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Staining and fixation of unsaturated membrane lipids by osmium tetroxide
Biochimica et Biophysica Acta, 354 (1974) 152--154
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA Report BBA 21393 STAINING AND FIXATION OF UNSATURATED MEMBRANE LIPIDS BY OSMIUM TETROXIDE CRYSTAL STRUCTURE OF A MODEL OSMIUM(VI) DI-ESTER
R. COLLIN, W.P. GRIFFITH, F.L. PHILLIPS and A.C. SKAPSKI Chemical Crystallography and Inorganic Chemistry Laboratories, Imperial College, London SW7 2A Y (U.K.)
(Received April 16th, 1974)
Summary The X-ray crystal structure of a cyclic osmium(VI)di-ester is reported and compared with that of a related mono-ester. The results are used to discuss the role of osmium tetroxide as a staining and fixation agent for unsaturated lipids.
We have recently reported the X-ray crystal structure of the osmium(VI) mono-ester, [OsO~ (02 C2 Me4)] 2 [ 1 ] and suggested that its unexpected dimeric structure (Fig. 1) might offer an explanation of the fixation properties of osmium tetroxide for membrane lipids. To carry this argument forward, however, it seemed desirable to examine the structure of an analogous di-ester since such species, it has been claimed, may be important in lipid fixation [2--4]. We first a t t e m p t e d to carry out an X-ray study of OSO(O2 C2 Me4)2 (tetragonal, a = 10.224, c = 7.265 A), but the sensitivity of the crystals to prolonged X-ray irradiation, and the apparent molecular disorder in the crystal lattice, prevented us from obtaining a clear-cut structural solution. We have however succeeded in obtaining the full structure of the closely related oxobis(ethane-l,2-diolato)osmium(VI), OsO(O2 C2 Ha )2. The black prismatic crystals were obtained by the prolonged action (over some eight weeks) of osmium tetroxide on ethylene glycol at room temperatures [5]. They are o r t h o r h o m b i c with unit-cell dimensions a = 10.950, b = 8.376, c = 7.735 A; space group P b c n (No. 60), and Z = 4. A total of 676 independent reflections were measured on a Siemens four-circle diffractometer (to 0 = 70 °, Cu-Ka radiation). The structure was solved by Patterson and
153
0
Me,C/O~ / %
/O~cMe =
0 Fig. 1. Schematic diagram of t h e s t r u c t u r e of [OsO~(O2C2Me4)] 2 i
Os
C
C
Fig. 2. T h e s t r u c t u r e of OsO(O2C2H4)2.
Fourier methods, and least-squares refinement has now reached R = 0.035. The molecule, shown in Fig. 2, lies on a crystallographic two-fold axis. The osmium has an essentially square-based pyramidal coordination, the four ester oxygen atoms constituting the basal plane (mean Os--O distance 1.89(1} £) and the terminal oxo ligand in the apical position (Os--O 1.66(1} A). The mean O(ester)--Os--O(terminal) angle is 110 °, suggesting that, as in the mono-ester [1] and in the related species Ph4As[OsNC14 ]6 the terminal ligand is strongly n-donating, depressing the basal plane well below the osmium atom. These structural parameters are very close to those observed for [OsO2 (O2 C2Me4 )]2, the major differences being the absence of the dioxo bridge and the distance between the midpoints of the C--C bonds (approx. 5.1 A for OsO(O2 C2 H4 )2 and approx. 7.7 A for [(OsO2 (02 C2 Me4 )] 2). These latter separations are unlikely to change significantly with a change of ester since they are closely controlled by the tight bonding environment of the osmium(VI) atom. Despite a number of attempts, we have not yet obtained crystals suitable for X-ray work either from aqueous media or from lipids of the size likely to be found in living systems. Nevertheless, we have shown elsewhere [ 5] by chemical and spectroscopic means that the esters formed by large unsaturated molecules (e.g., cholesterol, ergosterol} have structures similar to these model compounds. A number of structures have been proposed for osmium(VI) di-esters e.g., two incorrect proposals involving five coordination [3] and one involving octahedral osmium [7]. However, the original supposition of Criegee et al. [8, 9] of five coordination has been shown to be correct, although they did not specify an exact stereochemistry. Our elucidation of this di-ester structure is relevant to the continuing debate as to whether mono-esters [10, 11] or di-
154
esters [3, 4, 6] are primarily involved in the reaction of osmium tetrox~de with lipids (the evidence concerning both sides of this debate have been summarised by Riemersma [12]). Cross-linking in cyclic osmium esters was first suggested by Wigglesworth [3] as a possible reason for the important fixation properties of osmium tetroxide. It is clear, however, that in di-esters the erstwhile C=C bonds thus linked would have to be drawn (or even pushed apart) to within approx. 5.1 A of each other. The formation of mono-esters, which involve the interposition of a dioxo bridge (Fig. 1) imposes a larger separation of approx. 7.7 £. Thus, the formation of mono- or di-esters must in part depend on the original disposition of double bonds in the pre-fixed substrate and the degree to which the chains of which these bonds form a part can move relative to each other. We are now studying the interaction of osmium tetroxide in aqueous media with lipids and phospholipids containing polar groups. We thank the Science Research Council for a Postdoctoral Fellowship (to R.J.C.), the University of Ghana for a Postgraduate Scholarship (to F.L.P.), and Johnson, Matthey Ltd, for the loan of osmium tetroxide. References 1 Collin, R.J., Griffith, W.P., Phillips, F.L. and Skapski, A.C. (1973) Biochim. Biophys. Acta 320, 745--747 2 Korn, E.D. (1967) J. Cell Biol. 34, 627--638 3 Wigglesworth, H.B. (1957) Proc. R. Soc. B147, 185--199 4 Baker, J.R. (1958) J. Histochem. Cytoehem. 6, 303--308 5 Collin, R.J., Jones, J. and Griffith, W.P. (1974) J. Chem. Soc. (Dalton), in the press 6 Fletcher. S.R., Griffith, W.P., Pawson, D., Phillips, F.L. and Skapski, A.C. (1973) Inorg. NucL Chem. Lett. 9, 1 1 1 7 - - 1 1 2 0 7 Griffith, W.P. a n d R o s s e t t i , R. (1972) J. Chem. Soe. (Dalton) 1449--1453 8 Criegee, R., Marchand, B. and Wannowius, H. (1942) Ann. Chem. 550, 99--133 9 Criegee, R. (1936) Ann. Chem. 522, 75--96 10 Stoeckenius, W. and Mahr, S.C. (1965) Lab. Invest. 14, 1196--1207 11 Riemersma, J.C. (1968) Biochim. Biophys. Acta 152, 718--727 12 Riemersma, J.C. (1970) Some Biological Techniques in Electron Microscopy, p. 69, Academic Press, N e w York
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA Report BBA 21393 STAINING AND FIXATION OF UNSATURATED MEMBRANE LIPIDS BY OSMIUM TETROXIDE CRYSTAL STRUCTURE OF A MODEL OSMIUM(VI) DI-ESTER
R. COLLIN, W.P. GRIFFITH, F.L. PHILLIPS and A.C. SKAPSKI Chemical Crystallography and Inorganic Chemistry Laboratories, Imperial College, London SW7 2A Y (U.K.)
(Received April 16th, 1974)
Summary The X-ray crystal structure of a cyclic osmium(VI)di-ester is reported and compared with that of a related mono-ester. The results are used to discuss the role of osmium tetroxide as a staining and fixation agent for unsaturated lipids.
We have recently reported the X-ray crystal structure of the osmium(VI) mono-ester, [OsO~ (02 C2 Me4)] 2 [ 1 ] and suggested that its unexpected dimeric structure (Fig. 1) might offer an explanation of the fixation properties of osmium tetroxide for membrane lipids. To carry this argument forward, however, it seemed desirable to examine the structure of an analogous di-ester since such species, it has been claimed, may be important in lipid fixation [2--4]. We first a t t e m p t e d to carry out an X-ray study of OSO(O2 C2 Me4)2 (tetragonal, a = 10.224, c = 7.265 A), but the sensitivity of the crystals to prolonged X-ray irradiation, and the apparent molecular disorder in the crystal lattice, prevented us from obtaining a clear-cut structural solution. We have however succeeded in obtaining the full structure of the closely related oxobis(ethane-l,2-diolato)osmium(VI), OsO(O2 C2 Ha )2. The black prismatic crystals were obtained by the prolonged action (over some eight weeks) of osmium tetroxide on ethylene glycol at room temperatures [5]. They are o r t h o r h o m b i c with unit-cell dimensions a = 10.950, b = 8.376, c = 7.735 A; space group P b c n (No. 60), and Z = 4. A total of 676 independent reflections were measured on a Siemens four-circle diffractometer (to 0 = 70 °, Cu-Ka radiation). The structure was solved by Patterson and
153
0
Me,C/O~ / %
/O~cMe =
0 Fig. 1. Schematic diagram of t h e s t r u c t u r e of [OsO~(O2C2Me4)] 2 i
Os
C
C
Fig. 2. T h e s t r u c t u r e of OsO(O2C2H4)2.
Fourier methods, and least-squares refinement has now reached R = 0.035. The molecule, shown in Fig. 2, lies on a crystallographic two-fold axis. The osmium has an essentially square-based pyramidal coordination, the four ester oxygen atoms constituting the basal plane (mean Os--O distance 1.89(1} £) and the terminal oxo ligand in the apical position (Os--O 1.66(1} A). The mean O(ester)--Os--O(terminal) angle is 110 °, suggesting that, as in the mono-ester [1] and in the related species Ph4As[OsNC14 ]6 the terminal ligand is strongly n-donating, depressing the basal plane well below the osmium atom. These structural parameters are very close to those observed for [OsO2 (O2 C2Me4 )]2, the major differences being the absence of the dioxo bridge and the distance between the midpoints of the C--C bonds (approx. 5.1 A for OsO(O2 C2 H4 )2 and approx. 7.7 A for [(OsO2 (02 C2 Me4 )] 2). These latter separations are unlikely to change significantly with a change of ester since they are closely controlled by the tight bonding environment of the osmium(VI) atom. Despite a number of attempts, we have not yet obtained crystals suitable for X-ray work either from aqueous media or from lipids of the size likely to be found in living systems. Nevertheless, we have shown elsewhere [ 5] by chemical and spectroscopic means that the esters formed by large unsaturated molecules (e.g., cholesterol, ergosterol} have structures similar to these model compounds. A number of structures have been proposed for osmium(VI) di-esters e.g., two incorrect proposals involving five coordination [3] and one involving octahedral osmium [7]. However, the original supposition of Criegee et al. [8, 9] of five coordination has been shown to be correct, although they did not specify an exact stereochemistry. Our elucidation of this di-ester structure is relevant to the continuing debate as to whether mono-esters [10, 11] or di-
154
esters [3, 4, 6] are primarily involved in the reaction of osmium tetrox~de with lipids (the evidence concerning both sides of this debate have been summarised by Riemersma [12]). Cross-linking in cyclic osmium esters was first suggested by Wigglesworth [3] as a possible reason for the important fixation properties of osmium tetroxide. It is clear, however, that in di-esters the erstwhile C=C bonds thus linked would have to be drawn (or even pushed apart) to within approx. 5.1 A of each other. The formation of mono-esters, which involve the interposition of a dioxo bridge (Fig. 1) imposes a larger separation of approx. 7.7 £. Thus, the formation of mono- or di-esters must in part depend on the original disposition of double bonds in the pre-fixed substrate and the degree to which the chains of which these bonds form a part can move relative to each other. We are now studying the interaction of osmium tetroxide in aqueous media with lipids and phospholipids containing polar groups. We thank the Science Research Council for a Postdoctoral Fellowship (to R.J.C.), the University of Ghana for a Postgraduate Scholarship (to F.L.P.), and Johnson, Matthey Ltd, for the loan of osmium tetroxide. References 1 Collin, R.J., Griffith, W.P., Phillips, F.L. and Skapski, A.C. (1973) Biochim. Biophys. Acta 320, 745--747 2 Korn, E.D. (1967) J. Cell Biol. 34, 627--638 3 Wigglesworth, H.B. (1957) Proc. R. Soc. B147, 185--199 4 Baker, J.R. (1958) J. Histochem. Cytoehem. 6, 303--308 5 Collin, R.J., Jones, J. and Griffith, W.P. (1974) J. Chem. Soc. (Dalton), in the press 6 Fletcher. S.R., Griffith, W.P., Pawson, D., Phillips, F.L. and Skapski, A.C. (1973) Inorg. NucL Chem. Lett. 9, 1 1 1 7 - - 1 1 2 0 7 Griffith, W.P. a n d R o s s e t t i , R. (1972) J. Chem. Soe. (Dalton) 1449--1453 8 Criegee, R., Marchand, B. and Wannowius, H. (1942) Ann. Chem. 550, 99--133 9 Criegee, R. (1936) Ann. Chem. 522, 75--96 10 Stoeckenius, W. and Mahr, S.C. (1965) Lab. Invest. 14, 1196--1207 11 Riemersma, J.C. (1968) Biochim. Biophys. Acta 152, 718--727 12 Riemersma, J.C. (1970) Some Biological Techniques in Electron Microscopy, p. 69, Academic Press, N e w York