyan, E. G., Kuranova, 1. I’., Borisov. V. V., Sosf’enov, N. I.. l’avlovskii. A. G.. Grebenko, A. I., Konareva, N. V. & Nekrasov, Yu. V. (1977). Dokl. .-Ikad. Sauk.
#‘SRR,
223, 238S241.
W&brook. E. M. (1976). J. Mol. Biol. 103, 659464. Wilford Olsen. 0. (1974). Animal Parasittz. Their Life Cycles and Ecology, pp. 284-289, 3rd edit., University Park Press, Baltimore, London and Tokyo. Winslow. It. M. 8r Anderson, W. F. (1978). In The Metabolic Basis of Inherited Disease (Stanbury, J. B., Wyngaarden, J. B. & Fredrickson, D. S.. rds), 4th edit., pp. 1465 1.507, Mc(:raw-Hill Co.. New York. Wood. E. ,I. (1979). Satwe (London), 281, 341-342.
Rid.
(1981) 146, 641-647
Crystallographic Data for Haemoglobin from the Lanceolate Fluke Dicrocoelium dendriticum Two crystal modifications of the monomeric haemoglobin from the flatworm I)icrocoelium drvrdriticum have been obtained by vapour diffusion against buffered polyethylene glycol solutions. Both the triclinic and hexagonal crystals contain c~yanomethaemoglobin. The triclinic modification, space group PI, a = 37.1 A. h = 39.9 A. c = 49.0 A, I = 88%‘, /3 = 76%“, y = 646”. with two molecules. M, = IS.750 each. per unit, cell, has been selected for a detailed crystallographic st,lldy.
Structural information on the globin fold is now available to near-atomic resolution for three different animal phyla. The chordates are represented with mammalian haemoglobins from man, Homo sapiens (Baldwin, 1980: Fermi, 1976 : Frier & I’erutz, 1977). and horse, Equus caballus (Ladner et al., 1977), and myoglobins from Physeter catodon (Takano. 1977a,6) and seal. Phoca Gtulivxcr sperm whale, (Scouloudi & Baker, 1978), as well as haemoglobin from the agnathic sea lamprey. I’etromypott marinus (Hendrickson et al., 1973). Other phyla for which structural information on the globin fold has been presented are the annelids, with GZ~cyc~ dihranchiatn haemoglobin (Padlan & Love, 1974), and the arthropods, with larval haemoglobin 111 from Chirovzomus thummi thu,mmi (Steigemann $ Weber. 1979). Moreover. the three-dimensional structure of leghaemoglobin from the root nodules of the yellow lupin, Lupinus Euteus L., has been elucidated (Vainshtein et al.. 1977). Additiona,] information is likely to become available shortly for the chordates deer. Orlo~oil~rts virgiavvrts (Girling et al.. 198(I), and trout, Salmo irideus (L. (‘. Andrews. W. E:. Love. (:. MacNichol, (1. A. Reed & M. Brunori, Abstr. Nwv~mrr Meet. -4mrr. (‘rystallograph. .+Iss. p. Tl, 1977), for the mollusc Aplysia limacina (Ungaretti et al., 197X). and for the echinoderm Molpadia arenicolu. a sea cucumber (W. M. (“arson. RI. Prrntrup, I,. L. Reed & M. L. Hack&, .A bstr. Bummw Meet. =I mrr. ( ‘rysfnllogmph .-Iss. p. 71, 1977). Despite a growing interest in invertebrat)(l respiratory proteins (Wood. 1979), structural information on the globin fold from animals more primitive than annelids is still lacking. However. several of thtJsf> lower animals are known to possess haemoglobin (Lee & Smith. 1965). Amongst them is the lanceolate fluke Dicrocoelirrm devvdriticum. a flatworm that lives in the bile duct’s of certain mammals (Wilford Olsen. 1974). The monomeric haemoglobin f’rom the body fluid of this parasite is perhaps the most primitive animal haemoglobin functionally characterized to date (Tuchschmid it al.. 197X). It, rrpresents the phylum Platyhelminthes and has low sequence homology with other globins (K. J. Wilson, personal communication). This predestinates it for studies on strwctural prot>ein evolution. A recent, study on globin evolution by Lesk & (‘hothia
ti41
.I. D.G. SMIT AND K. H. WINTEKHALTEK
(1980) states that only those amino acid replacements that preserve the geometry of the haem pocket, even at the expense of the relative positions of the helices in the globin fold, are compatible with a functional molecule and thus with the survival of the individual. In Z). dendriticuwz haemoglobin. some unusual amino acid replacements are observed (K. J. Wilson, personal communication). Phenylalanine CD4, whose side-chain packs between helices B and (’ in all known globin structures, is replaced by leucine: and leucine BlO, a residue involved in the packing of helix B to E, is replaced by tyrosine. The structure of the evolutionary ancient haemoglobin from Zj. deudriticum could also shed more light on the convergent or divergent evolution of the haem pocket for different classes of haem proteins such as globins, cytochromes, catalases and peroxidases (Argos & Rossmann, 1979). The orientation of the haem is similar in all known chordate globin structures. Of the two non-chordate structures, the haem orientation in (:. haemoglobin III a dibranchiatu is similar to those for chordates but in Phirortomus 180” rotation around the ‘1, y-methylene axis of the haem group is observed predominantly (Steigemann Xr Weber. 1979; La Mar et al.. 1980). The haem orientation in D. de,/driticum haemoglobin should be revealed by our structure determination and thus will provide more information about the generality of haem orientation in non-chordates and its functional significance. The distal histidine has been observed in all chordate globin structures (Dayhoff, 1976), with the exception of some rare human mutants (for review, see Winslow & Anderson, 1978 ; Tucker et al., 1978). Non-chordates often have the distal histidine replaced, as in (:. dihranchiata by leucine and in -4. bimacin.a by valine (Dayhoff, 1976). Chironomws represents a special case: the distal histidine is present in the sequence but is forced out of the haem pocket by isoleucine El 1 (Steigemann &. Weber. 1979). Removal of this distal histidine from the haem pocket leads to a high partition coefficient between CO and 0, : M = (~02. [HbCO I)/(pCO. [HbO, I) (Tucker et al.. 1978). n. dendriticum haemoglobin represents an extreme example of it. since its distal histidine is replaced by a glycine but position El 1 is still occupied by the usual small valine. The oxygen affinity of haemoglobin depends on steric fact,ors: a restrained iron movement and hindrance of ligand binding in t,he haem pocket (Perutz, 1979; Baldwin & Chothia, 1979). For mammalian myoglobins and isolated haemoglobin chains, the p,, value is around @6 mmHg but invertebrates generally have higher p,, values (Antonini Rr.Brunori. 197 I ). I). dendriticuw~ haemoglobin has rt al.. 1978). which is still the much lower p,,, value of 0.085 mmHg (Tuchschmid considerably higher than the 0.004 mmHg observed for the perienteric fluid erythrocruorin from the nematode Ascaris Iumbricoidrs (Davenport, 1949). However, the oxygen affinity of ll. dendriticuwc haemoglobin is higher than that for any globin structure reported to date. In addition. the oxyhaemoglobin absorption spectrum shows an ad540 nm to A 570 nm ratio greater than unity (Tuchschmid ct al.. 1978), in marked contrast to the spectra of mammalian oxyhaemoglobins. It seems that this ligand can bind at different angles in different globin folds (Steigemann & Weber, 1979: Phillips, 1978) but more structural data are needed. We are presently determining the structure of haemoglobin from the lanceolate fluke Il. drntriticum in view of its remarkable position in the evolutionary scale and the properties mentioned above.
LETTER TO THE EDITOR
GKl
Purified /I. dent&cum haemoglobin, isolated from flukes obtained from infested livers of sheep. was kindly provided by Dr K. Wilson. University of Zurich and Dr P. Tuchschmid. Kinderspital, Zurich. This material represents haemoglobin II rechromatographed over diethylaminoethane-Sephadex as characterized 1)~ et al. (1978). Although this material splits into two components with Tuchschmid isoelectric points of 451 and 4.53 upon isoelectric focusing (Tuchschmid it nl.. 1!)7X). no separation attempt was made before crystallization. The haemoglobin was crystallized in the cyanomet form, using polyethylene glycol as precipitant. with a hanging drop vapour diffusion procedure (Reid et al., 1973) at room temperature. (‘rystals grow from 30 to 3S0/, (w/v) polyethylene glycol solutions. containing (Pl M-potassium acetate (pH 4 to 5) and 5 mM-KC%. The success of thr method depends to some extent on the molecular weight fraction of the polyethylene glycol used, and the best resultIs are obtained with polyethylene glycol 20(H). The nat,ure of the buffer is also important (Smit. 1979): use of potassium acetate instead of succinate consistently yields about tenfold thicker crystals. Init,ial crystallization is slow. often taking months, and mostly yields small rrystals. Therefore, we used small de no00 grown crystals as macroscopic seeds at concentrations of polyethylene glycol below those needed for initial crystallization grow larger well-shaped t,o to crystals. Large, crystals (up 1.7 mm x 0.5 mm x 0.4 mm) were then obtained by repeatedly exchanging (ovtar a period of one week) the polyethylene glycol solution in the equilibration reservoir against solutions of increasing concentration of polyethylene glycol approaching the concentration for dr no~o crystallization. Two crystal modifications. an hexagonal and a triclinic form, were obtained under apparently identical crystallization conditions. Since the low pH maintained during crystallization might) lead to loss of cyanide from the haemoglobin, we compared the absorption spectra. from 500 to 650 nm, of single. triclinic crystals at pH 4.5 in 400/, (w/v) polyethylene glycol 2000, and of cyanomethaemoglobin in solution at pH 7%. These spect,ra (Fig. 1) are identical, demonstrating the presence of cya”ometharmoglol)iIl in the crystalline state even at pH 45. Spectra of crystals were recorded wit,h a Zeiss lYVlSF’ I spectrophotometer as described (Smit et al.. 1977). (‘rystallographic data for the two crystal modifications are given in Table 1. We have chosen the triclinic crystals for a high-resolution study because they grow preferentiaIl>,. become larger, and diffract better. Moreover, their X-ray deterioration is slow : 21. precession photographs show a strong diffraction pattern out to the edge of t,ht> phot,ographs. even after a previous 100 hours exposure to CuKn radiation (1.5 kW. N-tittered, fine-focus tube). After 300 hours exposure on a diffractometer. the mean int’eljsitjy dropped by less than lOO/o. The hk0 zone of the triclinic crystals shows I)s”“dohrxagotlal symmetry (Fig. 2) but a relationship with the h/c0 zone of the hexagonal modification seems not apparent (Table 1). The density of the triclinica cryst,als is more than 1.20 g ml- ’ as determined from Ficoll-400 solutions (Westbrook, 1976). In a bromobenzene/orthoxylene gradient column (Lo\{, & Richards. I!I52). a density of 1.24 g ml-’ was observed, consistent with t’hr presence of two monomers per unit cell. An anomalous difference Patterson (Rossmann. 1961) for t)he native protein at 3.3 A nominal resolution confirmed the prest~nc~~ of t,\ro iron atoms (i.e. two monomers) per unit cell. The smallest iron--iroll
644
J. D. G. SMIT
AND
K. H. WINTERHALTER
0.1 I I I I 5x)540560580600 Wavelength
I 65 (nm)
Fro. 1. Comparison of absorption spectra (566 to 656 nm) of Hb’CNin solution and from a single triclinic crystal. () In solution; conditions: 5 mr+bis-Tris (pH 7.8) 1 mM-KCN. (------) Crystalline; conditions: 469, (w/v) polyethylene glycol 2606, 61 M-potassium acetate (pH 45), 5 mMKCN. The absorption spectrum of hemoglobin in solution has been raised by 062 O.D. unit for ease of comparison. Spectra of crystals were recorded on the universal microspectrophotometer of the Wissenschaftlicher Dienst/Kriminalpolizei Zurich. The wavelength scale is given by the dispersion of the quartz monochromator (MI&III).
FIG. 2. Precession photograph (3.4 A nominal resolution) of the hk6 zone of a triclinic cysnomethyaemoglobin crystal showing pseudohexagonal symmetry. Ni-filtered CuKo radiation, precession angle p = 13”.
I). dendriticum
37.1 91.5
39.9 91.5
49.0 28.3
88.8 90
TABLE
1
768 90
B (7 64.6 120
data for Dicrocoelium
63,600 205,206
dendriticum
‘33
2
z
1.90 2.04
v,t (A/dalton)
haemoglobin
t Calculated after Matthews (1968). -W, (U. dent&cum haemoglobin) = 16,750. calculated from Kunz (1975). 1 The V, value (Matthews. 1968) suggests that the asymmetric unit contains one molecule.
PI P62 or P64
Space group
Crystallographic
035 0.40
Solvent content (v/v)
12.15 12.5
Resolution (4 (h/2 sin 0)
ti4ti
.l. L). G. SMIT AND K. H. \I’INTERHALTEK
distance observed is about 28.5 A (Smit, 1980), a value within the range of intramolecular iron-iron distances in tetrameric haemoglobin (Yerutz. 1969). The occurrence of two monomers per asymmetric unit provides an opportunity to assess the significance of structural features observed for different liganded states with respect to differences due solely to crystal packing. As a first step to this goal. data collection on the cyanomethaemoglobin to higher resolution is in progress. We thank Professor J. Dunitz for providing technical equipment, Professor J. Drenth for the opportunity to collect 3-dimensional data, Dr R. Halonbrenner for recording spectra of crystalline material, and Miss U. Neininger for technical assistance. This work was supported by grant no. 0.330.077.98/3 from the EidgenGssische Technische Hochschule, Zurich.
Laboratorium fiir Biochemie I Eidgeniissische Technische Hochschule Ziirich, Switzerland
.JAN DEKK G. Sm-t KASPAR H. WINTERHALTER
Received 9 October 1980 i A4uthor to whom correspondence should be addressed.
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