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Structural proteins of adenovirus
J. LVOl. Biol.
(1971)
57, 383-395
Structural Proteins of Adenovirus V.$ Size and Structure of the Adenovirus BICHA~
Type 2 Hexon
M. FFIRANKLIN$,ULF PETTERSSON,KRISTER &CERVALI+ BROR STRANDBERGAND LENN~RT PHKGIFSON
Depurtment of Microbiology and Biological Xtructure Group The Wallenberg Laboratory University of Uppsala Uppsala, Sweden and Department of Molecular Biophysics The Public Health Researclh Institute of the City of New York, Inc. New York, N.Y., U.S.A. (Receiaed
30 March 1970, and in revised form
26 October 19?0)
The molecular weight of the adenovirus type 2 hexon was determined from both sedimentation-diffusion and equilibrium sedimentation experiments. The partial specific volume was calculated from the ammo acid composition (P = @719),dfrom sedimentation in Ha0 and Da0 solvents (7 = O-727), and from sedimentation equilibrium experiments in Hz0 and D,O solvents (P - 0.736). ih the latter two cases the amount of exchangeable hydrogen was measured by tritium exchange experiments. The diffusion constant was calculated from the Stokes’ radius determined by quantitative gel filtration on 6% Agarose. Using these experimentally determined parameters, the molecular weight from sedi.nentation velocity was 313,000 to 333,000 and from sedimentation equilibrium tt was 333,000 t.0 356,000. The adenovirus type 2 hexon crystallized into a bipyramidal-shaped crystal and the unit cell was cubic with a = 149.9 A. The space group was P2,3 and there were four hexons per unit cell. The molecular weight of the hexon, from the crystallographic data, was 330,000 to 356,000. Since there are 12 crystallographic asymmetric units per unit cell in P2,3, there must be three crystallographic asymmetric units per hexon, each with a molecular weight of 110,000 to 119,000.
1. Introduction A number of structural proteins have been isolated either from cells infected with adenovirnses, or from degraded adenovirus particles. These proteins can be related to the morphological subunits seen in the intact virion (Wilcox C%Ginsberg, 1963; Valentine & Pereira, 1965; Kijhler, 1965; Pettersson, Philipson & Hiiglund, I Rage, Pettersson, Hoglund, Lonberg-Holm & Philipson, 1970). The major morphologkal subunit of the icosahedrally-shaped virion is the hexon which is looated on axes of local 6-fold symmetry having the same direction as the particle 3-fold axis f Paper IV in this series is Prage, Pettersson, HBglund, Lonberg-Helm. & Philipson, 1970. $ Dedicated to the memory of Nicole Granboulan, dear friend and respeoted colleague. 16 888
384
R. FRANKLIN
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(Valentine & Pereira, 1965 ; Ginsberg, Pereira, Valentine & Wilcox: 1966). There are 240 hexons per virion. Besides the hexon, the surface morphology is characterized by having 12 pentons at the corners of the icosahedron; the penton is divided into a penton base and a fiber (Ginsberg et al., 1966). The hexon is of great interest to students of viral structure since it is one of the few examples of a native protein complex isolated from a spherical virus. Although it is not firmly established that the hexon cont’ains one single type of polypeptide, it appears evident that this structure is composed of several polypeptide subunits (Pettersson et al., 1967; Maizel, White & Scharff, 1968a,b). Maize1 and his colleagues have suggest’ed that the hexon is made up of three subunits of molecular weight 120,000 (Maize1 et al.; 19&3a,b; Horwitz, Maize1 & Scharff, 1970). Part of the purpose of the present st,udy is to attempt t’o describe the number and arrangement of polypeptides in the hexon. Although the molecular weight of hexon has been determined by a number of workers, the values reported have not been in good agreement. From the size of the hexon determined by electron microscopy, Valentine & Pereira (1965) estimated the molecular weight to be 210,000. Wasmuth & Tytell(l967) also reported the hexon molecular weight to be about 200,000 from gel filtration studies. Sedimentation and diffiision constants have been determined by several workers and the calculated molecular weights were 310,000 (Kohler, 1965), 400,000 (Pettersson et al., 1967) and 380,000 (Shortridge & Biddle, 1970). Values from equilibrium sedimentation were 288,000 to 311,000 using the Archibald method (KGhler, 1965) and 309,000 (P. A. Charlwood, quoted by Macint,yre, Pereira & Russell, 1969). The molecular weight estimated from crystallographic parameters was 260,000 to 330,000 (Macintyre et al., 1969). Thus values in a range 0f200,000 to 400,900 have been reported. In the present work, the sedimentation and diffusion constants have been remeasured at very low concentration, the pa,rtial specific volume has been recalculated, using the latest, available data on the amino acid composition, the sediment,ation coefficients in H,O and D,O (Martin, Cook & Winkler, 1956) and the sedimentation equilibrium distribution in Ha0 and I&O (Edelstein & Schachman, 1967) coupled with tritium-exchange experiments (Englander, 1963). From these data a molecular weight of 313,000 to 354,000 has been calculated. Furthermore, the hexon has been crystallized in a form different from that reported by Macintyre et al. (1969) and the cell parameters and space group, as well as the crystal density, have been determined. From these crystallographic data a molecular weight of 330,000 to 356,000 has been calculated. These vaIues are compared with earlier published values.
2. Materials
and Methods
(a) Pur$cation
of hexon antigen
Hexon purification was carried out according to the procedure of Pettersson et ul. The preparative polyacrylamide gel electro(1967) with one important modifioation. by a similar step using continuous phoresis according to Maize1 (1966) was replaced elution (Hjerten, Jerstedt & Tiselius, 1965). This modification has the advantage that the final rate zonal centrifugation may be omitted in the purification procedure. The preparative polyacrylamide gel electrophoresis was carried out as follows: acrylamide and N,N’-bismethylene acrylamide were recrystallized according to the method of Loening (1967). The monomer solution contained 5% acrylamide and 0.05% N,N’bismethylene acrylamide in 0.05 M-Tris-glycine buffer, pH 9~5. Polymerization was induced by 0~2% fresh ammonium peroxide sulfate and 0.1 oh N,N,N’,N’-tetramethylethylenediamine.
SIZE
AND
STRUCTURE
OF THE
HEXOX
386
The preparative electrophoresis was carried out in a continuous elution apparatus (Hjerten et al., 1966) using an 18 cm x 1 cm column with an elution chamber at the lower end (AB Stalprodukter, Uppsala, Sweden) filled with Pevikon powder (Fosfatbolaget, Stockholm, Sweden). The gels were submitted to an overnight prerun before the start of each separat;ion. The applied samples of 0.5 to 1.0 ml. were in 0.01 M-Tsis-glgcine (pII 9.5), containing 5% sucrose. The running buffer was 0.05 M-Tris-glycine, pII 9.5. The star-ting potential of 10 v/cm was raised to 30 v/cm after 1 hr. The elution rate was maintained at LO ml,/hr and t,he hexon elutcd after 12 hr running time. Kexon prepared by this method analytical polyacrylajmide gel was homogeneous according to the following criteria: olectrophoresis at pH 8.0 and at pH 9.5, immunoelectrophoresis, ultracentrifugation (see Results), N-terminal amino acid analysis (Pettersson, 1970 and manuscript in preparation), and electron microscopy. (b) Analytical
ultracentr$ugation
Hexons were dialyzed for at least 48 hr against 0.1 af-NaCl buffered with 0.91 ?a-Tris, pH $9. The buffer was prepared with II,0 or D,O. In the case of the D,O buffer, a volume of 0.60 to 0.70 ml. hexon in 1 x 10T2 M-ammonium acetate buffer, pH 6.5, was dialyzed against the NaCl-Tris buffer. The D,O was 99.8% pure and purchased from Sorsk Wydro, Norway. Centrifugation was carried out at 42,040 rev./mm in a Spinco model E centrifuge lrsing an An-D rotor. Epon double-sector centerpieces were used and the sedimenting boundary was measured at X = 280 nm using the automatic scanner. Equilibrium sedimentation was performed at low speed (approx. 4000 rev./mm) using the 4-place An-F rotor in a Spinco model E ultracentrifuge equipped with electronic sped control. For each run the speed was accurately measured by means of the revolution counter. Temperature regulation (20%) was used. The distributions were recorded on the photoelectric scanner at /\ = 280 nm and with outer dialysate in the solvent sector. Data seduction and molecular weight determinations were carried out on a General Electric mark XI computer. Details of the buffers are given in Table 4. In this series, D,O (99.8 mole %), was obtained from Bio-Rad Laboratories, Richmond, Calif. (c) Pycnometry
and viscometry
Solution densities were measured at room temperature in a pycnometer calibrated with freshly distilled water. Standard temperature corrections were applied. The relative viscosities of the H,O and D,O buffers were measured at 20°C in a Cannon-Ubbelhode capillary viscometer. (d) Analytical
gel chromatography
radius was determined by gel chromatography on 6% Agarose (Pharmacia Fine Chemicals, Uppsala, Sweden) according to the method of Laurent & Killander (1964). *P column, 1.5 cm x 90 cm, was equilibrated with O-01 M-Tris-HCl containing 0.2 ~-XaaCl. The void volume (V,) was determined from the peak of Blue Dextran 2000 (Pharmacia Fine Chemicals, Uppsala, Sweden) and the total volume (V,) from the peak of ?K20. The elution volume (V,) for the hexon was measured by use of 3H-labeled hexoo and the elution volume of human IgG (kindly supplied by Dr Hans Bennich, Uppsala, Sweden) was determined spectrophotometrically. The volume of each fraction was determined aecurately by weighing. The weights were not converted back to volumes since a ratio of volumes is used to calculate KAv (see under Results). Stokes'
(e) Hydrogen
exchange
The exchange of deuterium into hexon was estimated by measuring exchange wit,11 tritiated water according to Englander (1963). A 5.5 cm x 2 cm (diameter) column of coarse Sephadex G25 (Pharmacia Ltd) was equilibrated at 4°C with 0.1 ~r-N&l, 0.91~~ Tris-HCI, pH 8.0. Samples equilibrated for 48 hr at 4°C containing approximately 1 me “J&O and 0.8 to 1 mg hexon in 1 ml. of the same buffer were applied on top ofthe column and t,he void volume was collected in less than 3 min at 4°C. Fractions containing a constant ratio between radioactivity and optical density at 280 nm were separately used to determine moles of hydrogen per mole of hexon using the formula of Englander (1963) and
R. FRANKLIN
386
E!l’
AL.
a molecular weight of hexon of 360,000. A total number of 5852 exchangeable hydrogena per hexon was estimated from the amino acid composition (Pettersson, 1970). (f) Collection of X-ray dutw Data were collected photographically using Buerger-Supper precession cameras and G-filtered CuKar radiation from a Philips fine-focus X-ray tube run at 45 kv. A 0.6 mm collimator was used for the determination of cell dimensions and the space group, The crystals were mounted in sealed capillaries with mother liquor present in one end of the capillary. Details of crystallization are reported in the Results.
(g) Determination of cry&xl density Individual crystals in their mother liquor were placed on the side of small (4.2 cm x 0.50 cm) nitrocellulose tubes containing mixtures of bromobenzene and xylol of known density (Low & Richards, 1952&,1954). A fine glass fiber was used to remove a crystal from the surrounding mother liquor and to place it in the density solution. The bromobenzene and xylol had been equilibrated with the buffer used for crystallization (0.5 M-citrate, pH 3.2) but to prepare the density fluids, the density was assumed to be that reported for the individual reagents. This is a good approximation since the reagents are practically immiscible with aqueous reagents. To check this, however, the density of the mixtures of bromobenzene and xylol above and below the crystal density were deter-
mined directly by pycnometry.
3. Results (a) Hydrodynamic parameters and the determination of (i) Sedimentation in H,O and D,O buffers and the determination of 0 The first parameter to be calculated is P according to the formula
where Q and r/a are the viscosities of the aqueous and heavy water solutions, S, and S, the sedimentation coefficients in the aqueous and heavy water solutions, Pl and pz the densities of the aqueous and heavy water solutions, and K the ratio of the molecular weights in the two media; the increase in molecular weight in the Da0 buffer being due to deuterium exchange (Martin et al., 1956). In order to oalculate K it is necessary to know what percentage of the protons can exchange. In amino acids and small polypeptides all protons not oovalently bonded to carbon are exchangeable (Morowitz Lk Chapman, 19%) but this may not be true for proteins (Hvidt & Nielsen, 1966). Therefore we measured the amount of H exchange directly, using the tritium-exchange method of Englander (1963). Three separate experiments were carried out and in each experiment the two fractions in the middle of the most constant part of the curve of exchangeable 3H per molecule were used in the final average (Table 1). For reasons which will become a,pparent (see Discussion), a hexon molecular weight of 360,000 was assumed. The average percentage of exohangeable hydrogen was 18.2. With a maximum of 5852 exchangeable hydrogens per unit of 360,000 daltons, calculated from the latest amino-acid analysis of the adenovirus type 2 hexon (Pettersson, 1970), we find that there are 1065 exchangeable hydrogens and that K, the ratio of the molecular weights in D,O and H,O, is 1.00298. The uncorrected sedimentation coefficients for four runs in Ha0 buffer at 20.0% were 1255, 12.71, 12-85 and 12-32 and for three runs in Da0 buffer at 20°C were
SIZE
AND
STRUCTURE
OF THE
387
HEXON
‘T-36, T*67 and 7.64. In all cases the sedimenting boundary appeared to be homogeneous. The average values of X, = 12.61 and 8, = 7.56 were used to calculate P. Then using Q~, for the aqueous solution of 1.014 and qrel for the heavy water solution of 1.132 and the values of 0*010019 and 0.01099 poise for the respective absolute values of the viscosities of light and heavy water at 2O*O”C, we can calculate r to be o-727 g/cm3. TABLE 1
Tritium Experiment
1 2
no.
exchange of adenovirus type 2 hexon Exchanged H/molecule
Fraction
12
768.5
13
1008
11 12
1272 1208 1056
13
1094
12
3
Percentage exchange
13.1 17.2 21.7 20.6
18.0 18.7
This so-called hydrodynamic partial specific volume has been shown to be equivalent to the conventional partial specific volume when there are no selective solvation effects (Gagen & Holme, 1964; Gagen, 1966) and this should be the case with the solvent system used here. If we calculate P from the amino acid composition and the partial specific volumes of the individual amino acids (Schachman, 1957), the value is 0.719 g/cm3, which is in fairly good agreement with that calculated from t#he hydrodynamic data. It should be noted that the value of 7 of ribonuclease calculated from the amino-acid composition (Schachman, 1957) was in excellent agreement with that determined by pycnometry (Rothen, 1941). A third determination of v was made from sedimentation equilibrium distributions of hexons sedimented at 4000 rev./min in 0.1 M-sodium borate buffer, pH 8.0, in either H,O or D,O. The K determined by tritium-exchange experiments (see above) was also used in the formula of Edelstein & Schachman (1967). Two separate experiments gave V values of 0.734 and O-737 g/cm3, an average of 0.736 g/cm3. (ii) Determination of the d@usion constant of the hexon The experimental data obtained from four filtration experiments are shown in Table 2, and a typical experiment is shown in Figure 1. The partition coefficient.s (K,,) of IgG and hexon were calculated from such data according to the relationship K
_ ye AV
VI3
vt
-
(Laurent & Killander,
1964).
vcl
The Stokes’ radius (rJ of the hexon was calculated from the equation:
KAv = exp [-r
L (r, + r,)‘]
where I; is the concentration of gel rods in the solution measured as cm rod per cm3 and T, is the radius of the gel rods (Ogston, 1958). Although L may vary considerably
388
R. FRANKLIN
ET
AL.
with the concentration of Agarose used and also with the batch of Agarose of any single given concentration, this is not the case for rr and we may assume rr to be 25.0 A for 6% Agarose (Laurent, 1967 and personal communication). Then, using rs = 50 A for IgG (Pettersson & Berggard, personal communication) and rr = 25.0 8, we may calculate L in each experiment using the experimental value of K,v for IgG (Table 2). TABLE 2 Determination of the Stokes’ radius of the hexon Experiment IlO.
Hexon batch no.
1 2 3 4
& & & b
L
KdgG
0.552 0.555 0.552 0.557
3.36 3.33 3.67 3.31
x x x x
K*vhexon
1O’l 1011 lo= 1011
rs (4
0.464 0.469 0.461 0,470
60.25 60.05 60.61 60.18
I -4 I ’ ’ ’ 1 ’ ’ ‘----I Cts/min
‘4
(0)
0.7 700
0
i”i
A
IQ
20
30
40
50
60
70
80
7000 I
90
FIG. 1. Quantitative gel filtration experiment to determine the Stokes’ radius of the hexon. Experimental details are given in Materials and Methods and in Results. -x - x -, O.D.830 of the Blue De&ran 2000; -O-O-, “H radioactivity (cts/min) of the hexon; -a-@-, 0.0.~~~ of the IgG; -A-A-, 3H radioactivity (ots/min) of the 3H,0.
Now it is possible to use the KAv of the hexon to calculate the hexon Stokes’ radius (Table 2) and the average value for the four experiments was 60.27 A or 6.027 x 10W7 cm. From this value of Stokes’ radius we then calculate DzO,W= 3.56 x 10e7 om2/sec. (iii) The molecular weight of the hexon from hydrodynamic parameters and from sedimentation equilibrium With the range of v reported above we calculate X20,W(Svedberg $ Pedersen, 1940) from the average value of SaO.sOIV., then we can use DSOeW to calculate a molecular weight of 313,000 to 333,000 and finally we can use ~.!Yaa.~ and D2OSW to calculate according to the formula flfmin -f
= 10-e
1D2
7 Pz0.w lb3 x
t 20.W 20.W PJ (Svedberg & Pederson, 1940). These parameters are summarized in Table 3.
f mill
SIZE
AND
STRUCTURE TABLE
OF THE
380
HEXON
3
Hydrodynamic parameters of the hexoN
5.719 0.727 0.736
313,505 322,050 333,005
12.9 x lo- x3 12.9 x 15-la 12.9 x 15-13
1.34 1.31 1.30
The sedimentation equilibrium data are summarized in Table 4, giving the b&es used in the individual experiments and the calculated molecular weights using 7 = 0.727. From this set of data we calculated an average molecular weight of 342,600. Using 7 = O-719 or 0,736, we calculated average molecular weights of 33,900 or 354,000, respectively. TABLE
Sedimentation
Buffer 0.01 0.01 0.01 0.01 0.01 0.01 0.01
4
equilibrium molecular weight of adenovirus type 2 hexon
M-sodium M-sodium M-sodium M-sodium M-sodium M-sodium M-sodium
PH 9.2 9.2 8.8 8.0 8-O 8.0 7.6
borate borate borate borate borate borate phosphate 7 Calculated
Molecular
with
weight?
356,000 383,550 340,050 322,005 330,505 339,000 329,000
7 = 5.727.
(b) Crystallography of the hexon (i)
~0~d~t~0n~
jar crystallization
Cryst,als of tetrahedral shape were obtained by Pereira, Valentine & Russell (1968) from adenovirus type 5 hexon in 0.8 M-potassium dihydrogen phosphate at pH 4.4. Although it was possible to repeat this crystallization with adenovirns type 2 hexon, the crystals were only about O-1 mm on a side. Because of the small size, it was decided to explore a number of conditions for crystallization and soon it became evident that there were two distinct crystal forms, depending on the pH and ionic strength of the buffer. For example, in sodium acetate buffer at pH 5.0, tetrabedral crystals formed at 0.25 to 0.55 M; the second, bipyramidal crystal form was found at 1-O M; and transitional forms were found at 0.50 to 0.90 M. &though the bipyramidal-shaped crystals were very small in acetate buffer, it was possible to obtain excellent large crystals of this type in citrate buffer. The two different types of crystals formed within the narrow pH range of 3.2 to 4.1 (Table 5 and Plate I (a) to (e)). The bipyramidal-shaped crystals grew to O-45 to 0.60 mm in length, measured along the bipyramidal face, and all of the work reported here was done WitA such cryat&.
390
R. FRANKLIN
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AL.
5
Conditions for crystallization of adenovirus type 2 hexon using 0.5 M-sodium citrate buffer PH 2.9 or lower 3.2 3.4 3.5 3.7 to 4.1 4.4 or higher
Results No crystals Bipyramidal-shaped Transitional form Transitional form Tetrahedral No crystals
The crystal density was determined in bromobenzene-xylol mixtures. Crystals migrated upward in solutions of p = I.22 g/cm3 and downward, but at a slow rate, in solutions of p = 1.21 g/cm”. The nominal solution densities were found to be correct when the densities of the two mixtures (1.22 and 1.21) were checked by pycnometry. Therefore, we conclude that the crystal density lies between I.210 and l-215 g/cm3. If a is the volume fraction of the unit cell occupied by solvent, either free or bound, then the net crystal density is pC= up, + (1 - a) pP where pp is the protein density = I/P (cf. Matthews, 1968). Using pc = l-210 to 1.215 and p = 0.719 to 0.736 we calculate a to be 0.48 to 0.53. Matthews (1968) has shown that a lies between 0.27 and 0.65 for most proteins and there are a large number of proteins with a N 0.43. In general, a is higher than average for proteins of higher molecular weight (Matthews, 196$), but in our particular case the packing of the hexon in our unit cell seems to be similar to the packing of a number of the simpler proteins, being only slightly higher than average. (ii) Determination
of crystallographic parameters
Crystals up to 0.5 to 0.6 mm in length measured on the long axis of the bipyramid (Plate I(b)) were found to be optically inactive when viewed with plane polarized light using a Zeiss POL stereomicroscope. This indicated that the orystal might belong to the cubic system. A series of Laue photographs was taken to determine the shortest repetition distance in the real unit cell and then precession photographs were taken with the X-ray beam parallel to this direction. The [loo] and [OlO] photographs clearly showed that the unit cell was cubic. The cell length, 149.9 d, was determined using human carbonic anhydrase form C for calibration of the precession camera (Liljas et al., manuscript in preparation). Also, hexon crystal WM aligned with one axis parallel to the spindle axis of the camera; the other two axes were shown to be 90” apart by taking precession photographs at the two appropriate settings. Furthermore, the [llO] direction was found at 45” to these two axes. From the Laue photographs of the [IlO] direction, the face diagonal was measured to be 214& and from a [llO] precession photograph, the [110] spacing of 106.kwas measured. The unit cell dimensions are summarized in Table 6. Since 4-fold symmetery was absent in [loo] precession photographs (Plate II), the point group could be established as 23. The space group was identified by the systematic absence of reflections, and the only ones wbioh were found wera
!"L.ATE 1. (a) and (b) Hexon cqst~ah, bipyramldal form. U~ritht~o~l~ of cryxhahst~w?. iI d 1:. ~d1t11y1citrate buffer, pH 3.2. (c) Heson cryst&, tranaitlonal form. Conditions of cryst.allization, O-.5w-sodium citrate bi!‘S’?r? $3. 3.4. (cl! Hcxor~ crystjals, transitional form. Conditions of crystalhzahon, 0.5 wwdium cit~act~huffw, pff 3.5. (e) Wexon crystal+ tetrahedral form. L’onditions of cryst~allizarion, 0.5 xl-sodium citwie buffer’, p!T 3.7.
[jnrzil!!p. WJ
PLATE II. Zero-layer precession photograph of adenovirus type 2 hexon cryhtal taken with the X-ray beam parallel to a cube edge [lOO]. The precession angle wits 6” and the exposure time was I6 hr with a crystal-to-film distance of 75 mm and a 0.3 mm collimator. The edge of the photograph corresponds to spacings of about 8 .!L
[Toil
PLATE Ill. Zero-layer precession photograph of adenovirus typo 2 hexon crystal taken u.lth the -X-ray beam parallel to a cube face diagonal [loll. The precession angle was 5” and the exposure t)ime was 15 hr with a crystal-to-film distance of 60 mm and a 0.4 mm collimator. The edge of the 1:hotograph corresponds to spacings of about, 9 .&.
piO]
[OTT]
PLATE IV. Zero-layer precession photograph X-ray beam parallel to a cube body diagonal time was 15 hr with a crystal-to-film distance
r
taken with the of adenowrus type 2 hexon crystal [I 1 I]. The precession angle was 5” and the exposure of 60 mm and a 0.4 mm collimator.
SIZE
AND
STRUCTURE
OF THE
HEXON
391
TABLE 6 Unit cell dimensions Direction
s1001 [110] Cl111
Spacing from still photograph
Spacing from precession photograph
-
149.9A 106 B -
214 11 256 A
h = 2n + 1 for MO, k = 2n + 1 for Ok0 and 1 = 2% + 1 for 001, characteristic of a 2fold screw axis (Plate II). This can be seen especially well in the [llO] precession photographs (Plate III). In the cubic system with 23 point symmetry, the only possible space group with a 2fold screw axis is P2,3. I2,3 is ruled out since the general condition for extinction, iz + k + 1 = 2n + 1, was not found. The absence of body centering was confirmed by comparing zero and first-level precession photographs with the X-ray beam parallel to the flOO], [IlO] and [ill] directions. We may now apply the formula niM = (PC- PA NV (1 -
VP,)
to determine n, the number of proteins per unit cell; where M is the moIecular weight of the hexon = 313,000 to 354,000 from the sedimentation-diffusion and sedimentation equilibrium data, p. is the density of the crystal = l-210 to l-215 g/~rn.~, ps is the density of the solvent = l-052, N is Avogadro’s number, V is the volume of the unit cell = 3.368 x lo-l8 cm3 and v is the partial specific volume of the protein = 0.719 to O-736. Using these parameters we find n to lie between 3.78 and 455, and because there are 12 asymmetric units per unit cell in space group P2,3, we can oonclude that the only possible value of n is four. Since there must be 12 asymmetric units in the unit cell in the P2,3 space group and there are four hexons per unit cell, we conclude that there are three crystallographic asymmetric units in. the hexon crystallized in the P&3 space group. The special conditions for the P2,3 spaoe group will apply in this case and thus the co-ordinates of the four hexons are x,x,x; 112 + z: 112 - x, 5; 2, l/2 + x, l/2 - x; l/2 - x, 5, l/2 + x (International Tables jar X-ray Crystallography, 1969). These co-ordinates locate the centers of the four hexons on the body diagonals of four of the eight small cubes of side 75 d, obtained upon dividing the cubic unit cell into eights. The four body diagonals upon which the hexons lie are related by non-intersecting S-fold screw axes. We may now apply the above equation in a second way. Using n = 4, we may determine a molecular weight of the hexon and using the values of P = 0.719, 0~7.27 and 0.736, the calculated molecular weight will be 330,000, 342,000 and 356,006, respectively. A number of features of the reciprocal lattice are illustrated in the precession photographs. Features of the [loo] precession photograph (Plate II) have already been mentioned. The 2-fold symmetry of the [IlO] direction is illustrated in Plate III along wit,h “spikes” in the [ill] direction, the direction of the crystallographic 3-fold
392
R.
FRANKLIN
ET AL.
axis. The 3-fold symmetry of the [ill] precession photograph is illustrated in Plate IV, where actually 6-fold symmetery is evident in this zero layer due to the operation of Friedel’s law. Upper layers along [ill] show only S-fold symmetry. 4. Discussion As in the case with many proteins and other macromolecules available in very limited quantities, accurate determination of the partial specific volume of the hexon has been difficult since it has not been possible to obtain enough protein to make precise pycnometrio measurements. In using indirect methods we have obtained three different values for P (0719, 0727 and 0,736) and we prefer, therefore, to report the range of molecular weights for the hexon using the range of experimentally determined v. The hydrodynamic P was calculated using the average values of S, and S,, the only reasonable procedure since insufficient sedimentation data were taken to do a meaningful statistical analysis. The t&mm-exchange experiment, needed to obtain an accurate value for K, the increase in molecular weight due to exchanged deuterons, revealed that only 18.2% of the theoretically exchangeable protons did exchange. This suggests that there may be large solvent-inaccessible regions in the hexon, a suggestion supported by the presence of hydrophobic peptides in the tryptic digest of hexons (Pettersson, 19’70, and manuscript in preparation). The molecular weight range obtained by sedimentation equilibrium (333,000 to 354,000) agreed well with the range determined from the crystallographic parameters (333,000 to 356,000) but these values are slightly higher than those obtained from the hydrodynamic data (313,000 to 333,000). The estimate of the d.iffusion constant of the hexon may not be as reliable as our other parameters since the constants of the Agarose, particular r,, the radius of the gel rods, are not known precisely. The chemical studies of Pettersson (1970, and manuscript in preparation) indicate that there is a polypeptide repeat unit of molecular weight 60,000 daltons in the hexon, and the studies of Maize1 et al. (1968a,b) indicate that there may be a larger polypeptide subunit of molecular weight 120,000 daltons. Whereas Maize1 and his colleagues have suggested that the hexon is composed of three identical polypeptide chains of molecular weight 120,000 each (Maize1 et al., 1968a,b; Horwitz et al., 1970), Pettersson (1970) suggests that the hexon contains six identical polypeptide chains of molecular weight 60,000 each, Whatever the number of chains, our sedimentation equilibrium data and crystallographic data, coupled with the chemical data just mentioned, all suggest that the hexon must have a molecular weight of about 360,000 daltons. We may now comment on the earlier estimates of hexon molecular weight. It is generally accepted that molecular weight determinations from electron micrographs may be inaccurate due to shrinkage of structures after negative staining; this may explain the low value of 210,000 reported for the type 5 adenovirus hexon by Valentine $ Pereira (1965). KGhler (1965) measured the sedimentation and diffusion coefficients for hexons from several adenovirus types by analytical ultracentrifugatioa and arrived at values of 280,000 to 310,000 for the hexon molecular weight. The discrepancy between his values and our present estimates mainly depends on his low value for the sedimentation coehloient. Wasmuth & Tytell (1967) gave a range of 158,000 to 256,000 for hexons from six adenovirus types. Their estimates were made by direct comparison of the elution positions for hexons with those of other proteins of
SIZE
AND
STRUCTURE
OF THE
HEXON
3x3
known molecular weight. Such determinations may, however, be subject to considerable error since the elution position of a molecule is related to its Stokes’ radius rather than to its molecular weight (Siegel & Monty, 1966). Boulanger, Flamencourt & Biserte (1969) reported a molecular weight of 300,000 for type 2 and 5 hexons. This value was based on a sedimentation coefficient of 11.2 S, determined by analytica, nltracentrifugation, the diffusion coefficient determined by Kiihler (1965) (3.723 x 19-T cm2/sec) and our previous value of 0,720 cm3/g for 7, determined from the amino acid composition of the hexon (Pettersson et al., 1967). Recently, Shortridge & Biddle (1970) reported a value of 380,000 for hexons from adenovirus type 5. Although. their value of molecular weight is in reasonable agreement with our present determinations, their value for Stokes’ radius (64 8) is slightly higher than ours (SOA) ; this, however, was compensated for by their low estimate of 7. The different values obtained for Stokes’ radius may depend on the use of different gels for the analytical gel chromatography. Since hexon elutes rather close to the void volume on Scphadex G200, estimates made on 6% Agarose should be more accurate because the hexon elutes in the area of maximum separation capacity of the gel in this case. ur previously reported vahe of 400,000 daltons for type 2 hexons is higher than that obtained in the present study. This depends mainly on a low value for the diffusion coefficient (2.6 x 10m7 cm2/sec). The X-value was also slightly below the actual S,,,, ettersson et al., 1967). The molecular weight range given by Maeintyre et al. (1969) cannot be discussed since even his crystallographic unit cell dimensions are probably incorrect (see below). Finally, the value of 88,000 + 8000 for the crystallographic asymmetric unit, calculated by Corniek, Sigler & Ginsberg (1971) gives a hexon molecular weight of 240,000 to 290,000, below most reported values. Since the unit cell was identical to ours (see below), this difference appears to be due to the difference in measured crystal densities, and since similar techniques were used in both laboratories it is n.ot possible at this time to explain this discrepancy. Preliminary results from low-angle X-ray scattering indicate that the hexon is a cylinder of diameter 76 A and height 119 A (I. Lindqvist, personal communication). Electron microscopy of the hexon has indicated that the particles are 80 to 110 A in diameter (Pettersson et al., 1967) and we may interpret such electron micrographs in terms of the two views of the cylinder. Then we may approximate the hexon to a prolate ellipsoid with an axial ratio p = b/a of 1.57. The frictional ratio of such an unhydrate ellipsoid would be approximately 1.019 (Svedberg & Pedersen, 1940). e may now use our experimental frictional ratio (flfmin) to estimate the solvation from the equation
f -=-
f
f min fo (
-PP* + 6 l/3
PP, )
where f/f,, is the frictional ratio due to shape alone (Tanford, 1961). Using the range of P obtained for the hexon and f/fminof 1.30 to 1.34, we may estimate 6 to be about -80, an unusually high degree of solvation, when compared to many non-viral proteins (Tanford, 1961). Hydration estimates of this type have usually led to very high values for viruses (Lauffer & Bendet, 1954), but usually one did not distinguish between bound water and solvent spaces, as pointed out in the elegant discussion of this problem by Harrison (1969). Indeed, when the bound water is correctly calculated for tomato bushy stunt virus (0.35 to 0.43 g/g), it is very similar to that for
394
R. FRANKLIN
ET AL.
hemoglobin (Harrison, 1969). Thus future extended hydrodynamic studies on the hexon are necessary before we can obtain a true estimate of bound water. Although the space group of the tetrahedral crystals of type 5 adenovirus hexon was claimed to be I23 or I2,3 by Macintyre et al. (1969), the careful study of Cornick et al. (1971) has shown that these crystals are isomorphous with our type 2 hexon bipyramidal crystals. Thus at present we can only state that the hexon has three crystallographic asymmetric units. Three-dimensional data to 10 A resolution have been collect,ed and hopefully we will be able to detect local symmetry by analysis of this data. A large number of colleagues made important contributions to this work in the form of criticisms, as well as suggestions for carrying out the det,ermination of some parameters such as P, D, and pC. The authors are greatly indebted to these individuals who were: Professor T. C. Laurent and Dr P. A. Peterson, Institute of Medical Chemistry, University of Uppsala; Drs D. L. D. Caspar and S. Harrison, The Children’s Cancer Research Institute, Boston; Drs K. K. Kannan and A. Liljas, Biological Structure Group, The Wallenberg Laboratory, University of Uppsala; and Dr C. I. Brand&r, Institute of Chemistry, I, Lantbrukshogskolan, Ultuna. Mrs Eva Hjertson provided excellent technical assistance. One of us (R. M. F.) was on leave of absence from the Public Health Research Institute of the City of New York, New York, N.Y. during the course of this research. This work was supported by grants from the Damon Runyon Memorial Fund, the Swedish Cancer Society, the Swedish Delegation for Applied Medical Defense Research, the Swedish Medical Research Council, the Swedish Natural Science Research Council, and U. S. Public Health Service (AI GM 07382).
REFEREKCES
Boulanger, P. A., Flamencourt, P. & Biserte, 6. (1969). Europ. J. Biochem. 10, 116. Cornick, G., Sigler, P. B. & Ginsberg, H. S. (1971). J. Mol. Biol. 57, 397. Edelstein, S. J. & Schachman, H. K. (1967). J. Biol. Chem. 242, 306. Englander, S. W. (1963). Biochemistry, 2, 798. Gagen, W. L. (1966). Biochemistry, 5, 2553. Gagen, W. L. & Holme, J. (1964). J. Phys. Chem. 68, 723. Ginsberg, H. S., Pereira, H. G., Valentine, R. C. & Wilcox, W. 6. (1966). Virology, 28, 782. Harrison, S. C. (1969). J. Mol. Biol. 42, 457. Hjerten, S., Jerstedt, S. & Tiselius, A. (1965). Anulyt. Biochem. 11, 211. Horwitz, M. S., Maizel, J. V., Jr. & Scharff, M. D. (1970). J. Viral. 6, 569. Hvidt, A. & Nielson, S. 0. (1966). AcZuanc. Protein Chem. 21, 287. International Tables for X-ray Crystatlography, (1969). vol. 1, ed. by N. F. M. Henry & K. Lonsdale. Kohler, K. (1965). 2. Naturf. 20b, 747. Lauffer, M. A. & Bendet, I. J. (1954). Adoanc. Virus Res. 2, 241. Laurent, T. C. (1967). Biochim. biophyls. Acta, 136, 199. Laurent, T. C. & Killander, 5. (1964). J. Chromatog. 14, 317. Loening, U. E. (1967). Biochem. J. 102, 251. Low, B. W. & Riohards, F. M. (1952a). Nature, 170, 412. Low, B. W. & Richards, F. M. (19523). J. Amer. C%em. Sot. 74,166O. Low, B. W. & Richards, F. M. (1954). J. Amer. Chem. Sot. 76, 2511. Macintyre, W. M., Pereira, H. G. & Russell, W. C. (1969). Nature, 222, 1166. Maizel, J. V., Jr. (1966). Science, 151, 988. Maizel, J. V., Jr., White, D. 0. & Scharff, M. D. (1968a). Virology, 36, 115. Maizel, 5. V., Jr., White, D. 0. & Scharff, M. D. (1968b). Viirology, 36, 126. Martin, W. G., Cook, W. H. & Winkler, C. A. (1956). Canad. J. Chem. 34, 809. Matthews, B. W. (1968). J. Mol. BioZ. 33, 491. Morowitz, H. J. & Chapman, M. W. (1955). Arch. Biochem. Biophys. 56, 110.
SIZE
AND
STRUCTURE
OF THE
HEXON
396
Ogston, A. G. (19tiS). Trans. Faraday Sot. 54, 1754. Pereiaa, II. 6.: Valentine, R. C. & Russell, W. C. (1968). Xatz~re, 219, 946. Pettersson, U. (1970). Ph.D. Thesis, University of Uppsala. Pettersson, U., Philipson, L. & Hoglund, S. (1967). Virology, 33, 575. &age, L., Pettersson, U., Hoglund, S., Lonberg-Helm, K. & Philipson, L. (1970). T/irolocrg, 42, 341. Rothen, A. (1941). J. Gen. Physiol. 24, 203. Schachman, H. K. (1957). In Methods in Enzynzology, ed. by S. P. Colowick Cy:N. 0. Kaplan, vol. 4, p, 32. New York: Academic Press. Shortridge, K. F. & Biddle, F. (1970). Arch. ges. Virmfowch. 29, 1. Siegel, L. M. & Monty, K. J. (1966). Biochim. biophya. Actn, 112, 346. Svedberg, T. & Pedersen, K. 0. (1940). The Ultracentrifuge. Oxford: The Clarendon Press. Tanford, C. (1961). The Physical Chemistry of ikfacromolecules. New York, London: J. Wiley S: Sons. Valentine, R. C. & Pereira, H. G. (1965). J. MoZ. Biol. 13, 13. Wasmuth, E. H. S; Tytell, A. A. (1967). Life Sciences, 6, 1063. W7ilcox, W. C. & Ginsberg, H. S. (1963). J. Eqx Med. 118, 295.
(1971)
57, 383-395
Structural Proteins of Adenovirus V.$ Size and Structure of the Adenovirus BICHA~
Type 2 Hexon
M. FFIRANKLIN$,ULF PETTERSSON,KRISTER &CERVALI+ BROR STRANDBERGAND LENN~RT PHKGIFSON
Depurtment of Microbiology and Biological Xtructure Group The Wallenberg Laboratory University of Uppsala Uppsala, Sweden and Department of Molecular Biophysics The Public Health Researclh Institute of the City of New York, Inc. New York, N.Y., U.S.A. (Receiaed
30 March 1970, and in revised form
26 October 19?0)
The molecular weight of the adenovirus type 2 hexon was determined from both sedimentation-diffusion and equilibrium sedimentation experiments. The partial specific volume was calculated from the ammo acid composition (P = @719),dfrom sedimentation in Ha0 and Da0 solvents (7 = O-727), and from sedimentation equilibrium experiments in Hz0 and D,O solvents (P - 0.736). ih the latter two cases the amount of exchangeable hydrogen was measured by tritium exchange experiments. The diffusion constant was calculated from the Stokes’ radius determined by quantitative gel filtration on 6% Agarose. Using these experimentally determined parameters, the molecular weight from sedi.nentation velocity was 313,000 to 333,000 and from sedimentation equilibrium tt was 333,000 t.0 356,000. The adenovirus type 2 hexon crystallized into a bipyramidal-shaped crystal and the unit cell was cubic with a = 149.9 A. The space group was P2,3 and there were four hexons per unit cell. The molecular weight of the hexon, from the crystallographic data, was 330,000 to 356,000. Since there are 12 crystallographic asymmetric units per unit cell in P2,3, there must be three crystallographic asymmetric units per hexon, each with a molecular weight of 110,000 to 119,000.
1. Introduction A number of structural proteins have been isolated either from cells infected with adenovirnses, or from degraded adenovirus particles. These proteins can be related to the morphological subunits seen in the intact virion (Wilcox C%Ginsberg, 1963; Valentine & Pereira, 1965; Kijhler, 1965; Pettersson, Philipson & Hiiglund, I Rage, Pettersson, Hoglund, Lonberg-Holm & Philipson, 1970). The major morphologkal subunit of the icosahedrally-shaped virion is the hexon which is looated on axes of local 6-fold symmetry having the same direction as the particle 3-fold axis f Paper IV in this series is Prage, Pettersson, HBglund, Lonberg-Helm. & Philipson, 1970. $ Dedicated to the memory of Nicole Granboulan, dear friend and respeoted colleague. 16 888
384
R. FRANKLIN
ET
AL.
(Valentine & Pereira, 1965 ; Ginsberg, Pereira, Valentine & Wilcox: 1966). There are 240 hexons per virion. Besides the hexon, the surface morphology is characterized by having 12 pentons at the corners of the icosahedron; the penton is divided into a penton base and a fiber (Ginsberg et al., 1966). The hexon is of great interest to students of viral structure since it is one of the few examples of a native protein complex isolated from a spherical virus. Although it is not firmly established that the hexon cont’ains one single type of polypeptide, it appears evident that this structure is composed of several polypeptide subunits (Pettersson et al., 1967; Maizel, White & Scharff, 1968a,b). Maize1 and his colleagues have suggest’ed that the hexon is made up of three subunits of molecular weight 120,000 (Maize1 et al.; 19&3a,b; Horwitz, Maize1 & Scharff, 1970). Part of the purpose of the present st,udy is to attempt t’o describe the number and arrangement of polypeptides in the hexon. Although the molecular weight of hexon has been determined by a number of workers, the values reported have not been in good agreement. From the size of the hexon determined by electron microscopy, Valentine & Pereira (1965) estimated the molecular weight to be 210,000. Wasmuth & Tytell(l967) also reported the hexon molecular weight to be about 200,000 from gel filtration studies. Sedimentation and diffiision constants have been determined by several workers and the calculated molecular weights were 310,000 (Kohler, 1965), 400,000 (Pettersson et al., 1967) and 380,000 (Shortridge & Biddle, 1970). Values from equilibrium sedimentation were 288,000 to 311,000 using the Archibald method (KGhler, 1965) and 309,000 (P. A. Charlwood, quoted by Macint,yre, Pereira & Russell, 1969). The molecular weight estimated from crystallographic parameters was 260,000 to 330,000 (Macintyre et al., 1969). Thus values in a range 0f200,000 to 400,900 have been reported. In the present work, the sedimentation and diffusion constants have been remeasured at very low concentration, the pa,rtial specific volume has been recalculated, using the latest, available data on the amino acid composition, the sediment,ation coefficients in H,O and D,O (Martin, Cook & Winkler, 1956) and the sedimentation equilibrium distribution in Ha0 and I&O (Edelstein & Schachman, 1967) coupled with tritium-exchange experiments (Englander, 1963). From these data a molecular weight of 313,000 to 354,000 has been calculated. Furthermore, the hexon has been crystallized in a form different from that reported by Macintyre et al. (1969) and the cell parameters and space group, as well as the crystal density, have been determined. From these crystallographic data a molecular weight of 330,000 to 356,000 has been calculated. These vaIues are compared with earlier published values.
2. Materials
and Methods
(a) Pur$cation
of hexon antigen
Hexon purification was carried out according to the procedure of Pettersson et ul. The preparative polyacrylamide gel electro(1967) with one important modifioation. by a similar step using continuous phoresis according to Maize1 (1966) was replaced elution (Hjerten, Jerstedt & Tiselius, 1965). This modification has the advantage that the final rate zonal centrifugation may be omitted in the purification procedure. The preparative polyacrylamide gel electrophoresis was carried out as follows: acrylamide and N,N’-bismethylene acrylamide were recrystallized according to the method of Loening (1967). The monomer solution contained 5% acrylamide and 0.05% N,N’bismethylene acrylamide in 0.05 M-Tris-glycine buffer, pH 9~5. Polymerization was induced by 0~2% fresh ammonium peroxide sulfate and 0.1 oh N,N,N’,N’-tetramethylethylenediamine.
SIZE
AND
STRUCTURE
OF THE
HEXOX
386
The preparative electrophoresis was carried out in a continuous elution apparatus (Hjerten et al., 1966) using an 18 cm x 1 cm column with an elution chamber at the lower end (AB Stalprodukter, Uppsala, Sweden) filled with Pevikon powder (Fosfatbolaget, Stockholm, Sweden). The gels were submitted to an overnight prerun before the start of each separat;ion. The applied samples of 0.5 to 1.0 ml. were in 0.01 M-Tsis-glgcine (pII 9.5), containing 5% sucrose. The running buffer was 0.05 M-Tris-glycine, pII 9.5. The star-ting potential of 10 v/cm was raised to 30 v/cm after 1 hr. The elution rate was maintained at LO ml,/hr and t,he hexon elutcd after 12 hr running time. Kexon prepared by this method analytical polyacrylajmide gel was homogeneous according to the following criteria: olectrophoresis at pH 8.0 and at pH 9.5, immunoelectrophoresis, ultracentrifugation (see Results), N-terminal amino acid analysis (Pettersson, 1970 and manuscript in preparation), and electron microscopy. (b) Analytical
ultracentr$ugation
Hexons were dialyzed for at least 48 hr against 0.1 af-NaCl buffered with 0.91 ?a-Tris, pH $9. The buffer was prepared with II,0 or D,O. In the case of the D,O buffer, a volume of 0.60 to 0.70 ml. hexon in 1 x 10T2 M-ammonium acetate buffer, pH 6.5, was dialyzed against the NaCl-Tris buffer. The D,O was 99.8% pure and purchased from Sorsk Wydro, Norway. Centrifugation was carried out at 42,040 rev./mm in a Spinco model E centrifuge lrsing an An-D rotor. Epon double-sector centerpieces were used and the sedimenting boundary was measured at X = 280 nm using the automatic scanner. Equilibrium sedimentation was performed at low speed (approx. 4000 rev./mm) using the 4-place An-F rotor in a Spinco model E ultracentrifuge equipped with electronic sped control. For each run the speed was accurately measured by means of the revolution counter. Temperature regulation (20%) was used. The distributions were recorded on the photoelectric scanner at /\ = 280 nm and with outer dialysate in the solvent sector. Data seduction and molecular weight determinations were carried out on a General Electric mark XI computer. Details of the buffers are given in Table 4. In this series, D,O (99.8 mole %), was obtained from Bio-Rad Laboratories, Richmond, Calif. (c) Pycnometry
and viscometry
Solution densities were measured at room temperature in a pycnometer calibrated with freshly distilled water. Standard temperature corrections were applied. The relative viscosities of the H,O and D,O buffers were measured at 20°C in a Cannon-Ubbelhode capillary viscometer. (d) Analytical
gel chromatography
radius was determined by gel chromatography on 6% Agarose (Pharmacia Fine Chemicals, Uppsala, Sweden) according to the method of Laurent & Killander (1964). *P column, 1.5 cm x 90 cm, was equilibrated with O-01 M-Tris-HCl containing 0.2 ~-XaaCl. The void volume (V,) was determined from the peak of Blue Dextran 2000 (Pharmacia Fine Chemicals, Uppsala, Sweden) and the total volume (V,) from the peak of ?K20. The elution volume (V,) for the hexon was measured by use of 3H-labeled hexoo and the elution volume of human IgG (kindly supplied by Dr Hans Bennich, Uppsala, Sweden) was determined spectrophotometrically. The volume of each fraction was determined aecurately by weighing. The weights were not converted back to volumes since a ratio of volumes is used to calculate KAv (see under Results). Stokes'
(e) Hydrogen
exchange
The exchange of deuterium into hexon was estimated by measuring exchange wit,11 tritiated water according to Englander (1963). A 5.5 cm x 2 cm (diameter) column of coarse Sephadex G25 (Pharmacia Ltd) was equilibrated at 4°C with 0.1 ~r-N&l, 0.91~~ Tris-HCI, pH 8.0. Samples equilibrated for 48 hr at 4°C containing approximately 1 me “J&O and 0.8 to 1 mg hexon in 1 ml. of the same buffer were applied on top ofthe column and t,he void volume was collected in less than 3 min at 4°C. Fractions containing a constant ratio between radioactivity and optical density at 280 nm were separately used to determine moles of hydrogen per mole of hexon using the formula of Englander (1963) and
R. FRANKLIN
386
E!l’
AL.
a molecular weight of hexon of 360,000. A total number of 5852 exchangeable hydrogena per hexon was estimated from the amino acid composition (Pettersson, 1970). (f) Collection of X-ray dutw Data were collected photographically using Buerger-Supper precession cameras and G-filtered CuKar radiation from a Philips fine-focus X-ray tube run at 45 kv. A 0.6 mm collimator was used for the determination of cell dimensions and the space group, The crystals were mounted in sealed capillaries with mother liquor present in one end of the capillary. Details of crystallization are reported in the Results.
(g) Determination of cry&xl density Individual crystals in their mother liquor were placed on the side of small (4.2 cm x 0.50 cm) nitrocellulose tubes containing mixtures of bromobenzene and xylol of known density (Low & Richards, 1952&,1954). A fine glass fiber was used to remove a crystal from the surrounding mother liquor and to place it in the density solution. The bromobenzene and xylol had been equilibrated with the buffer used for crystallization (0.5 M-citrate, pH 3.2) but to prepare the density fluids, the density was assumed to be that reported for the individual reagents. This is a good approximation since the reagents are practically immiscible with aqueous reagents. To check this, however, the density of the mixtures of bromobenzene and xylol above and below the crystal density were deter-
mined directly by pycnometry.
3. Results (a) Hydrodynamic parameters and the determination of (i) Sedimentation in H,O and D,O buffers and the determination of 0 The first parameter to be calculated is P according to the formula
where Q and r/a are the viscosities of the aqueous and heavy water solutions, S, and S, the sedimentation coefficients in the aqueous and heavy water solutions, Pl and pz the densities of the aqueous and heavy water solutions, and K the ratio of the molecular weights in the two media; the increase in molecular weight in the Da0 buffer being due to deuterium exchange (Martin et al., 1956). In order to oalculate K it is necessary to know what percentage of the protons can exchange. In amino acids and small polypeptides all protons not oovalently bonded to carbon are exchangeable (Morowitz Lk Chapman, 19%) but this may not be true for proteins (Hvidt & Nielsen, 1966). Therefore we measured the amount of H exchange directly, using the tritium-exchange method of Englander (1963). Three separate experiments were carried out and in each experiment the two fractions in the middle of the most constant part of the curve of exchangeable 3H per molecule were used in the final average (Table 1). For reasons which will become a,pparent (see Discussion), a hexon molecular weight of 360,000 was assumed. The average percentage of exohangeable hydrogen was 18.2. With a maximum of 5852 exchangeable hydrogens per unit of 360,000 daltons, calculated from the latest amino-acid analysis of the adenovirus type 2 hexon (Pettersson, 1970), we find that there are 1065 exchangeable hydrogens and that K, the ratio of the molecular weights in D,O and H,O, is 1.00298. The uncorrected sedimentation coefficients for four runs in Ha0 buffer at 20.0% were 1255, 12.71, 12-85 and 12-32 and for three runs in Da0 buffer at 20°C were
SIZE
AND
STRUCTURE
OF THE
387
HEXON
‘T-36, T*67 and 7.64. In all cases the sedimenting boundary appeared to be homogeneous. The average values of X, = 12.61 and 8, = 7.56 were used to calculate P. Then using Q~, for the aqueous solution of 1.014 and qrel for the heavy water solution of 1.132 and the values of 0*010019 and 0.01099 poise for the respective absolute values of the viscosities of light and heavy water at 2O*O”C, we can calculate r to be o-727 g/cm3. TABLE 1
Tritium Experiment
1 2
no.
exchange of adenovirus type 2 hexon Exchanged H/molecule
Fraction
12
768.5
13
1008
11 12
1272 1208 1056
13
1094
12
3
Percentage exchange
13.1 17.2 21.7 20.6
18.0 18.7
This so-called hydrodynamic partial specific volume has been shown to be equivalent to the conventional partial specific volume when there are no selective solvation effects (Gagen & Holme, 1964; Gagen, 1966) and this should be the case with the solvent system used here. If we calculate P from the amino acid composition and the partial specific volumes of the individual amino acids (Schachman, 1957), the value is 0.719 g/cm3, which is in fairly good agreement with that calculated from t#he hydrodynamic data. It should be noted that the value of 7 of ribonuclease calculated from the amino-acid composition (Schachman, 1957) was in excellent agreement with that determined by pycnometry (Rothen, 1941). A third determination of v was made from sedimentation equilibrium distributions of hexons sedimented at 4000 rev./min in 0.1 M-sodium borate buffer, pH 8.0, in either H,O or D,O. The K determined by tritium-exchange experiments (see above) was also used in the formula of Edelstein & Schachman (1967). Two separate experiments gave V values of 0.734 and O-737 g/cm3, an average of 0.736 g/cm3. (ii) Determination of the d@usion constant of the hexon The experimental data obtained from four filtration experiments are shown in Table 2, and a typical experiment is shown in Figure 1. The partition coefficient.s (K,,) of IgG and hexon were calculated from such data according to the relationship K
_ ye AV
VI3
vt
-
(Laurent & Killander,
1964).
vcl
The Stokes’ radius (rJ of the hexon was calculated from the equation:
KAv = exp [-r
L (r, + r,)‘]
where I; is the concentration of gel rods in the solution measured as cm rod per cm3 and T, is the radius of the gel rods (Ogston, 1958). Although L may vary considerably
388
R. FRANKLIN
ET
AL.
with the concentration of Agarose used and also with the batch of Agarose of any single given concentration, this is not the case for rr and we may assume rr to be 25.0 A for 6% Agarose (Laurent, 1967 and personal communication). Then, using rs = 50 A for IgG (Pettersson & Berggard, personal communication) and rr = 25.0 8, we may calculate L in each experiment using the experimental value of K,v for IgG (Table 2). TABLE 2 Determination of the Stokes’ radius of the hexon Experiment IlO.
Hexon batch no.
1 2 3 4
& & & b
L
KdgG
0.552 0.555 0.552 0.557
3.36 3.33 3.67 3.31
x x x x
K*vhexon
1O’l 1011 lo= 1011
rs (4
0.464 0.469 0.461 0,470
60.25 60.05 60.61 60.18
I -4 I ’ ’ ’ 1 ’ ’ ‘----I Cts/min
‘4
(0)
0.7 700
0
i”i
A
IQ
20
30
40
50
60
70
80
7000 I
90
FIG. 1. Quantitative gel filtration experiment to determine the Stokes’ radius of the hexon. Experimental details are given in Materials and Methods and in Results. -x - x -, O.D.830 of the Blue De&ran 2000; -O-O-, “H radioactivity (cts/min) of the hexon; -a-@-, 0.0.~~~ of the IgG; -A-A-, 3H radioactivity (ots/min) of the 3H,0.
Now it is possible to use the KAv of the hexon to calculate the hexon Stokes’ radius (Table 2) and the average value for the four experiments was 60.27 A or 6.027 x 10W7 cm. From this value of Stokes’ radius we then calculate DzO,W= 3.56 x 10e7 om2/sec. (iii) The molecular weight of the hexon from hydrodynamic parameters and from sedimentation equilibrium With the range of v reported above we calculate X20,W(Svedberg $ Pedersen, 1940) from the average value of SaO.sOIV., then we can use DSOeW to calculate a molecular weight of 313,000 to 333,000 and finally we can use ~.!Yaa.~ and D2OSW to calculate according to the formula flfmin -f
= 10-e
1D2
7 Pz0.w lb3 x
t 20.W 20.W PJ (Svedberg & Pederson, 1940). These parameters are summarized in Table 3.
f mill
SIZE
AND
STRUCTURE TABLE
OF THE
380
HEXON
3
Hydrodynamic parameters of the hexoN
5.719 0.727 0.736
313,505 322,050 333,005
12.9 x lo- x3 12.9 x 15-la 12.9 x 15-13
1.34 1.31 1.30
The sedimentation equilibrium data are summarized in Table 4, giving the b&es used in the individual experiments and the calculated molecular weights using 7 = 0.727. From this set of data we calculated an average molecular weight of 342,600. Using 7 = O-719 or 0,736, we calculated average molecular weights of 33,900 or 354,000, respectively. TABLE
Sedimentation
Buffer 0.01 0.01 0.01 0.01 0.01 0.01 0.01
4
equilibrium molecular weight of adenovirus type 2 hexon
M-sodium M-sodium M-sodium M-sodium M-sodium M-sodium M-sodium
PH 9.2 9.2 8.8 8.0 8-O 8.0 7.6
borate borate borate borate borate borate phosphate 7 Calculated
Molecular
with
weight?
356,000 383,550 340,050 322,005 330,505 339,000 329,000
7 = 5.727.
(b) Crystallography of the hexon (i)
~0~d~t~0n~
jar crystallization
Cryst,als of tetrahedral shape were obtained by Pereira, Valentine & Russell (1968) from adenovirus type 5 hexon in 0.8 M-potassium dihydrogen phosphate at pH 4.4. Although it was possible to repeat this crystallization with adenovirns type 2 hexon, the crystals were only about O-1 mm on a side. Because of the small size, it was decided to explore a number of conditions for crystallization and soon it became evident that there were two distinct crystal forms, depending on the pH and ionic strength of the buffer. For example, in sodium acetate buffer at pH 5.0, tetrabedral crystals formed at 0.25 to 0.55 M; the second, bipyramidal crystal form was found at 1-O M; and transitional forms were found at 0.50 to 0.90 M. &though the bipyramidal-shaped crystals were very small in acetate buffer, it was possible to obtain excellent large crystals of this type in citrate buffer. The two different types of crystals formed within the narrow pH range of 3.2 to 4.1 (Table 5 and Plate I (a) to (e)). The bipyramidal-shaped crystals grew to O-45 to 0.60 mm in length, measured along the bipyramidal face, and all of the work reported here was done WitA such cryat&.
390
R. FRANKLIN
ET
TABLE
AL.
5
Conditions for crystallization of adenovirus type 2 hexon using 0.5 M-sodium citrate buffer PH 2.9 or lower 3.2 3.4 3.5 3.7 to 4.1 4.4 or higher
Results No crystals Bipyramidal-shaped Transitional form Transitional form Tetrahedral No crystals
The crystal density was determined in bromobenzene-xylol mixtures. Crystals migrated upward in solutions of p = I.22 g/cm3 and downward, but at a slow rate, in solutions of p = 1.21 g/cm”. The nominal solution densities were found to be correct when the densities of the two mixtures (1.22 and 1.21) were checked by pycnometry. Therefore, we conclude that the crystal density lies between I.210 and l-215 g/cm3. If a is the volume fraction of the unit cell occupied by solvent, either free or bound, then the net crystal density is pC= up, + (1 - a) pP where pp is the protein density = I/P (cf. Matthews, 1968). Using pc = l-210 to 1.215 and p = 0.719 to 0.736 we calculate a to be 0.48 to 0.53. Matthews (1968) has shown that a lies between 0.27 and 0.65 for most proteins and there are a large number of proteins with a N 0.43. In general, a is higher than average for proteins of higher molecular weight (Matthews, 196$), but in our particular case the packing of the hexon in our unit cell seems to be similar to the packing of a number of the simpler proteins, being only slightly higher than average. (ii) Determination
of crystallographic parameters
Crystals up to 0.5 to 0.6 mm in length measured on the long axis of the bipyramid (Plate I(b)) were found to be optically inactive when viewed with plane polarized light using a Zeiss POL stereomicroscope. This indicated that the orystal might belong to the cubic system. A series of Laue photographs was taken to determine the shortest repetition distance in the real unit cell and then precession photographs were taken with the X-ray beam parallel to this direction. The [loo] and [OlO] photographs clearly showed that the unit cell was cubic. The cell length, 149.9 d, was determined using human carbonic anhydrase form C for calibration of the precession camera (Liljas et al., manuscript in preparation). Also, hexon crystal WM aligned with one axis parallel to the spindle axis of the camera; the other two axes were shown to be 90” apart by taking precession photographs at the two appropriate settings. Furthermore, the [llO] direction was found at 45” to these two axes. From the Laue photographs of the [IlO] direction, the face diagonal was measured to be 214& and from a [llO] precession photograph, the [110] spacing of 106.kwas measured. The unit cell dimensions are summarized in Table 6. Since 4-fold symmetery was absent in [loo] precession photographs (Plate II), the point group could be established as 23. The space group was identified by the systematic absence of reflections, and the only ones wbioh were found wera
!"L.ATE 1. (a) and (b) Hexon cqst~ah, bipyramldal form. U~ritht~o~l~ of cryxhahst~w?. iI d 1:. ~d1t11y1citrate buffer, pH 3.2. (c) Heson cryst&, tranaitlonal form. Conditions of cryst.allization, O-.5w-sodium citrate bi!‘S’?r? $3. 3.4. (cl! Hcxor~ crystjals, transitional form. Conditions of crystalhzahon, 0.5 wwdium cit~act~huffw, pff 3.5. (e) Wexon crystal+ tetrahedral form. L’onditions of cryst~allizarion, 0.5 xl-sodium citwie buffer’, p!T 3.7.
[jnrzil!!p. WJ
PLATE II. Zero-layer precession photograph of adenovirus type 2 hexon cryhtal taken with the X-ray beam parallel to a cube edge [lOO]. The precession angle wits 6” and the exposure time was I6 hr with a crystal-to-film distance of 75 mm and a 0.3 mm collimator. The edge of the photograph corresponds to spacings of about 8 .!L
[Toil
PLATE Ill. Zero-layer precession photograph of adenovirus typo 2 hexon crystal taken u.lth the -X-ray beam parallel to a cube face diagonal [loll. The precession angle was 5” and the exposure t)ime was 15 hr with a crystal-to-film distance of 60 mm and a 0.4 mm collimator. The edge of the 1:hotograph corresponds to spacings of about, 9 .&.
piO]
[OTT]
PLATE IV. Zero-layer precession photograph X-ray beam parallel to a cube body diagonal time was 15 hr with a crystal-to-film distance
r
taken with the of adenowrus type 2 hexon crystal [I 1 I]. The precession angle was 5” and the exposure of 60 mm and a 0.4 mm collimator.
SIZE
AND
STRUCTURE
OF THE
HEXON
391
TABLE 6 Unit cell dimensions Direction
s1001 [110] Cl111
Spacing from still photograph
Spacing from precession photograph
-
149.9A 106 B -
214 11 256 A
h = 2n + 1 for MO, k = 2n + 1 for Ok0 and 1 = 2% + 1 for 001, characteristic of a 2fold screw axis (Plate II). This can be seen especially well in the [llO] precession photographs (Plate III). In the cubic system with 23 point symmetry, the only possible space group with a 2fold screw axis is P2,3. I2,3 is ruled out since the general condition for extinction, iz + k + 1 = 2n + 1, was not found. The absence of body centering was confirmed by comparing zero and first-level precession photographs with the X-ray beam parallel to the flOO], [IlO] and [ill] directions. We may now apply the formula niM = (PC- PA NV (1 -
VP,)
to determine n, the number of proteins per unit cell; where M is the moIecular weight of the hexon = 313,000 to 354,000 from the sedimentation-diffusion and sedimentation equilibrium data, p. is the density of the crystal = l-210 to l-215 g/~rn.~, ps is the density of the solvent = l-052, N is Avogadro’s number, V is the volume of the unit cell = 3.368 x lo-l8 cm3 and v is the partial specific volume of the protein = 0.719 to O-736. Using these parameters we find n to lie between 3.78 and 455, and because there are 12 asymmetric units per unit cell in space group P2,3, we can oonclude that the only possible value of n is four. Since there must be 12 asymmetric units in the unit cell in the P2,3 space group and there are four hexons per unit cell, we conclude that there are three crystallographic asymmetric units in. the hexon crystallized in the P&3 space group. The special conditions for the P2,3 spaoe group will apply in this case and thus the co-ordinates of the four hexons are x,x,x; 112 + z: 112 - x, 5; 2, l/2 + x, l/2 - x; l/2 - x, 5, l/2 + x (International Tables jar X-ray Crystallography, 1969). These co-ordinates locate the centers of the four hexons on the body diagonals of four of the eight small cubes of side 75 d, obtained upon dividing the cubic unit cell into eights. The four body diagonals upon which the hexons lie are related by non-intersecting S-fold screw axes. We may now apply the above equation in a second way. Using n = 4, we may determine a molecular weight of the hexon and using the values of P = 0.719, 0~7.27 and 0.736, the calculated molecular weight will be 330,000, 342,000 and 356,006, respectively. A number of features of the reciprocal lattice are illustrated in the precession photographs. Features of the [loo] precession photograph (Plate II) have already been mentioned. The 2-fold symmetry of the [IlO] direction is illustrated in Plate III along wit,h “spikes” in the [ill] direction, the direction of the crystallographic 3-fold
392
R.
FRANKLIN
ET AL.
axis. The 3-fold symmetry of the [ill] precession photograph is illustrated in Plate IV, where actually 6-fold symmetery is evident in this zero layer due to the operation of Friedel’s law. Upper layers along [ill] show only S-fold symmetry. 4. Discussion As in the case with many proteins and other macromolecules available in very limited quantities, accurate determination of the partial specific volume of the hexon has been difficult since it has not been possible to obtain enough protein to make precise pycnometrio measurements. In using indirect methods we have obtained three different values for P (0719, 0727 and 0,736) and we prefer, therefore, to report the range of molecular weights for the hexon using the range of experimentally determined v. The hydrodynamic P was calculated using the average values of S, and S,, the only reasonable procedure since insufficient sedimentation data were taken to do a meaningful statistical analysis. The t&mm-exchange experiment, needed to obtain an accurate value for K, the increase in molecular weight due to exchanged deuterons, revealed that only 18.2% of the theoretically exchangeable protons did exchange. This suggests that there may be large solvent-inaccessible regions in the hexon, a suggestion supported by the presence of hydrophobic peptides in the tryptic digest of hexons (Pettersson, 19’70, and manuscript in preparation). The molecular weight range obtained by sedimentation equilibrium (333,000 to 354,000) agreed well with the range determined from the crystallographic parameters (333,000 to 356,000) but these values are slightly higher than those obtained from the hydrodynamic data (313,000 to 333,000). The estimate of the d.iffusion constant of the hexon may not be as reliable as our other parameters since the constants of the Agarose, particular r,, the radius of the gel rods, are not known precisely. The chemical studies of Pettersson (1970, and manuscript in preparation) indicate that there is a polypeptide repeat unit of molecular weight 60,000 daltons in the hexon, and the studies of Maize1 et al. (1968a,b) indicate that there may be a larger polypeptide subunit of molecular weight 120,000 daltons. Whereas Maize1 and his colleagues have suggested that the hexon is composed of three identical polypeptide chains of molecular weight 120,000 each (Maize1 et al., 1968a,b; Horwitz et al., 1970), Pettersson (1970) suggests that the hexon contains six identical polypeptide chains of molecular weight 60,000 each, Whatever the number of chains, our sedimentation equilibrium data and crystallographic data, coupled with the chemical data just mentioned, all suggest that the hexon must have a molecular weight of about 360,000 daltons. We may now comment on the earlier estimates of hexon molecular weight. It is generally accepted that molecular weight determinations from electron micrographs may be inaccurate due to shrinkage of structures after negative staining; this may explain the low value of 210,000 reported for the type 5 adenovirus hexon by Valentine $ Pereira (1965). KGhler (1965) measured the sedimentation and diffusion coefficients for hexons from several adenovirus types by analytical ultracentrifugatioa and arrived at values of 280,000 to 310,000 for the hexon molecular weight. The discrepancy between his values and our present estimates mainly depends on his low value for the sedimentation coehloient. Wasmuth & Tytell (1967) gave a range of 158,000 to 256,000 for hexons from six adenovirus types. Their estimates were made by direct comparison of the elution positions for hexons with those of other proteins of
SIZE
AND
STRUCTURE
OF THE
HEXON
3x3
known molecular weight. Such determinations may, however, be subject to considerable error since the elution position of a molecule is related to its Stokes’ radius rather than to its molecular weight (Siegel & Monty, 1966). Boulanger, Flamencourt & Biserte (1969) reported a molecular weight of 300,000 for type 2 and 5 hexons. This value was based on a sedimentation coefficient of 11.2 S, determined by analytica, nltracentrifugation, the diffusion coefficient determined by Kiihler (1965) (3.723 x 19-T cm2/sec) and our previous value of 0,720 cm3/g for 7, determined from the amino acid composition of the hexon (Pettersson et al., 1967). Recently, Shortridge & Biddle (1970) reported a value of 380,000 for hexons from adenovirus type 5. Although. their value of molecular weight is in reasonable agreement with our present determinations, their value for Stokes’ radius (64 8) is slightly higher than ours (SOA) ; this, however, was compensated for by their low estimate of 7. The different values obtained for Stokes’ radius may depend on the use of different gels for the analytical gel chromatography. Since hexon elutes rather close to the void volume on Scphadex G200, estimates made on 6% Agarose should be more accurate because the hexon elutes in the area of maximum separation capacity of the gel in this case. ur previously reported vahe of 400,000 daltons for type 2 hexons is higher than that obtained in the present study. This depends mainly on a low value for the diffusion coefficient (2.6 x 10m7 cm2/sec). The X-value was also slightly below the actual S,,,, ettersson et al., 1967). The molecular weight range given by Maeintyre et al. (1969) cannot be discussed since even his crystallographic unit cell dimensions are probably incorrect (see below). Finally, the value of 88,000 + 8000 for the crystallographic asymmetric unit, calculated by Corniek, Sigler & Ginsberg (1971) gives a hexon molecular weight of 240,000 to 290,000, below most reported values. Since the unit cell was identical to ours (see below), this difference appears to be due to the difference in measured crystal densities, and since similar techniques were used in both laboratories it is n.ot possible at this time to explain this discrepancy. Preliminary results from low-angle X-ray scattering indicate that the hexon is a cylinder of diameter 76 A and height 119 A (I. Lindqvist, personal communication). Electron microscopy of the hexon has indicated that the particles are 80 to 110 A in diameter (Pettersson et al., 1967) and we may interpret such electron micrographs in terms of the two views of the cylinder. Then we may approximate the hexon to a prolate ellipsoid with an axial ratio p = b/a of 1.57. The frictional ratio of such an unhydrate ellipsoid would be approximately 1.019 (Svedberg & Pedersen, 1940). e may now use our experimental frictional ratio (flfmin) to estimate the solvation from the equation
f -=-
f
f min fo (
-PP* + 6 l/3
PP, )
where f/f,, is the frictional ratio due to shape alone (Tanford, 1961). Using the range of P obtained for the hexon and f/fminof 1.30 to 1.34, we may estimate 6 to be about -80, an unusually high degree of solvation, when compared to many non-viral proteins (Tanford, 1961). Hydration estimates of this type have usually led to very high values for viruses (Lauffer & Bendet, 1954), but usually one did not distinguish between bound water and solvent spaces, as pointed out in the elegant discussion of this problem by Harrison (1969). Indeed, when the bound water is correctly calculated for tomato bushy stunt virus (0.35 to 0.43 g/g), it is very similar to that for
394
R. FRANKLIN
ET AL.
hemoglobin (Harrison, 1969). Thus future extended hydrodynamic studies on the hexon are necessary before we can obtain a true estimate of bound water. Although the space group of the tetrahedral crystals of type 5 adenovirus hexon was claimed to be I23 or I2,3 by Macintyre et al. (1969), the careful study of Cornick et al. (1971) has shown that these crystals are isomorphous with our type 2 hexon bipyramidal crystals. Thus at present we can only state that the hexon has three crystallographic asymmetric units. Three-dimensional data to 10 A resolution have been collect,ed and hopefully we will be able to detect local symmetry by analysis of this data. A large number of colleagues made important contributions to this work in the form of criticisms, as well as suggestions for carrying out the det,ermination of some parameters such as P, D, and pC. The authors are greatly indebted to these individuals who were: Professor T. C. Laurent and Dr P. A. Peterson, Institute of Medical Chemistry, University of Uppsala; Drs D. L. D. Caspar and S. Harrison, The Children’s Cancer Research Institute, Boston; Drs K. K. Kannan and A. Liljas, Biological Structure Group, The Wallenberg Laboratory, University of Uppsala; and Dr C. I. Brand&r, Institute of Chemistry, I, Lantbrukshogskolan, Ultuna. Mrs Eva Hjertson provided excellent technical assistance. One of us (R. M. F.) was on leave of absence from the Public Health Research Institute of the City of New York, New York, N.Y. during the course of this research. This work was supported by grants from the Damon Runyon Memorial Fund, the Swedish Cancer Society, the Swedish Delegation for Applied Medical Defense Research, the Swedish Medical Research Council, the Swedish Natural Science Research Council, and U. S. Public Health Service (AI GM 07382).
REFEREKCES
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SIZE
AND
STRUCTURE
OF THE
HEXON
396
Ogston, A. G. (19tiS). Trans. Faraday Sot. 54, 1754. Pereiaa, II. 6.: Valentine, R. C. & Russell, W. C. (1968). Xatz~re, 219, 946. Pettersson, U. (1970). Ph.D. Thesis, University of Uppsala. Pettersson, U., Philipson, L. & Hoglund, S. (1967). Virology, 33, 575. &age, L., Pettersson, U., Hoglund, S., Lonberg-Helm, K. & Philipson, L. (1970). T/irolocrg, 42, 341. Rothen, A. (1941). J. Gen. Physiol. 24, 203. Schachman, H. K. (1957). In Methods in Enzynzology, ed. by S. P. Colowick Cy:N. 0. Kaplan, vol. 4, p, 32. New York: Academic Press. Shortridge, K. F. & Biddle, F. (1970). Arch. ges. Virmfowch. 29, 1. Siegel, L. M. & Monty, K. J. (1966). Biochim. biophya. Actn, 112, 346. Svedberg, T. & Pedersen, K. 0. (1940). The Ultracentrifuge. Oxford: The Clarendon Press. Tanford, C. (1961). The Physical Chemistry of ikfacromolecules. New York, London: J. Wiley S: Sons. Valentine, R. C. & Pereira, H. G. (1965). J. MoZ. Biol. 13, 13. Wasmuth, E. H. S; Tytell, A. A. (1967). Life Sciences, 6, 1063. W7ilcox, W. C. & Ginsberg, H. S. (1963). J. Eqx Med. 118, 295.