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Unit cell dimensions of crystalline horseradish peroxidase
J.
.lloZ.
Hid.
(1076)
106,
229 -230
Unit Cell Dimensions
of Crystalline Horseradish
Peroxidase
Horseradish peroxidase is a protein of molecular weight 40,000 containing one probohaematin IX per molecule (Saunders et al., 1964; Saunders, 1973), the same as in haemoglobin. It also contains 18% ( w / wr) carbohydrate, making it a glycoprotein. We wish to determine its structure in order t.o establish how the protein moiety gives t’he prosthetic group a chemical reactivity quite different from that of haemoglobin. Horseradish peroxidase was prepared from its source using a method based on t,hat described by Shannon et al. (1966). Approximately 200 mg of horseradish peroxidase isozyme C were obtained from 50 kg of horseradishes (A4,,a/A2a,, = 3-O). Crystallizausing an ammonium sulphate solution as suggested by t,ion was first attempted Shannon et al. (1966). Theorell (1943) and Kenten & Mann (1954) but these methods along with vapour diffusion and “pulsing” (Koeppe II of aZ.. 1975), only produced globules of very small microcrystals. Equilibrium dialysis techniques were also used : 25 III of solution of bct’wcen 0.5 and loA p erosidase solution was first dialgsed t’o a
I.‘I(:. 1. (a) Sod rrystals in solut~ion I ion All I,
crydaIs fomwtl (magnificatjion
at tsho base of a tlialysin 75 * ) and (inset) crystals
cell. Magnification rrYno\-cd for S-ray
50~. work,
(b) Larger tnagnifica-
A.
23u
BlCAI’l’HWAITE
salt concentration of 1.8 M. crystallization using various buffer solutions then being at,tempted at slightly higher salt concentrations. After exhaust’ive tests with ammonium sulphate using different) buffers (from pH 4.!i t’o 8+), various ot)her salt solut(ions were tried. Finally, crystals appeared from dialysis against, a Tris-buffered magnesium sulphat’e solution (2.61 M) at pW 7.7. The light brown crystals so formed were square-based pyramids, measuring up t’o 180 111~1from cap t)o cap (Fig. 1). Precession photographs showed that the crystals were well-ordered, but, t,hr, diffraction patterns were weak. The crystals were almost certainly tetragonal a-it,h 001 absent for 1 # 4n, indicating a 4, screw axis, but the exact space group is uncertain. The axial lengths were a 27 0 = 96.8(S) A. c = 371(2) A. a == /3 :- y == 90”. wit,h a volume of 3.48 x lo6 A3, suggesting approximately 32 molecules per unit cell (based on a solvent content of riOO/, (Matthews, 1968)). This large number would make it extremely difficult to determine the positions of heavy atoms, and it’ was therefore decided t’o search for another form of peroxidasr more suitable for detailed X-rajr analysis. I wish to thank Dr M. F. J. Kilmartin and R. Henderson Medical Laboratory Received
Research Council of Molecular 25 May
Perutz for suggesting for helpful discussion.
and
attvising
in
this
work,
A.
Biology,
Cambridge,
and
Drs
HRAITHwAITE
England
1976 REFERENCES
Kenten,
R. H.
Koeppe
II R. E., Stroud,
155-160. Matthews, B. Saunders, B. Saunders, B. London. Shannon, L. Theorell, H.
& Mann,
P. J. G. (1954).
&o&em.
J. 57,
R. M., Pena, V. A. & Santi.
347.-348. D. V. (1975).
W. (1968). J. Mol. Biol. 33, 491-497. C. (1973). In Inorganic Biochemistry, pp. 988-1021, Elsevier, C., Holmes-Siedle, A. G. & Stark, B. P. (1964). In Peroxidase, M., Kay, E. & Lew, (1943). Arkiv. Kerni.
J. W. (1966). J. Biol. 16A (no. 2), l-11.
Chem.
241,
J.
&lol.
Riol.
New York. Butterworths,
2166-2172.
98,
.lloZ.
Hid.
(1076)
106,
229 -230
Unit Cell Dimensions
of Crystalline Horseradish
Peroxidase
Horseradish peroxidase is a protein of molecular weight 40,000 containing one probohaematin IX per molecule (Saunders et al., 1964; Saunders, 1973), the same as in haemoglobin. It also contains 18% ( w / wr) carbohydrate, making it a glycoprotein. We wish to determine its structure in order t.o establish how the protein moiety gives t’he prosthetic group a chemical reactivity quite different from that of haemoglobin. Horseradish peroxidase was prepared from its source using a method based on t,hat described by Shannon et al. (1966). Approximately 200 mg of horseradish peroxidase isozyme C were obtained from 50 kg of horseradishes (A4,,a/A2a,, = 3-O). Crystallizausing an ammonium sulphate solution as suggested by t,ion was first attempted Shannon et al. (1966). Theorell (1943) and Kenten & Mann (1954) but these methods along with vapour diffusion and “pulsing” (Koeppe II of aZ.. 1975), only produced globules of very small microcrystals. Equilibrium dialysis techniques were also used : 25 III of solution of bct’wcen 0.5 and loA p erosidase solution was first dialgsed t’o a
I.‘I(:. 1. (a) Sod rrystals in solut~ion I ion All I,
crydaIs fomwtl (magnificatjion
at tsho base of a tlialysin 75 * ) and (inset) crystals
cell. Magnification rrYno\-cd for S-ray
50~. work,
(b) Larger tnagnifica-
A.
23u
BlCAI’l’HWAITE
salt concentration of 1.8 M. crystallization using various buffer solutions then being at,tempted at slightly higher salt concentrations. After exhaust’ive tests with ammonium sulphate using different) buffers (from pH 4.!i t’o 8+), various ot)her salt solut(ions were tried. Finally, crystals appeared from dialysis against, a Tris-buffered magnesium sulphat’e solution (2.61 M) at pW 7.7. The light brown crystals so formed were square-based pyramids, measuring up t’o 180 111~1from cap t)o cap (Fig. 1). Precession photographs showed that the crystals were well-ordered, but, t,hr, diffraction patterns were weak. The crystals were almost certainly tetragonal a-it,h 001 absent for 1 # 4n, indicating a 4, screw axis, but the exact space group is uncertain. The axial lengths were a 27 0 = 96.8(S) A. c = 371(2) A. a == /3 :- y == 90”. wit,h a volume of 3.48 x lo6 A3, suggesting approximately 32 molecules per unit cell (based on a solvent content of riOO/, (Matthews, 1968)). This large number would make it extremely difficult to determine the positions of heavy atoms, and it’ was therefore decided t’o search for another form of peroxidasr more suitable for detailed X-rajr analysis. I wish to thank Dr M. F. J. Kilmartin and R. Henderson Medical Laboratory Received
Research Council of Molecular 25 May
Perutz for suggesting for helpful discussion.
and
attvising
in
this
work,
A.
Biology,
Cambridge,
and
Drs
HRAITHwAITE
England
1976 REFERENCES
Kenten,
R. H.
Koeppe
II R. E., Stroud,
155-160. Matthews, B. Saunders, B. Saunders, B. London. Shannon, L. Theorell, H.
& Mann,
P. J. G. (1954).
&o&em.
J. 57,
R. M., Pena, V. A. & Santi.
347.-348. D. V. (1975).
W. (1968). J. Mol. Biol. 33, 491-497. C. (1973). In Inorganic Biochemistry, pp. 988-1021, Elsevier, C., Holmes-Siedle, A. G. & Stark, B. P. (1964). In Peroxidase, M., Kay, E. & Lew, (1943). Arkiv. Kerni.
J. W. (1966). J. Biol. 16A (no. 2), l-11.
Chem.
241,
J.
&lol.
Riol.
New York. Butterworths,
2166-2172.
98,