J. Mol. Biol. (1990) 211, 691-692
Crystallization
of the Rainbow Trout (Salvo gairdrrerz] Haemoglobin IV
G. G. Dodson,
V. R. Richard,
S. P. Tolley,
D. A. Wallerf-
Department of Chemistry, University of York Heslington, York YOl 5DD, U.K.
and R. E. Weber Department of Zoophysiology University of Aarhus DK-8000 Aarhus C, Denmark (Received 6’ October 1989; accepted 14 November
1989)
Crystals of rainbow trout (Salmo gairdneri) haemoglobin IV were grown in mini batches from a solution of ammonium sulphate. Large single crystals grew over five days and were up to 2 mm in lengt,h. X-ray diffraction experiments indicated a space group of C222, with unit cell dimensions of a = 85.3 A, b = 94.6 A and c = 105.7 A. The crystals diffract to better than 2.5 A but exhibit some mosaicity along the c axis.
Trout use haemoglobin (Hb$) for both respiratory oxygen transport and secretion of oxygen into the swim bladder (Wittenberg & Haedrich, 1974). In t,he gas gland (a localized modification of the epithelial cells lining the swimbladder), lactate secretion acidifies the blood, which reduces its oxygenbinding capacity (the Root effect) and causes oxygen secretion into the swimbladder. Only one of the major components found in the trout blood, trout IV Hb, is characterized by a Root effect. The B-chains of trout IV are 52% homologous to the P-chains of human Hb A (Petruzzelli et al., 1984). Like human Hb A, oxygen binding to trout IV is associated with quaternary structural changes (Brunori et al., 1973). However, the response of trout IV to heterotropic ligands is markedly different from that of human Hb A. At low pH, its oxygen affinity becomes so low that it is impossible to saturate the molecule even with pure oxygen at atmospheric pressure. This is thought to provide a molecular mechanism for pumping oxygen into the swim bladder. Is there a structural parameter that may be related to the Root effect? X-ray crystallography is the only tool for investigating the structural basis of this phenomenon. We report here the crystallization of trout IV Hb and preliminary X-ray diffraction experiments.
(a) Lysis Blood was drawn from the caudal blood vessels into heparinized syringes. Red blood cells were washed twice in cold 1 y. (w/v) NaCl and spun down. These were lysed in three volumes of ice-cold 1 mM-Tris. HCl (pH 8). One-tenth of the final volume of 1 M-NaCl was added and the lysate was centrifuged for 15 minutes at 4°C at 28,000 g. After centrifugation, the supernatant was removed and dialysed against 50 mM-Tris. HCl (pH 8.4) at 4°C. (b) PuriJication The supernatant was loaded onto a Pharmacia mono Q HRlO/lO column previously equilibrated with 50 m&r-Tris.HCl (pH 8.4). The protein was eluted by employing a linear gradient of 0 M to 95 M-NaCl in the same buffer. Four peaks were eluted. The major peaks were trout I, which did not bind to the column and was eluted in the void volume, and trout IV, which bound to the column and was eluted at 250 mM-NaCl. Both Hbs were collected separately and were concentrated fourfold for crystallization and desalted into 20 mM-Tris . HCl (pH %4). (c) Crystallization
tPresent address: Astbury
Department of Biophysics, llniversity of Leeds, Leeds LS2 9JT, U.K. IAbbreviation used: Hb, haemoglobin. 0022-2836/90/040691~2
$03.00/O
Crystals of trout IV Hb were grown in mini batches (50 to 100 ~1) from 40 to 42% saturated 691
0
1990 Academic
Press Limited
G. G. Dodson et al.
692
sulphate in 20 mlrr-Tris * HCl (pH 8.4) at 18°C. Large crystals (up to 2 mm) grew over four to five days. The quality of the crystals was improved by using fresh blood, crystals grown from old blood ammonium
exhibit
strong
mosaicity
in every
direction.
cell volume of 852,933 A3. The molecular mass for an c~J?~tetramer is about 63.5 kDa (estimated from the protein sequence of the P-chains). Assuming a mass per unit volume within the normal range for protein crystals (Matthews, 1968), each of the eight asymmetric units of C222, contains one ap dimer, the crystals
(d) X-ray diffraction Using CuKcl radiation (1 = 1*5418A;l A = 0.1 nm), initial still photographs showed the crystals to diffract beyond 2.5 A, with some mosaicity along the shortest growth axis. Two 12” precession photographs have been obtained, using a sealed tube operating at 40 kV and 20 mA. The crystals were very stable, with a beam life-time in excess of 48 hours. The results of X-ray diffraction experiments revealed the space group to be C222,; this is different from both human and horse haemoglobins, which crystallize in P2, and P4,2,2 for deoxygenated (Fermi et al., 1984) and oxygenated (Shaanan, 1983) forms. The unit cell dimensions are a = 85.3 A, b = 94.6 A and c = 1057 A (mosaic). This gives a unit
Edited
containing
60%
(w/v)
solvent.
These results are the preliminaries to data collection. The problem of the mosaic spread can be reduced by mounting the crystal along its c axis.
References Brunori, M., Bonaventura, J., Bonaventura, C., Giardina. B., Bossa, F. & Antonini. E. (1973). Mol. CrZZ. Biochem. 1, 189-196. Fermi: G., Perutz, M. F., Shaanan, B. & Fourme. R. (1984). J. Mol. BioZ. 175, 159174. Matthews, B. W. (1968). J. Mol. BioZ. 33, 491-497. Petruzzelli, R., Barra. D., Goffredo, M. B., Bossa, F., Coletta, M. & Brunori, M. (1984). Biochim. Biophys. ilcta, 789, 6S-73. Shaanan, B. (1983). J. Mol. BioZ. 171, 31-59. Wittenberg, J. B. & Haedrich, R. L. (1974). BioZ. Bull.
146, 137-146.
by R. Huber
Crystallization
of the Rainbow Trout (Salvo gairdrrerz] Haemoglobin IV
G. G. Dodson,
V. R. Richard,
S. P. Tolley,
D. A. Wallerf-
Department of Chemistry, University of York Heslington, York YOl 5DD, U.K.
and R. E. Weber Department of Zoophysiology University of Aarhus DK-8000 Aarhus C, Denmark (Received 6’ October 1989; accepted 14 November
1989)
Crystals of rainbow trout (Salmo gairdneri) haemoglobin IV were grown in mini batches from a solution of ammonium sulphate. Large single crystals grew over five days and were up to 2 mm in lengt,h. X-ray diffraction experiments indicated a space group of C222, with unit cell dimensions of a = 85.3 A, b = 94.6 A and c = 105.7 A. The crystals diffract to better than 2.5 A but exhibit some mosaicity along the c axis.
Trout use haemoglobin (Hb$) for both respiratory oxygen transport and secretion of oxygen into the swim bladder (Wittenberg & Haedrich, 1974). In t,he gas gland (a localized modification of the epithelial cells lining the swimbladder), lactate secretion acidifies the blood, which reduces its oxygenbinding capacity (the Root effect) and causes oxygen secretion into the swimbladder. Only one of the major components found in the trout blood, trout IV Hb, is characterized by a Root effect. The B-chains of trout IV are 52% homologous to the P-chains of human Hb A (Petruzzelli et al., 1984). Like human Hb A, oxygen binding to trout IV is associated with quaternary structural changes (Brunori et al., 1973). However, the response of trout IV to heterotropic ligands is markedly different from that of human Hb A. At low pH, its oxygen affinity becomes so low that it is impossible to saturate the molecule even with pure oxygen at atmospheric pressure. This is thought to provide a molecular mechanism for pumping oxygen into the swim bladder. Is there a structural parameter that may be related to the Root effect? X-ray crystallography is the only tool for investigating the structural basis of this phenomenon. We report here the crystallization of trout IV Hb and preliminary X-ray diffraction experiments.
(a) Lysis Blood was drawn from the caudal blood vessels into heparinized syringes. Red blood cells were washed twice in cold 1 y. (w/v) NaCl and spun down. These were lysed in three volumes of ice-cold 1 mM-Tris. HCl (pH 8). One-tenth of the final volume of 1 M-NaCl was added and the lysate was centrifuged for 15 minutes at 4°C at 28,000 g. After centrifugation, the supernatant was removed and dialysed against 50 mM-Tris. HCl (pH 8.4) at 4°C. (b) PuriJication The supernatant was loaded onto a Pharmacia mono Q HRlO/lO column previously equilibrated with 50 m&r-Tris.HCl (pH 8.4). The protein was eluted by employing a linear gradient of 0 M to 95 M-NaCl in the same buffer. Four peaks were eluted. The major peaks were trout I, which did not bind to the column and was eluted in the void volume, and trout IV, which bound to the column and was eluted at 250 mM-NaCl. Both Hbs were collected separately and were concentrated fourfold for crystallization and desalted into 20 mM-Tris . HCl (pH %4). (c) Crystallization
tPresent address: Astbury
Department of Biophysics, llniversity of Leeds, Leeds LS2 9JT, U.K. IAbbreviation used: Hb, haemoglobin. 0022-2836/90/040691~2
$03.00/O
Crystals of trout IV Hb were grown in mini batches (50 to 100 ~1) from 40 to 42% saturated 691
0
1990 Academic
Press Limited
G. G. Dodson et al.
692
sulphate in 20 mlrr-Tris * HCl (pH 8.4) at 18°C. Large crystals (up to 2 mm) grew over four to five days. The quality of the crystals was improved by using fresh blood, crystals grown from old blood ammonium
exhibit
strong
mosaicity
in every
direction.
cell volume of 852,933 A3. The molecular mass for an c~J?~tetramer is about 63.5 kDa (estimated from the protein sequence of the P-chains). Assuming a mass per unit volume within the normal range for protein crystals (Matthews, 1968), each of the eight asymmetric units of C222, contains one ap dimer, the crystals
(d) X-ray diffraction Using CuKcl radiation (1 = 1*5418A;l A = 0.1 nm), initial still photographs showed the crystals to diffract beyond 2.5 A, with some mosaicity along the shortest growth axis. Two 12” precession photographs have been obtained, using a sealed tube operating at 40 kV and 20 mA. The crystals were very stable, with a beam life-time in excess of 48 hours. The results of X-ray diffraction experiments revealed the space group to be C222,; this is different from both human and horse haemoglobins, which crystallize in P2, and P4,2,2 for deoxygenated (Fermi et al., 1984) and oxygenated (Shaanan, 1983) forms. The unit cell dimensions are a = 85.3 A, b = 94.6 A and c = 1057 A (mosaic). This gives a unit
Edited
containing
60%
(w/v)
solvent.
These results are the preliminaries to data collection. The problem of the mosaic spread can be reduced by mounting the crystal along its c axis.
References Brunori, M., Bonaventura, J., Bonaventura, C., Giardina. B., Bossa, F. & Antonini. E. (1973). Mol. CrZZ. Biochem. 1, 189-196. Fermi: G., Perutz, M. F., Shaanan, B. & Fourme. R. (1984). J. Mol. BioZ. 175, 159174. Matthews, B. W. (1968). J. Mol. BioZ. 33, 491-497. Petruzzelli, R., Barra. D., Goffredo, M. B., Bossa, F., Coletta, M. & Brunori, M. (1984). Biochim. Biophys. ilcta, 789, 6S-73. Shaanan, B. (1983). J. Mol. BioZ. 171, 31-59. Wittenberg, J. B. & Haedrich, R. L. (1974). BioZ. Bull.
146, 137-146.
by R. Huber
