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Trinodular structure of fibrinogen
J. Mol. Biol.
(1979) 134, 241-249
Trinodular Confirmation
Structure of Fibrinogen
by both Shadowing and Negative Stain Electron Microscopy WALTER
E. FOWLER
Departments
AND HAROLD P. ERICKSON
of Pharmacology and Anatomy Duke University
Durham,
N.C. 27710, U.S.A.
(Received 21 February
1979)
Fibrinogen molecules sprayed on mica, dried in vacua and shadowed with platinum appear as trinodular rods. We describe here the flotation technique of negative staining by which we were able to visualize individual fibrinogen molehave a cules. The fibrinogen molecules in our negatively stained preparations trinodular structure identical to that of shadowed molecules. We believe that the 20 nm diameter globules seen in previous negative staining studies are aggregates of these molecules. This is the first study in which both shadowed and negatively stained preparations of fibrinogen support a single, consistent model for the structure of fibrinogen. The molecules in our preparation are 45 nm long (k2.5 nm, our overall estimate of accuracy), the two outer nodules are 6 to 7 nm in diameter and the middle nodule is 4 to 5 nm in diameter.
1. Introduction Fibrinogen is a plasma protein of about 340,000 molecular weight which, upon enzymatic removal of small peptides or by manipulation of the ionic environment, polymerizes to form an anastomosing network of striated fibers, the fibrin clot. The structure of fibrinogen, a dimer with two copies each of three different polypeptides, has been studied for many years by a variety of techniques (for reviews, see Doolittle, 1973,1975). The most widely accepted model for the fibrinogen molecule is the trinodular structure, which was proposed by Hall & Slayter (1959) on the basis of the electron microscopy of shadowed fibrinogen preparations. They described a molecule about 47 nm long consisting of three interconnected globular domains 5 to 6 nm in diameter. Alternative models have been proposed on the basis of electron microscopy and each can claim some support from the diverse biochemical data reported for this protein. Confirmation of Hall and Slayter’s results with unidirectionally shadowed fibrinogen has been reported by Krakow et al. (1972) using freeze-dried specimens. Bachmann et al. (1975) interpreted shadowed specimens of freeze-etched fibrinogen as demonstrating a sausage-like molecule, 45 nm x 9 nm, with no nodules. Stewart (1971) and Blakey et al. (1977) report quite different results from the use of Hall and Slayter’s techniques. These contradictory results have been obtained with the use of virtually the same technique in the hands of different investigators. Negative staining has been widely used in the electron microscopy of other proteins 241 0022-2836/79/300241-09
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Press Inc. (London)
Ltd.
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W. E. FOWLER
AND
H.
1’. ERICKSOS
is regarded as a higher resolution technique than shadow casting. Rod-like or trinodular structures have never been reported in fibrinogen studies utilizing negative stain techniques. Previous attempts at visualization of the fibrinogen molecule by negative staining have led to the conclusion that the results are uninterpretable (Kay & Cuddigan, 1967 ; Haschemeyer, 1970) or that negatively stained fibrinogen is a globular structure about 20 nm in diameter (Koppel, 1967 ; Pouit et al.. 1972). Kiippel interprets this globular structure as a pentagonal dodecahedron wit’11 polypeptide chains located only on the edges whereas Pouit et al. and other groups have suggested that a conformational change during activation by thrombin or other agents causes a globular fibrinogen molecule to unfold to a more rod-like structure (Tranqui-Pouit, et al., 1975; Hudry-Clergeon et al., 1975; Belitser et aE., 1973). It should be noted that hydrodynamic and solution data can fit either model which has been proposed on the basis of electron microscopy: if the molecule is a narrou rod or trinodular structure 45 nm long it will be a relatively compact protein, whereas if the molecule is a sphere 20 nm in diameter it, must be very highly hydrated (approximately 8 g water per g protein). To settle this controversy and to establish the correct model for the structure of the fibrinogen molecule, consistent images of the molecule as visualized by different, electron microscopic techniques are needed. We present here the results of such studies in which two types of shadowed preparations and negatively stained specimens all show an identical trinodular structure for the fibrinogen molecule. and
2. Materials and Methods (a) Preparation
of jibrinogen
Fibrinogen (Grade L, Kabi, Stockholm) was a gift from Dr Patrick McKee, as was plasmin-free fibrinogen which had been purified by passage through a lysine-affinity column (Matsuta et al., 1972). Kabi fibrinogen was dissolved in 50 rnM-ammonium formate, pH 7, at 1 to 5 mg/ml and dialyzed overnight at 4°C against 2 changes of the same buffer. This stock fibrinogen solution was stored at 4°C and used within 2 days. The plasmin-free fibrinogen solution was stored frozen at 4 mg/ml in Tris-buffered saline and portions were thawed immediately before use. These solutions were either diluted and used directly or further purified by sedimenting 200-~1 samples through &ml glycerol gradients (8% to 30% in 50 mM-ammonium formate, pH 7) in an SW 50.1 rotor at 35,000 revs/min for 11 11 at 25°C. These gradients were collected in 500-~1 fractions, the absorbance at 280 nm was determined, and the peak fractions were used for experiments. There was no apparent difference in the structures observed whether Kabi fibrinogen or plasmin-free fibrinogen was used or whether or not gradient purification was performed. Figure 1 and Figure 2(a) are direct dilutions of Kabi fibrinogen and of plasmin-free fibrinogen, respectively. Figure 3(a) is from a preparation of gradient-purified plasmin-free fibrinogen. (b ) Shadowirbg Fibrinogen solutions at 3 to 1000 pg/ml in 50 m&f-ammonium formate, pH 7, were mixed with glycerol and a 100 pg/ml solution of tobacco mosaic virus (TMV) in a 1: 1: 1 ratio. A 150-~1 portion of the mixture was sprayed on a freshly cleaved mica strip (1 cm x 4 cm) using a bulb-nebulizer (Fisons Corp., Bedford, Mass.). The sprayed mica was placed immediately in an Edwards vacuum evaporator (model E12E, Edwards High Vacuum Ltd., Crawley, England) and dried in uacuo at room temperature for 1 to 2 h. In one case sprayed mica was frozen onto a copper block and freeze-dried in a Denton model DV-502. In all cases, the vacuum maintained was better than 2x low6 t,orr and liquid nitrogen cold traps were used. Specimens were shadowed with platinum at an angle of 1 in 10 and a carbon film was deposited on the replica from directly above. The replicas were floated off onto distilled water and were picked up from below with 200-mesh copper grids.
TRINODULAR
STRUCTURE
OF FIBRINOGEN
243
Rotary shadowing of identical specimens was performed either in the Edwards vacuum evaporator or ‘in a Balzers model BAE 120 (Leichtenstein) equipped with an electron beam evaporation source and a carbon/platinum electrode. The shadowing angle was again nominally 1 in 10. (c) Negative ekzinkg Negative staining of fibrinogen was performed using the flotation technique of Valentine et al. (1968) as modified by Wrigley et al. (1977). Briefly, a carbon film was floated off of a piece of mica (2 mm x 2 mm) onto the surface of a solution of fibrinogen molecules at 3 to 25 pg/ml in 50 IIIM-amIUOniUm formate, pH 7. After 10 s, the carbon film was picked up on the same piece of mica and then floated off onto a drop of 2% uranyl acetate. The carbon film was finally picked up from above on a 200-mesh grid and blotted dry with filter paper. (d) Electron microscopy Specimens were examined in a Philips 301 electron microscope using a 50ym objective aperture at 80 kV accelerating voltage. Micrographs were taken at 34,000~ nominal magnification and photographically enlarged to 100,000 x and 200,000 x final magnification. Magnification was calibrated using micrographs of negatively stained cat&se crystals (Wrigley, 1968) which were taken at the beginning and end of a series of exposures.
3. Results Shadowing of fibrinogen which had been sprayed on mica at concentrations from 1 pg/ml to 350 pg/ml always showed a predominance of trinodular structures. Even at 1 pg/ml where individual molecules are very sparsely distributed, the trinodular structure was virtually the only species observed. Thus there is no indication that the trinodular structure is an aggregate, The optimal concentration of fibrinogen for spraying was determined to be about 50 pg/ml. Initially, samples sprayed onto mica were freeze-dried by the method of Elliot et al. (1976) in IL Denton device, but no difference was seen between specimens prepared in this way and specimens dried at, room temperature in a conventional vacuum evaporator. In addition, the trinodular structure was the same whether prepared in 50 mM or 500 mM-ammonium formate, both at pH 7. The structure of molecules dried in the presence of glycerol seemed to be better preserved relative to those dried with no glycerol present. Figure 1 is a representative field of fibrinogen molecules shadowed unidirectionally. Figure 2(a) is a micrograph of a similar preparation which was rotary shadowed. Selected molecules which have been rotary shadowed are shown in Figure 2(b) and unidirectionally shadowed molecules are shown in Figure 2(c) and (d). The same trinodular shape and similar overall molecular lengths are apparent from Figures 1 and 2 and from Table 1. The data in Table 1 are determined by averaging the dimensions of at least 20 molecules from each type of preparation. Shadow lengths in unidirectionally shadowed specimens were corrected for variations in shadowing angle by using the shadou length of TMV rods which were mixed into the fibrinogen/glycerol solution prior to spraying. The height of the TMV rod was taken to be 17 nm (Champness et al., 1976). The dimensions of rotary shadowed fibrinogen molecules are given as measured and also after correction is made for the uniform shell of platinum which must surround the molecule. This shell is estimated in these preparations to be 2 nm thick. Conventional negative staining methods, in which a drop of a protein solution is
244
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P. ERICKSON
FIG. 1. Kabi fibrinogen in 60 mx-ammonium formate, 33 9; glycerol, sprayed on freshly cleaved mica and dried ilz VWUO. Shadowed unidirectionally at an angle of 1 in 10. The concentration of fibrinogen in the sprayed solution was 67 pg/ml. Magnification, 100,000 x .
applied to a carbon film followed by several drops of uranyl acetate or other electron dense stain, failed to give interpretable images. Low concentrations of protein (5 to 50 pg/ml) left the carbon film hydrophobic, which resulted in lack of sufficient stain on the film. A higher protein concentration rendered the carbon film hydrophilic and the amount of stain deposited was increased. However, images now showed complicated networks of fibers and irregular globules. We believe that these are aggregates of many molecules produced when a solution containing a high concentration of fibrinogen is dried down onto the film. The only convincing negatively stained images of fibrinogen we have obtained
FIG. 2. (a) Kabi fibrinogen prepared as in Fig. 1 and rotary shadowed at an angle of about 1 in 10 in a Balzers BAE 120. Concentration of fibrinogen was 40 pg/ml. Magnification, 100,000 x . (b) Selected fibrinogen molecules from rotary shadowed preparations such as in (a), but with varying amounts of Pt shadow. Magniiication, 200,000 x . (c) Fibrinogen molecules unidirectionally shadowed as in Fig. 1. Note the 3 shadows (white) resulting from the 3 globular domains of each molecule. Magnification, 200,000 x . (d) Curved fibrinogen molecules prepared as in Fig. 1 demonstrating the flexibility of some molecules in such preparations. Magnification, 200,000 x .
W. E. FOWLER
246
AND
H. P. ERICKSON
TABLE 1 Molecule dimensions
Unidire&ional Rotary
shadowing
shadowing?
Negative
stain
Fibrinogen length
End nodule diameter$
(-1
(-1
(-)
4.0&0.3
2.7 $0.2
(11.2hl.l) 7.25 l-1
(8+&1’1) 4.8 + 1.1
444f3.9 (48.91-1.1) 44.9; 1.1 45062.8
6.7+0.9
Middle nodule diameter$
4.9tO.6
Dimensions in nanometers were determined by averaging at least 20 molecules for each technique. Each value is followed by & standard deviation. Our overall estimate for the length determination and its accuracy is 45k2.5 nm. The accuracy is limited by the observed variation in measured lengths, not by the absolute accuracy of the magnification calibration ( +2.5”&, Wrigley, 1968). t The value in parenthesis is the actual subtracting 4.0 nm from each measurement, surrounding the molecule. $ Diameter
indicates
height
for unidirectional
measured assuming
value. The lower number is corrected by a 2 nm thick layer of platinum completely
shadowing
and width
for rotary
shadowing.
have been through the use of the flotation technique of Valentine et al. (1968) as described in Materials and Methods. The optimal concentration for visualizing fibrinogen molecules using this technique was found to be 10 pg/ml or less. Figure 3(a) is a representative field of a negatively stained preparation of fibrinogen and Figure 3(b) shows selected molecules at higher magnification. These trinodular structures are the same length as those seen in shadowed preparations (Table 1). The flexibility of the molecule is also similar under shadowing and negative stain conditions, as can be seen from a comparison of Figure 2(d) and Figure 3(b), although the vast majority of the molecules in both cases are straight. The widths of the outer nodules of negatively stained fibrinogen molecules are compatible with the diameters of these nodules in shadowed preparations (Table 1).
4. Discussion The trinodular structure of fibrinogen was first proposed on the basis of electron microscopy of shadowed preparations (Hall & Slayter, 1959). Globular structures, seen in some negatively stained fibrinogen preparations, have led some investigators to propose a very different type of model (Koppel, 1967; Pouit et al., 1972). Each of these models has previously been supported by only a single technique of electron microscopy: shadowing or negative staining. The present study, by showing virtually identical structures in both shadowed and negatively stained specimens, confirms the trinodular model of fibrinogen and establishes consistent molecular dimensions for the molecule. We conclude that the irregular globular structures 20 nm in diameter described in previous studies of fibrinogen by negative staining (Koppel, 1967; Pouit et ul., 1972) are not individual fibrinogen molecules. We have never seen these structures using the flotation technique as described in Materials and Methods and very low concentrations of fibrinogen. These specimens consistently show trinodular structures
TRINODULAR
STRUCTURE
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FIBRINOGEN
247
Fro. 3. (a) Gradient-purified fibrinogen molecules negatively stained with 2% uranyl acetate using the flotation technique. Note that molecules are apparent only in the area with the correct level of staining. The fibrinogen concentration was 3 pg/ml. Magnification, 100,000 x , (b) Selected negatively stained fibrinogen molecules. Note trinodular structure and the appareht, flexibility in some of the examples. Magnification, 200,000 x .
identical to those seen in shadowed specimens. Since these previous investigators used very high fibrinogen concentrations, 50 to 1000 times higher than that used in the present study, the globular structures almost certainly result from the superposition of several layers of fibrinogen molecules in their specimens, as previously suggested by Krakow et al. (1972).
248
W. E. FOWLER
ANL,
H. I’. ERICKSON
Unidirectionally shadowed fibrinogen has been studied in several laboratories and most results agree at least approximately with the original study of Hall & Slayter (1959) (Bang, 1964; Krakow et al., 1972). Bachmann et al. (1975), in a study of freezeetched fibrinogen, proposed a sausage-shaped molecule, 45 nm x 9 nm. These dimensions are roughly compatible with the trinodular model, though this technique apparently lacks sufficient resolution to identify the individual nodules. Our micrographs of rotary shadowed fibrinogen (Fig. 2(a) and (b)) are similar to those obtained by Slayter (1976,1978) and show the trinodular structure in a somewhat different aspect than in unidirectionally shadowed preparations. Interpretation of the dimensions of shadowed molecules is complicated by the thickness of the platinum deposits and by possible drying effects. Both undirectionally and rotary shadowed specimens show that the nodules at either end of the fibrinogen molecule are equal in size and that the central nodule is smaller, again confirming the results of Hall & Slayter (1959). The heights of the nodules (Table l), as calculated from the shadow lengths of unidirectionally shadowed molecules, may indicate a flattening of the molecule during drying. Both the length and the nodule diameter of rotary shadowed molecules are about 4 nm greater than unidirectionally shadowed or negatively stained molecules, which implies that rotary shadowed molecules are completely surrounded by a shell of platinum. We estimate this shell to be 2 nm thick. The dimensions given for these molecules in Table 1 agree very well with the other values after correction is made for this thickness of the platinum shell. The apparent round shape of the outer nodules in these preparations may also be due to this platinum shell, since these nodules appear elongated in negatively stained molecules. Slayter (1978) has also suggested that the nodules appear somewhat elongated in certain unidirectionally shadowed specimens. Both shadowing and negative stain give similar values for the average length of the fibrinogen molecule, which indicates that no drastic molecular changes occur during the two quite different preparatory procedures. It is worth noting that t,he flexibility of the molecule is likewise apparently independent of the technique used. Although the fibrinogen molecule demonstrates flexibility, the large majority of the molecules in these preparations are straight. This argues against a suggestion that a 35” bend is an intrinsic structural feature of the molecule (Doolittle, 1977). In conclusion, this study provides confirmation of the trinodular structure of fibrinogen in shadowed preparations and presents the first evidence of the same structure in negatively stained preparations, thus resolving the controversy regarding the shape of the molecule as visualized in the electron microscope. This result will allow the interpretation of previously ambiguous biochemical data, as well as stimulating further study of the fibrinogen molecule and other large proteins using a combination of these electron microscopic techniques. This work was supported by National Science Foundation grant PCM7723592 and National Institute of Health grant GMN623911. One of the authors (W. E. F.) is supported by a National Research Service Award predoctoral fellowship. REFERENCES Bachmann, lekulare Bang, N. U. Belitser, V.
L., Schmitt-Fumian, W., Hammel, R. & Lederer, K. (1975). Die fMakromoChemie, 176, 2603-2618. (1964). Thromb. Diath. Haemorrh. (Suppl.), 13, 73-80. A., Varetska, T. V. & Manjakov, V. Ph. (1973). Thrombosis Res. 2, 567-578.
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Blakey, P., Groom, M. & Turner, R. (1977). B&t. J. Haematol. 35, 437-440. Champness, J., Bloomer, A. C., Bricogne, G., Butler, P. & Klug, A. (1976). Nature
(London),
259, 2&24.
Doolittle, R. (1973). Achan. Protein Chem. 27, l-109. Doolittle, R. F. (1975). In The Plasma Proteins (Putnam, F. W., ed.), vol. 2, pp. 1099161, Academic Press, New York. Doolittle, R. (1977). Horizolzs Biochem. Biophys. 3, 164-191. Elliot, A. & Offer, G. (1978). J. Mol. Biol. 123, 505-519. Elliot, A., Offer, G. & Burridge, K. (1976). Proc. Roy. Sot. ser. B, 193, 45-53. Hall, C. & Slayter, H. (1959). J. B&hem. Biophys. Cytol. 5, 11-15. Haschemeyer, R. (1970). Advan. Enzymol. 33, 71-117. Hudrey-Clergeon, G., Marguerie, G., Pouit, L. & Suscillon, M. (1975). Thrombosis Rea. 6, 533-541. Kay, D. & Cuddigan, B. (1967). Brit. J. Haematol. 13, 341-347. Koppel, G. (1967). 2. ZeZZforschung, 77, 443-517. Krakow, W., Endres, G., Siegel, B. & Scheragtt, W. (1972). J. Mol. BioZ. 71, 95-103. Marguerie, G. (1977). Biochem. Biophys. Acta, 494, 172-182. Metsuta, M., Iwanaga, S. & Nakamura, S. (1972). Thrombosis Res. 1, 619-630. Pouit, L., Marcille, G., Suscillon, M. & Hollard, D. (1972). Thromb. Diath. Huemorrh. 27, 559-572. Slayter, H. (1976). Ultramicroscopy, 1, 341-357. Slayter, H. (1978). In Principles and Techniques of Electron Microscopy (Hayat, M., ed.), vol. 9, pp. 175245, Van Nostrand, New York. Stewart, G. (1971). Comments and Discussion in Stand. J. Haematol. 13, 63-67. Tranqui-Pouit, L., Marder, V., Suscillon, M., Budzynski, A. & Hudry-Clergeon, G. (1975). Biochim. Biophys. Acta, 400, 189-199. Valentine, R. C., Shapiro, B. & Stadtman, E. (1968). Biochemistry, 7, 2143-2152. Wrigley, N. G. (1968). J. Ultrastruct. Res. 24, 454-464. Wrigley, N., Laver, W. & Downie, J. (1977). J. Mol. BioZ. 109, 405-421.
from the length of the Note Added in Proof: The diameter of the nodules determined shadows is smaller than that measured from negatively stained or rotary shadowed molecules. The same discrepancy has been noted by Elliot & Offer (1978) with myosin and by W. Voter and H. P. Erickson (unpublished) with aldolase. It seems generally true that the height of small globular protein molecules is significantly underestimated by the shadow length determinations. It has been suggested by Marguerie (1977) that calcium may stabilize a more compact structure of fibrinogen. C&I, (2.5 mM) or EDTA (2.5 mM) have no effect on the trinodular structure as seen in the electron microscope by our techniques.
(1979) 134, 241-249
Trinodular Confirmation
Structure of Fibrinogen
by both Shadowing and Negative Stain Electron Microscopy WALTER
E. FOWLER
Departments
AND HAROLD P. ERICKSON
of Pharmacology and Anatomy Duke University
Durham,
N.C. 27710, U.S.A.
(Received 21 February
1979)
Fibrinogen molecules sprayed on mica, dried in vacua and shadowed with platinum appear as trinodular rods. We describe here the flotation technique of negative staining by which we were able to visualize individual fibrinogen molehave a cules. The fibrinogen molecules in our negatively stained preparations trinodular structure identical to that of shadowed molecules. We believe that the 20 nm diameter globules seen in previous negative staining studies are aggregates of these molecules. This is the first study in which both shadowed and negatively stained preparations of fibrinogen support a single, consistent model for the structure of fibrinogen. The molecules in our preparation are 45 nm long (k2.5 nm, our overall estimate of accuracy), the two outer nodules are 6 to 7 nm in diameter and the middle nodule is 4 to 5 nm in diameter.
1. Introduction Fibrinogen is a plasma protein of about 340,000 molecular weight which, upon enzymatic removal of small peptides or by manipulation of the ionic environment, polymerizes to form an anastomosing network of striated fibers, the fibrin clot. The structure of fibrinogen, a dimer with two copies each of three different polypeptides, has been studied for many years by a variety of techniques (for reviews, see Doolittle, 1973,1975). The most widely accepted model for the fibrinogen molecule is the trinodular structure, which was proposed by Hall & Slayter (1959) on the basis of the electron microscopy of shadowed fibrinogen preparations. They described a molecule about 47 nm long consisting of three interconnected globular domains 5 to 6 nm in diameter. Alternative models have been proposed on the basis of electron microscopy and each can claim some support from the diverse biochemical data reported for this protein. Confirmation of Hall and Slayter’s results with unidirectionally shadowed fibrinogen has been reported by Krakow et al. (1972) using freeze-dried specimens. Bachmann et al. (1975) interpreted shadowed specimens of freeze-etched fibrinogen as demonstrating a sausage-like molecule, 45 nm x 9 nm, with no nodules. Stewart (1971) and Blakey et al. (1977) report quite different results from the use of Hall and Slayter’s techniques. These contradictory results have been obtained with the use of virtually the same technique in the hands of different investigators. Negative staining has been widely used in the electron microscopy of other proteins 241 0022-2836/79/300241-09
$02.00/O
0 1979 Academic
Press Inc. (London)
Ltd.
242
W. E. FOWLER
AND
H.
1’. ERICKSOS
is regarded as a higher resolution technique than shadow casting. Rod-like or trinodular structures have never been reported in fibrinogen studies utilizing negative stain techniques. Previous attempts at visualization of the fibrinogen molecule by negative staining have led to the conclusion that the results are uninterpretable (Kay & Cuddigan, 1967 ; Haschemeyer, 1970) or that negatively stained fibrinogen is a globular structure about 20 nm in diameter (Koppel, 1967 ; Pouit et al.. 1972). Kiippel interprets this globular structure as a pentagonal dodecahedron wit’11 polypeptide chains located only on the edges whereas Pouit et al. and other groups have suggested that a conformational change during activation by thrombin or other agents causes a globular fibrinogen molecule to unfold to a more rod-like structure (Tranqui-Pouit, et al., 1975; Hudry-Clergeon et al., 1975; Belitser et aE., 1973). It should be noted that hydrodynamic and solution data can fit either model which has been proposed on the basis of electron microscopy: if the molecule is a narrou rod or trinodular structure 45 nm long it will be a relatively compact protein, whereas if the molecule is a sphere 20 nm in diameter it, must be very highly hydrated (approximately 8 g water per g protein). To settle this controversy and to establish the correct model for the structure of the fibrinogen molecule, consistent images of the molecule as visualized by different, electron microscopic techniques are needed. We present here the results of such studies in which two types of shadowed preparations and negatively stained specimens all show an identical trinodular structure for the fibrinogen molecule. and
2. Materials and Methods (a) Preparation
of jibrinogen
Fibrinogen (Grade L, Kabi, Stockholm) was a gift from Dr Patrick McKee, as was plasmin-free fibrinogen which had been purified by passage through a lysine-affinity column (Matsuta et al., 1972). Kabi fibrinogen was dissolved in 50 rnM-ammonium formate, pH 7, at 1 to 5 mg/ml and dialyzed overnight at 4°C against 2 changes of the same buffer. This stock fibrinogen solution was stored at 4°C and used within 2 days. The plasmin-free fibrinogen solution was stored frozen at 4 mg/ml in Tris-buffered saline and portions were thawed immediately before use. These solutions were either diluted and used directly or further purified by sedimenting 200-~1 samples through &ml glycerol gradients (8% to 30% in 50 mM-ammonium formate, pH 7) in an SW 50.1 rotor at 35,000 revs/min for 11 11 at 25°C. These gradients were collected in 500-~1 fractions, the absorbance at 280 nm was determined, and the peak fractions were used for experiments. There was no apparent difference in the structures observed whether Kabi fibrinogen or plasmin-free fibrinogen was used or whether or not gradient purification was performed. Figure 1 and Figure 2(a) are direct dilutions of Kabi fibrinogen and of plasmin-free fibrinogen, respectively. Figure 3(a) is from a preparation of gradient-purified plasmin-free fibrinogen. (b ) Shadowirbg Fibrinogen solutions at 3 to 1000 pg/ml in 50 m&f-ammonium formate, pH 7, were mixed with glycerol and a 100 pg/ml solution of tobacco mosaic virus (TMV) in a 1: 1: 1 ratio. A 150-~1 portion of the mixture was sprayed on a freshly cleaved mica strip (1 cm x 4 cm) using a bulb-nebulizer (Fisons Corp., Bedford, Mass.). The sprayed mica was placed immediately in an Edwards vacuum evaporator (model E12E, Edwards High Vacuum Ltd., Crawley, England) and dried in uacuo at room temperature for 1 to 2 h. In one case sprayed mica was frozen onto a copper block and freeze-dried in a Denton model DV-502. In all cases, the vacuum maintained was better than 2x low6 t,orr and liquid nitrogen cold traps were used. Specimens were shadowed with platinum at an angle of 1 in 10 and a carbon film was deposited on the replica from directly above. The replicas were floated off onto distilled water and were picked up from below with 200-mesh copper grids.
TRINODULAR
STRUCTURE
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243
Rotary shadowing of identical specimens was performed either in the Edwards vacuum evaporator or ‘in a Balzers model BAE 120 (Leichtenstein) equipped with an electron beam evaporation source and a carbon/platinum electrode. The shadowing angle was again nominally 1 in 10. (c) Negative ekzinkg Negative staining of fibrinogen was performed using the flotation technique of Valentine et al. (1968) as modified by Wrigley et al. (1977). Briefly, a carbon film was floated off of a piece of mica (2 mm x 2 mm) onto the surface of a solution of fibrinogen molecules at 3 to 25 pg/ml in 50 IIIM-amIUOniUm formate, pH 7. After 10 s, the carbon film was picked up on the same piece of mica and then floated off onto a drop of 2% uranyl acetate. The carbon film was finally picked up from above on a 200-mesh grid and blotted dry with filter paper. (d) Electron microscopy Specimens were examined in a Philips 301 electron microscope using a 50ym objective aperture at 80 kV accelerating voltage. Micrographs were taken at 34,000~ nominal magnification and photographically enlarged to 100,000 x and 200,000 x final magnification. Magnification was calibrated using micrographs of negatively stained cat&se crystals (Wrigley, 1968) which were taken at the beginning and end of a series of exposures.
3. Results Shadowing of fibrinogen which had been sprayed on mica at concentrations from 1 pg/ml to 350 pg/ml always showed a predominance of trinodular structures. Even at 1 pg/ml where individual molecules are very sparsely distributed, the trinodular structure was virtually the only species observed. Thus there is no indication that the trinodular structure is an aggregate, The optimal concentration of fibrinogen for spraying was determined to be about 50 pg/ml. Initially, samples sprayed onto mica were freeze-dried by the method of Elliot et al. (1976) in IL Denton device, but no difference was seen between specimens prepared in this way and specimens dried at, room temperature in a conventional vacuum evaporator. In addition, the trinodular structure was the same whether prepared in 50 mM or 500 mM-ammonium formate, both at pH 7. The structure of molecules dried in the presence of glycerol seemed to be better preserved relative to those dried with no glycerol present. Figure 1 is a representative field of fibrinogen molecules shadowed unidirectionally. Figure 2(a) is a micrograph of a similar preparation which was rotary shadowed. Selected molecules which have been rotary shadowed are shown in Figure 2(b) and unidirectionally shadowed molecules are shown in Figure 2(c) and (d). The same trinodular shape and similar overall molecular lengths are apparent from Figures 1 and 2 and from Table 1. The data in Table 1 are determined by averaging the dimensions of at least 20 molecules from each type of preparation. Shadow lengths in unidirectionally shadowed specimens were corrected for variations in shadowing angle by using the shadou length of TMV rods which were mixed into the fibrinogen/glycerol solution prior to spraying. The height of the TMV rod was taken to be 17 nm (Champness et al., 1976). The dimensions of rotary shadowed fibrinogen molecules are given as measured and also after correction is made for the uniform shell of platinum which must surround the molecule. This shell is estimated in these preparations to be 2 nm thick. Conventional negative staining methods, in which a drop of a protein solution is
244
W. E. FOWLER
AND
H.
P. ERICKSON
FIG. 1. Kabi fibrinogen in 60 mx-ammonium formate, 33 9; glycerol, sprayed on freshly cleaved mica and dried ilz VWUO. Shadowed unidirectionally at an angle of 1 in 10. The concentration of fibrinogen in the sprayed solution was 67 pg/ml. Magnification, 100,000 x .
applied to a carbon film followed by several drops of uranyl acetate or other electron dense stain, failed to give interpretable images. Low concentrations of protein (5 to 50 pg/ml) left the carbon film hydrophobic, which resulted in lack of sufficient stain on the film. A higher protein concentration rendered the carbon film hydrophilic and the amount of stain deposited was increased. However, images now showed complicated networks of fibers and irregular globules. We believe that these are aggregates of many molecules produced when a solution containing a high concentration of fibrinogen is dried down onto the film. The only convincing negatively stained images of fibrinogen we have obtained
FIG. 2. (a) Kabi fibrinogen prepared as in Fig. 1 and rotary shadowed at an angle of about 1 in 10 in a Balzers BAE 120. Concentration of fibrinogen was 40 pg/ml. Magnification, 100,000 x . (b) Selected fibrinogen molecules from rotary shadowed preparations such as in (a), but with varying amounts of Pt shadow. Magniiication, 200,000 x . (c) Fibrinogen molecules unidirectionally shadowed as in Fig. 1. Note the 3 shadows (white) resulting from the 3 globular domains of each molecule. Magnification, 200,000 x . (d) Curved fibrinogen molecules prepared as in Fig. 1 demonstrating the flexibility of some molecules in such preparations. Magnification, 200,000 x .
W. E. FOWLER
246
AND
H. P. ERICKSON
TABLE 1 Molecule dimensions
Unidire&ional Rotary
shadowing
shadowing?
Negative
stain
Fibrinogen length
End nodule diameter$
(-1
(-1
(-)
4.0&0.3
2.7 $0.2
(11.2hl.l) 7.25 l-1
(8+&1’1) 4.8 + 1.1
444f3.9 (48.91-1.1) 44.9; 1.1 45062.8
6.7+0.9
Middle nodule diameter$
4.9tO.6
Dimensions in nanometers were determined by averaging at least 20 molecules for each technique. Each value is followed by & standard deviation. Our overall estimate for the length determination and its accuracy is 45k2.5 nm. The accuracy is limited by the observed variation in measured lengths, not by the absolute accuracy of the magnification calibration ( +2.5”&, Wrigley, 1968). t The value in parenthesis is the actual subtracting 4.0 nm from each measurement, surrounding the molecule. $ Diameter
indicates
height
for unidirectional
measured assuming
value. The lower number is corrected by a 2 nm thick layer of platinum completely
shadowing
and width
for rotary
shadowing.
have been through the use of the flotation technique of Valentine et al. (1968) as described in Materials and Methods. The optimal concentration for visualizing fibrinogen molecules using this technique was found to be 10 pg/ml or less. Figure 3(a) is a representative field of a negatively stained preparation of fibrinogen and Figure 3(b) shows selected molecules at higher magnification. These trinodular structures are the same length as those seen in shadowed preparations (Table 1). The flexibility of the molecule is also similar under shadowing and negative stain conditions, as can be seen from a comparison of Figure 2(d) and Figure 3(b), although the vast majority of the molecules in both cases are straight. The widths of the outer nodules of negatively stained fibrinogen molecules are compatible with the diameters of these nodules in shadowed preparations (Table 1).
4. Discussion The trinodular structure of fibrinogen was first proposed on the basis of electron microscopy of shadowed preparations (Hall & Slayter, 1959). Globular structures, seen in some negatively stained fibrinogen preparations, have led some investigators to propose a very different type of model (Koppel, 1967; Pouit et al., 1972). Each of these models has previously been supported by only a single technique of electron microscopy: shadowing or negative staining. The present study, by showing virtually identical structures in both shadowed and negatively stained specimens, confirms the trinodular model of fibrinogen and establishes consistent molecular dimensions for the molecule. We conclude that the irregular globular structures 20 nm in diameter described in previous studies of fibrinogen by negative staining (Koppel, 1967; Pouit et ul., 1972) are not individual fibrinogen molecules. We have never seen these structures using the flotation technique as described in Materials and Methods and very low concentrations of fibrinogen. These specimens consistently show trinodular structures
TRINODULAR
STRUCTURE
OF
FIBRINOGEN
247
Fro. 3. (a) Gradient-purified fibrinogen molecules negatively stained with 2% uranyl acetate using the flotation technique. Note that molecules are apparent only in the area with the correct level of staining. The fibrinogen concentration was 3 pg/ml. Magnification, 100,000 x , (b) Selected negatively stained fibrinogen molecules. Note trinodular structure and the appareht, flexibility in some of the examples. Magnification, 200,000 x .
identical to those seen in shadowed specimens. Since these previous investigators used very high fibrinogen concentrations, 50 to 1000 times higher than that used in the present study, the globular structures almost certainly result from the superposition of several layers of fibrinogen molecules in their specimens, as previously suggested by Krakow et al. (1972).
248
W. E. FOWLER
ANL,
H. I’. ERICKSON
Unidirectionally shadowed fibrinogen has been studied in several laboratories and most results agree at least approximately with the original study of Hall & Slayter (1959) (Bang, 1964; Krakow et al., 1972). Bachmann et al. (1975), in a study of freezeetched fibrinogen, proposed a sausage-shaped molecule, 45 nm x 9 nm. These dimensions are roughly compatible with the trinodular model, though this technique apparently lacks sufficient resolution to identify the individual nodules. Our micrographs of rotary shadowed fibrinogen (Fig. 2(a) and (b)) are similar to those obtained by Slayter (1976,1978) and show the trinodular structure in a somewhat different aspect than in unidirectionally shadowed preparations. Interpretation of the dimensions of shadowed molecules is complicated by the thickness of the platinum deposits and by possible drying effects. Both undirectionally and rotary shadowed specimens show that the nodules at either end of the fibrinogen molecule are equal in size and that the central nodule is smaller, again confirming the results of Hall & Slayter (1959). The heights of the nodules (Table l), as calculated from the shadow lengths of unidirectionally shadowed molecules, may indicate a flattening of the molecule during drying. Both the length and the nodule diameter of rotary shadowed molecules are about 4 nm greater than unidirectionally shadowed or negatively stained molecules, which implies that rotary shadowed molecules are completely surrounded by a shell of platinum. We estimate this shell to be 2 nm thick. The dimensions given for these molecules in Table 1 agree very well with the other values after correction is made for this thickness of the platinum shell. The apparent round shape of the outer nodules in these preparations may also be due to this platinum shell, since these nodules appear elongated in negatively stained molecules. Slayter (1978) has also suggested that the nodules appear somewhat elongated in certain unidirectionally shadowed specimens. Both shadowing and negative stain give similar values for the average length of the fibrinogen molecule, which indicates that no drastic molecular changes occur during the two quite different preparatory procedures. It is worth noting that t,he flexibility of the molecule is likewise apparently independent of the technique used. Although the fibrinogen molecule demonstrates flexibility, the large majority of the molecules in these preparations are straight. This argues against a suggestion that a 35” bend is an intrinsic structural feature of the molecule (Doolittle, 1977). In conclusion, this study provides confirmation of the trinodular structure of fibrinogen in shadowed preparations and presents the first evidence of the same structure in negatively stained preparations, thus resolving the controversy regarding the shape of the molecule as visualized in the electron microscope. This result will allow the interpretation of previously ambiguous biochemical data, as well as stimulating further study of the fibrinogen molecule and other large proteins using a combination of these electron microscopic techniques. This work was supported by National Science Foundation grant PCM7723592 and National Institute of Health grant GMN623911. One of the authors (W. E. F.) is supported by a National Research Service Award predoctoral fellowship. REFERENCES Bachmann, lekulare Bang, N. U. Belitser, V.
L., Schmitt-Fumian, W., Hammel, R. & Lederer, K. (1975). Die fMakromoChemie, 176, 2603-2618. (1964). Thromb. Diath. Haemorrh. (Suppl.), 13, 73-80. A., Varetska, T. V. & Manjakov, V. Ph. (1973). Thrombosis Res. 2, 567-578.
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Blakey, P., Groom, M. & Turner, R. (1977). B&t. J. Haematol. 35, 437-440. Champness, J., Bloomer, A. C., Bricogne, G., Butler, P. & Klug, A. (1976). Nature
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Doolittle, R. (1973). Achan. Protein Chem. 27, l-109. Doolittle, R. F. (1975). In The Plasma Proteins (Putnam, F. W., ed.), vol. 2, pp. 1099161, Academic Press, New York. Doolittle, R. (1977). Horizolzs Biochem. Biophys. 3, 164-191. Elliot, A. & Offer, G. (1978). J. Mol. Biol. 123, 505-519. Elliot, A., Offer, G. & Burridge, K. (1976). Proc. Roy. Sot. ser. B, 193, 45-53. Hall, C. & Slayter, H. (1959). J. B&hem. Biophys. Cytol. 5, 11-15. Haschemeyer, R. (1970). Advan. Enzymol. 33, 71-117. Hudrey-Clergeon, G., Marguerie, G., Pouit, L. & Suscillon, M. (1975). Thrombosis Rea. 6, 533-541. Kay, D. & Cuddigan, B. (1967). Brit. J. Haematol. 13, 341-347. Koppel, G. (1967). 2. ZeZZforschung, 77, 443-517. Krakow, W., Endres, G., Siegel, B. & Scheragtt, W. (1972). J. Mol. BioZ. 71, 95-103. Marguerie, G. (1977). Biochem. Biophys. Acta, 494, 172-182. Metsuta, M., Iwanaga, S. & Nakamura, S. (1972). Thrombosis Res. 1, 619-630. Pouit, L., Marcille, G., Suscillon, M. & Hollard, D. (1972). Thromb. Diath. Huemorrh. 27, 559-572. Slayter, H. (1976). Ultramicroscopy, 1, 341-357. Slayter, H. (1978). In Principles and Techniques of Electron Microscopy (Hayat, M., ed.), vol. 9, pp. 175245, Van Nostrand, New York. Stewart, G. (1971). Comments and Discussion in Stand. J. Haematol. 13, 63-67. Tranqui-Pouit, L., Marder, V., Suscillon, M., Budzynski, A. & Hudry-Clergeon, G. (1975). Biochim. Biophys. Acta, 400, 189-199. Valentine, R. C., Shapiro, B. & Stadtman, E. (1968). Biochemistry, 7, 2143-2152. Wrigley, N. G. (1968). J. Ultrastruct. Res. 24, 454-464. Wrigley, N., Laver, W. & Downie, J. (1977). J. Mol. BioZ. 109, 405-421.
from the length of the Note Added in Proof: The diameter of the nodules determined shadows is smaller than that measured from negatively stained or rotary shadowed molecules. The same discrepancy has been noted by Elliot & Offer (1978) with myosin and by W. Voter and H. P. Erickson (unpublished) with aldolase. It seems generally true that the height of small globular protein molecules is significantly underestimated by the shadow length determinations. It has been suggested by Marguerie (1977) that calcium may stabilize a more compact structure of fibrinogen. C&I, (2.5 mM) or EDTA (2.5 mM) have no effect on the trinodular structure as seen in the electron microscope by our techniques.