Studies on the molecular weight of the adenovirus type 2 hexon and its subunit

J. Mol. Biol. (1974) 89, 163-178

Studies on the Molecular Weight of the Adenovirus Type 2 Hexon and its Subunit M. GRUTTER AND R. M. FRANKLIN Abteilung fiir Strukturbiologie Biozentrum der Universit&t Base1 CH-4056 Ba.sel, Switzerland (Received 22 April 1974, and in revised form 1 July 1974) The molecular weight of the adenovirus type 2 hexon was calculated from sedimentation equilibrium, light scattering and sedimentation and diffusion experiments. The extinction coefficient, Ei”j,,,, was determined to be 14.3 at 279 nm, from quantitative nitrogen and carbon analyses combined with the N,C content calculated from the amino acid composition. Other parameters determined were: the partial specific volume, # = O-738 cm3 g-r ; the refractive index increment, (&zJ&),,,, Ir = O-193 cm3 g-l at 435.8 nm; the sedimentation coefficient, s!&* = 13.0 S; and the diffusion constant, II:,,, = 3.32 x lob7 cma s-l. All molecular weights were between 355,000 and 363,000. Crystal density measurements were made on native and glutaraldehyde cross-linked crystals and the molecular weights calculated from these data were compared with the precise molecular weight determined by physico-chemical methods. Only one polypeptide of molecular weight 120,000 was found in reduced, or reduced and alkylated, hexon. Four or six organomercurial molecules were bound per 120,000 molecular weight of native hexon upon titration with 2-chloromercuri-4nitrophenol and 2-chloromercuri-4,6-dinitrophenol, respectively. With 55’.dithiobis (2nitrobenzoic acid) only one SH-group per 120,000 could be titrated in native hexon, but after denaturation in 1% sodium dodeoyl sulphate five more SH-groups reacted per 120,000 molecular weight. Thus there are three identical polypeptides of molecular weight 120,000 per hexon of total molecular weight 360,000.

1. Introduction The adenovirus hexon? is the major structural subunit of the viron. There are 240 hexons per virion and these are located at the local g-fold symmetry axes of the capsid (Valentine & Pereira, 1965). Along with the 12 pentons located at the 5-fold axes, the hexons form the outer shell of the virion. The hexon is one of the few viral capsid proteins which can be isolated in a native form (Pettersson et al., 1967). Since this protein can be crystallized (Pereira et al., 1968; Macintyre et al., 1969; Franklin et al., 1971a; Con&k et al., 1971; DGhner & Hudemann, 1972) the threedimensional structure of a viral capsid protein may be determined to high resolution. Knowledge of this structure should aid our understanding of the self-assembly of proteins

to form

spherical

virus

shells.

Unfortunately, there is still no general agreement on the molecular weight of the hexon or the number of subunits. Maize1 and co-workers determined the hexon 5 The hexon referred wise.

to in this paper is from adenovirus 163

type 2 unless explicitly

stated other-

164

M. GRUTTER

AND

R. M. FRANKLIN

polypeptide molecular weight to be 120,000 by sodium dodecyl sulphate gel electrophoresis (Maize1 et al., 1968a,b) and ultracentrifugation in guanidine*HCl (Horwitz et d., 1970). Pettersson (1970) suggested a subunit molecular weight of 60,000 from dodecyl sulphate gel electrophoresis and gel filtration of reduced and alkylated hexons (cf. Franklin et al., 19715). Franklin et al. (1971a) obtained molecular weights of 313,000 to 356,000 for type 2 hexon from equilibrium sedimentation, sedimentation coefficients and diffusion constants, and from density measurements of crystals. Cornick et al. (1973) obtained a molecular weight of 309,000 for type 5 hexon from crystal density measurements on glutaraldehyde cross-linked crystals. From an estimate of at least seven unique cysteinelhalf cystine residues, a molecular weight of 100,000 was calculated for type 2 hexon polypeptide (Jornvall et al., 1974o,b). Assuming a trimer, the hexon would then have a minimum molecular weight of 300,000.

We felt, therefore, that it was imperative to redetermine the molecular weight of the native hexon and its polypeptide. In the present work, all necessary physicochemical parameters : extinction coefficient (E: &, 279 nm), partial specific volume (G), sedimentation coefficient (s!&,.), diffusion constant (D&,) and refractive index were determined directly. Molecular weights were calculated increment ((an/&),,,,,) from equilibrium sedimentation, light scattering, and the precise values of s&~ and %,w~ and were also estimated from density measurements on native and glutaraldehyde cross-linked crystals. The molecular weight of the polypeptide subunit was determined from polyacrylamide gel electrophoresis. The subunit size was also confirmed by titration experiments using 5,5’-dithiobis (2-nitrobenzoic acid), 2-chloromercuri-4-nitrophenol and 2-chloromercuri-4,6-dinitrophenol. Furthermore, the total number of free SH-groups was obtained from titration experiments using 5,5’-dithiobis (2-nitrobenzoic acid) after sodium dodecyl sulphate treatment of the protein.

2. Materials and Methods (a) Virus production Adenovirus type 2 was propagated in suspension cultures of HeLa cells (from the laboratory of Dr D. A. Wolff, Ohio State University) grown in Eagle’s spinner medium (Eagle, 1959) with double amounts of amino acids plus 5% foetal calf serum and 5% calf serum. The cells were infected at a multiplicity of 20 plaque-forming units per cell in spinner medium at a cell density of 3 x lo5 cells/ml. The cells were harvested 48 h after infection (cf. Pettersson et al., 1967). (b) Pur$cation of hexon Hexon purification was carried out according to the procedure of Pettersson et al. (1967) with the following modifications which gave higher yields. The salt gradient in the DEAE-cellulose chromatography step was halved in order to obtain a better separation of penton fibrea, pentons and hexons (see Fig. 1). The preparative gel electrophoresis step was omitted and instead the hexon fraction obtained after DEAE-cellulose chromatography was crystallized using conditions described by Franklin et al. (1971a). Crystallization was carried out as follows. The hexon fraction from the DEAE-cellulose column was concentrated by ultrafiltration with Diaflo membranes (XM50 from Amicon Corp., 21 Hartwell Avenue, Lexington, Mass.) to about 10 mg/ml and then crystallized from O-5 M-sodium citrate buffer (pH 3.2) at room temperature for about 8 days. When crystal growth was completed, the crystals were washed twice with ice-cold distilled water and then dissolved in 0.01 M-sodium phosphate buffer, 0.02% sodium azide at pH 7.0. Hexon

protein prepared by this method was homogeneous according to the following criteria:

MOLECULAR

ill Oo

IO

I 20

WEIGHT

I 30

I 40

OF THE

I I 60 50 Fraction no

I 70

166

HEXON

I 00

I 90

I 100

FIG. 1. DEAE-cellulose chromatography of the soluble antigens. Optical density at 280 nm was recorded for each fraction. Fractions of 2 ml were oollected. A linear salt gradient from 0 M to 0.6 M-N&I was used. Peak I corresponds to fibre, peak II to penton and peak III to hexon.

analytical dodecyl sulphate polyacrylamide gel electrophoresis according to Weber & Osborn (1969), analytical ultracentrifugation, light scattering, and intensity fluctuation spectroscopy. The recovery from the crystallization step was about 60 to 75%. The overall yield was 60 to 75 mg hexon protein from 6 x 10’ infected cells (cf. Boulanger 8t Puvion, 1973). (c) Spectra

and carbon, nitrogen deteminution

Hexons were dialysed for at least 48 h against 0.01 M-sodium phosphate buffer at pH 7.0 before recording the spectra (230 nm to 400 nm) on a Cary 17 spectrophotometer with the base-line checked before and after measurement. O.l-cm cuvettes were used for high protein concentrations. No corrections were made for either light scattering or for Duysens flattening (Duysens, 1956) of the peaks (Day et al., 1972). The optical densities of test samples recorded on the Cary 17 were the same as those given by a Zeiss PMQII singlebeam spectrophotometer and both instruments were used throughout this work. The N,C analyses were made by the microanalytical laboratory of Ciba Geigy, AG, Basel. The N,C yields were determined with a Perkin-Elmer 240 C,H,N analyser, after concentrating the solution for 12 h at 40°C. (d) Partial

sveci$c

volume

Hexons were dialysed for at least 48 h agamst 0.01 M-sodium phosphate buffer, 0.02% sodium azide, 0.2 M-NaCl or against 3.01 M-SOdiUm phosphate buffer, 0.02% sodium azide, both buffers at pH 7.0. The measurements were carried out in a densimeter DMAO 2 C(A.Paar, Graz, Austria). A description of the apparatus and the technique is given in Kratky et al. (1973). For each determination of the partial specific volume, the density of four protein solutions at different protein concentrations was measured; each was dialysed to equilibrium before the density measurement. The apparatus constant was determined from the densities obtained with air and freshly distilled water. The temperature in the measuring cell was very carefully controlled at 2500°C 5 0.01 deg. C. The protein concentrations were determined spectrophotometrically.

(e) Analytical

ultracentrifugation

Hexons were dialysed for 48 h against 0.01 M-SOdiUm phosphate buffer (pH 7-O), 0.02% sodium azide, with and without 0.2 M-NaCl. Centrifugation was carried out in a Beckman

166

M. GRtfTTER

AND

R. M. FRANKLIN

model E analytical centrifuge runnin g at 52,000 revs/n& at 2O.O”C. Epon double-sector centrepieces were used. For low concentrations of protein the sedimenting boundary was recorded at h = 279 nm with the automatic scanner; schlieren optics were used for higher protein concentrations. Equilibrium sedimentation was carried out at 4400 or 4800 revs/min in an AN-J rotor using electronic speed control. The temperature was held constant at lO.O”C. The protein distribution in the cell was measured with the photoelectric scanner at h = 279 nm with outer dialysate in the solvent sector. The molecular weight was found from a least-squares fit of log O.D. vemus r2. (f) Refmctive index increments Hexon protein was equilibrium dialysed at room temperature against 0.01 M-SOdhUn phosphate buffer (pH 7*0), 0.02% sodium azide in closed glass flasks. Refractive index differences between solution and outer dialysate were measured at known concentrations in a Raleigh interferometer L13 (Zeiss, Jena) at three different wavelengths: 436.8 nm, 646.1 nm and 589.0 mn. All measurements were made at 23°C in a 2*0027-cm long cuvette, which had been calibrated with standard KC1 solutions. (g) Light scattering For light scattering experiments the hexons were dialysed for 48 h against 0.01 M-sodium phosphate buffer (pH 7-O), 0.02% sod ium azide. The measurements were performed with unpolarized light at 23.0% at angles of observation from 45’ to 150” at he = 436.8 nm with a Fica model 50 (A. R. L. France-Fica, F-78 Le Mesnil-Saint-Den&, France) light scattering photometer. For all measurements cylindrical optical cells (about 2 ml sample volume) were used. They were closed with a well-cleaned Teflon stopper and half-immersed in a benzene bath ; no corrections for reflection or refraction were necessary. The solutions were freed of dust as follows. The protein solution was centrifuged for 1 h at 36,000 revs/min in an SW40 rotor (Christ). A sample of protein solution was removed from the middle of the tube and transfered to the cylindrical scattering cell. The pipette was cleaned before use with chromic acid, then washed and dried with filtered nitrogen gas. The cylindrical cuvette wes treated in the same way. The cuvette and the pipette were closed with Parafilm (Parafihn “M”, Marathon products, Neenah, Wisconsin) which was removed just before use. Sample transfer was carried out on a dust-free sterile bench (CEAG, Envirco, Dortmund, Germany). This method is preferred over filtration through Millipore filters since sign&ant amounts of hexon were lost in the latter process. Scattering was measured over a wide series of protein concentrations. At the higher concentrations, neutral filters were used to attenuate the scattered intensity. The attenuation factor for each filter was determined with a standard factory-provided glass etalon. At each angle, the scattered intensity was recorded as a function of time for several minutes. Isolated peaks (especially at small angles) were interpreted as scattering from dust, and these were discarded when averaging the scattered intensity. In order to calculate the absolute value of this average, benzene was measured at 90°C and the following value for the absolute scattering cross section was used: I/?’ (do/dS)),,. (coumou,

= k(h), k(435.8 nm) = 46.6 x 10m6 cm-i

1960).

(h) Intensity j&&&ion apectromopy The light source for these measurements was a Coherent Radiation Laboratories model 52K krypton ion laser usually operated at the red (647.1 nm) line which was dimmed by crossed polarizers and compressed to 5 mm. The correlation was performed with a 24channel Malvern Digital Autocorrelator. The cuvette was held at 2O*O”C 4 0.06 deg. C with circulating water filtered through a 0.45-w Millipore filter. The sample and cuvette preparation were the same as in the light scattering experiments. A square sample cell was used, being superior to a cylindrical cell in minimizing stray light scattered from the cell surface. The correlator output was recorded on punch tape and later processed on a Hewlett-Packard 98lOA computer.

MOLECULAR

WEIGHT

(i) Crystal

OF THE

den&y

167

HEXOX

determin&m

Hexon protein was crystallized in 05 ~-sodium citrate, pH 3.2 (Franklin et a.!., 1971a). Crystals up to O-4 mm in length were used for the measurements. In some experiments crystals were cross-linked by soaking them for 12 to 14 h in 0.25% glutaraldehyde (electron microscopy grade, purified by distillation; 25% aqueous solution, Schucherdt Inc., et al. (1973). The cross-linked crystals were Munich, Germany) according to Cornick repeatedly rinsed with water in order to convert the solvent to water. Native crystals were equilibrated with 0.5 M-sodium citrate, pH 3.2, for at least 12 h. The cell dimensions of cross-linked crystals were determined from appropriate X-ray diffraction photographs. Individual crystals were placed in small nitrocellulose tubes containing mixtures of bromobenzene and xylene of known and increasing density. A fine glass fibre was used to remove the crystal either from a wet filter pa.per or from a microscope glsss slide and place it into the mixtures. The adjacent tubes, in which the crystals ascended and descended when pushed into the solvent, were found and the densities of the solvent in these tubes were determined directly by pycnometry. (j) Polyacrylamide

gel electrophortis

and

enzynaatic

digestion

Gel electrophoresis using 5% acrylamide was performed according to Weber & Osborn (1969). Alkylation (carboxymethylation) after reduction of the hexon was carried out according to Crestfield et al. (1963). Hexon was dissociated at 37°C for 4 h in 1 Ivr-Tris buffer (pH 8.5) in the presence of 6 M-guanidine*HCl with 0.01 M-EDTA and 1% 2-mercaptoethanol and then the protein was alkylated in the dark for 15 min at room temperature by the addition of recrystallized iodoacetic acid to a molar concentration three times more than that of the 2-mercaptoethanol. The iodoacetic acid had been recrystallized six times just before use from an ethyl ether/petroleum ether mixture. The protein wss then dialysed extensively against 0.01 aa-sodium phosphate buffer (pH 7.0) containing 0.1% sodium dodecyl sulphate and subsequently treated with 1% dodecyl sulphate before electrophoresis. Purified hexon in 0.2 M-sodium phosphate buffer (pH 7.2) was incubated for 30 min with either pronase or chymotrypsin in a substrate to enzyme ratio of 100: 1, followed directly by electrophoresis with control gels of pronase, chymotrypsin and untreated hexon. For molecular weight determinations we used the following proteins : T-layer, 140,000 (Henry, 1972); bovine serum albumin, 67,000; ovalbumin, 45,000; chymotrypsinogen, 25,000; and cytochrome c, 17,000. (k) Titrations

with

organomercurial

WmpOUnd8

(d-nitrobenzoic

and

with

$5’~dithiobti

acid)

Titrations of the hexons were carried out with 2-chloromercuri-4-nitrophenol and 2-chloromercuri-4,6-dinitrophenol (Whatman Biochemicals, Ltd., England). 1 ml of hexon protein (1 mg/ml) was titrated in each experiment with the organomercurial reagent in a l-cm spectrophotometer cuvette. The titration was performed as follows. The zero optical density was checked before titration, then 2 ~1 of organomercurial compound were added to the sample cuvette and the reference cuvette with a 50-~1 motor-driven Hamilton syringe. The solutions in each ouvette were then mixed and the difference in O.D., as measured with a Zeiss PMQII spectrophotometer, was recorded after 15 min. The procedure was repeated until the O.D. difference remained constant. The titration with 2-chloromercuri-4-nitrophenol was recorded at 410 nm in O-1 Mtriethanolamine buffer (pH 7*4), containing 0.01 M-N&~, and with 2-chloromercuri-4, 6-dinitrophenol at 371 nm in O-1 M-SOdiUm citrate buffer (pH 5.5), containing 0.1 r,rEDTA. The concentration of the organomercurial stock solutions was determined in 0.1 M-sodium hydroxide using l = 1.74~ lo* mol-i cm-l st 405 nm for 2-chloromercuri-4nitrophenol and B = 1.57~ lo* mol-i cm-l at 371 nm for 2-chloromercuri-4,6-dinitrophenol (McMurray & Trentham, 1969). Titration of the free SH-groups with 5,6’-dithiobis (2-nitrobenzoic acid) (Fluka AG, Buchs, Switzerland) was done according to Ellman (1969), using a stock solution in 0.1 Mphosphate buffer (pH 7.0) which was prepared before each experiment. Titrations were done in 0.1 M-sodium phosphate buffer (pH 8.0) in a 1 -ml cuvette at room temperature. The c’ourse of the reaction was followed at 412 nm on a Gary 17 recording spectrophotometer.

1.14 1.01 1.11 1.19 1.20

N bxdml)

3.66 3.23 3.67 3.83 3.86

C (mg/ml) 7.22 6.39 7.03 7.53 7.59

Protein (mg/ml) from N

Calculated ratio N/C from amino acid composition is N/C = 0.305. Average ratio N/C from above N,C determination is N/C = 0.311. Mean value for E::m at 279 nm is 14.3 f 0.3 (& 2%).

1 2 3 4 6

Experiment

Extinction

I

7-06 6.23 6.89 7.39 7.44

Protein (mg/mI) from C

coefficient of hexon

TABLE

E 1cmat

IO.46 8.80 9.86 10.70 10.60

279 nm

1cmat

El%

14.6 13-s 14.0 14.2 14.0

279 mn from N

14.8 14.1 14.3 14.6 14.3

E:‘& at 279 nm from C

Cyst

t From Jbrnvall

=‘rp

TF Phe

Gls Ala Half Val Met Ile Leu

ASP Thr SW Glu Pro

LYS His -4%

Amino acid

128.17 137.14 166.18 116.08 lOl*lO 87.07 129.11 97-11 67.06 71.07 103.14 99.13 131.19 113.16 113.16 163.17 147.17 186.21

4.4 1.7 4.7 14.4 7.3 7.2 9.7 6-6 7-8 7-b 0.8 6.4 2.3 3.4 7.6 6.0 4.3 0.9

et al. (1974~).

Molecular weight less water

acid composition

Amino acid in heson (%)

Amino

56.22 62.64 46.14 41.74 47.62 41.38 46.61 61.84 42.10 60.70 34-93 60.68 46.77 63.68 63.68 66.24 73.44 70.96

c per amino acid (%I

according

TABLE 2

62.26% 61.86%

2.4737 0.8932 2-1686 6.0106 3.4690 2.9794 4.6116 4.0196 3.2838 3.8026 0.2794 3.2713 1.0627 2.1661 4.7760 3.3120 3.1679 0.6386

C content (o/o)

0.9618 0.6209 1.6864 1.7626 1.0118 1.1686 1.0626 o-9373 1.9149 1.4783 0.1086 0.7630 0.2466 0.4209 0.9286 0.4290 0.4089 0.1366

21.86 30.64 36.88 12.17 13.86 16.09 10.86 14.42 24.66 19.71 13.68 14.13 10.68 12.38 12.38 8.68 9.61 16.06

15.92% 16.80%

S content (%I

N per amino acid (%)

to Pettersson et al. (1967)

170

M. GRifTTER

AND

R. M. FRANKLIN

A molar extinction coefficient for the thionitrobenzoate ion of 1.36 x IO4 mol-l cm-l at 412 run was used in calculations of the extent of reaction (Elhnann, 1959). Titration of SH-groups not accessible to 5,5’-dithiobis (2-nitrobenzoic acid) in the native state was performed in 0.1 M-E?OdiUm phosphate buffer at pH 8.0 in the presence of 1% sodium dodecyl sulphate from Serva Feinbiochemica, Heidelberg, Germany. The presence of dodecyl sulphate does not change the extinction coefficient of the thionitrobenzoate ion (cf. Acharya & Moore, 1973).

3. Results (a) Determination of the extinction coegicient The contents of nitrogen and carbon in the hexon, as calculated from the amino acid composition given in Table 2, were Kv~O~~ and 5135%, respectively. The N,C contents of protein solutions were determined on samples from which spectra (230 to 400 run) had been recorded. From five independent measurements (Table 1) on different hexon preparations the extinction coetkient in 0.01 M-socliura phosphate buffer (pH 7.0) with or without O-2 M-NaCI, was calculated to be E:& = 14.3 f 0.3 at 279 nm, which agrees very well with the value calculated from the amino acid composition and is within the error range of the value from Day et al. (1972). The accuracy of our value is 2% compared with 4% from Day et al. (1972), based on a mean value for globular proteins of (an/%),,,,, = O-185 f 0905 cm3 g-l at 546 nm (cf. compilation by Timasheff, 1970). (b) The partial specijk volume The most important parameter in most of our molecular weight determinations is 6, the partial specific volume. Since the concentration of the protein was determined by N,C analysis, the B measured is related to the salt-free form of the protein. Since

0.28 ‘Y 0.27 -

.l

.

.

0.26 0.255-d 0

I

2

3

4

5

6

7

8

Concn (mg/ml)

FIQ. 2. Extrepolation of the value (I-$) from four different protein aonoentrations to zero concentration in order to obtain the partial specific volume. Measurements were done in 0.01 ~-sodium phosphate (pH 7.0) plus O*O2o/o sodium azide. There was only a slight dependenoe of (1 -Cp) on the concentration. The density of the solvent was 0.9990 g/ems.

we performed our measurements in a buffer similar to water, our calculated molecular weights will be for this material. Low salt concentrations should change this value but the change is within the standard deviation of the measurements. Figure 2 shows a typical experiment with extrapolation to c = 0 (c is the concentration of the protein). Table 3 shows the conditions and results from four different measurements.

MOLECULAR

WEIGHT

OF THE

171

HEXON

TABLE 3

Partial specifi volume of hexon Experiment

Buffer 1 2

1 2 3 4

P (Buffer)

1-

0.9982

0.269 0.261 0.265 0.257

1

1.0066 0.9990

2

1.0064

Bp

Mean value for the partial specifio volume fi = 0.738 f 0.004 cm3 g-l. Buffer 1: 0.01 ~-sodium phosphate (pH 7.0), 0.02% sodium azide. Buffer 2: 0.01 aa-sodium phosphate (pH 7.0), 0.02% sodium azide, 0.2 M-sodium

B 0.742 0.736 0.736 0.739

chloride.

From these data we calculated a mean value for the partial specific volume, d = O-738 f 0904 cm3 g-l. (c) Sedimentation of hexon protein 20,W)in buffer with 0.2 M-sodium chloride (20°C) The sedimentation coefficients (so and without sodium chloride (20°C) were 13.1 and 13.0, respectively, the latter in good agreement with that of Franklin et al. (1971a) measured under similar conditions. Figure 3 shows the dependence of the sedimentation coefficient on hexon concentration. The sedimentation varies in buffer without NaCl as for a normal globular protein (Schachman, 1959).

I2 II IO

:

90:

-I E Concn (mg/ml)

Fm. 3. (a) Sedimentation aa a fun&ion of concentration in 0.01 ~-sodium phosphate, pH 7.0. (b) Sedimentation ae a function of concentration in 0.01 M-sodium phosphate (pH 7.0), 0.2 M-N&l. -m-m--, Absorption method with photoelectrio scanner; -e-O--, schlieren optics.

172

M. GROTTER

AND

R. M. FRANKLIN

(d) Sedimentation equilibrium The linearity of the plots of the logarithm O.D. versus ra demonstrated the homogeneity of the protein. In Table 4 the molecular weights from sedimentation equilibrium experiments are summarized. The mean value from these data was 356,000 f 20,000. TABLE 4

Summay of the molecular weight determinations 012hexon protein Equilibrium

336,000’ 372,000’

sedimentation

346,000a 343,000s 37a,oooa

Light

scattering

Fluctuation spectrosaopy and sedimentation

360,000~ 360,000’

Solvents used : 1 0.01 ~-sodium phosphate, pH 7.0; a 0.01 ar-sodium phosphate (pH 7*0), 0.2 ~-sodium 8 0.6 ~-sodium citrate, pH 3.2; 4 water.

363,000’

Crystal

cross-linked in Ha0 3f32,0004

density

413,0003

ohloride;

(e) Light scattering The refractive index increment was determined at three different wavelengths: 4353 nm, 546.1 nm and 589 nm. The accuracy in these measurements is 2%, mainly due to the uncertainty of the concentration. Values are summarized in Table 5. TABLE 5

Refractive index increments at @feerent wavelengths

436.8 646.1 689

0.193 0.189 0.190

The Zimm plots were calculated and plotted on a Hewlett Packard 9180A computer. Figure 4 shows a typical example of these plots. Extrapolation of the data points to zero concentration and zero angle was done by ordinary least-squares fitting and the unbiased variance was calculated. The next least-squares fits were done with the inverse relative variances as weighting factors, in order to get the molecular weight, .M, the radius of gyration, rg, and the second virial ooet?icient, A, with their appropriate errors. The results from two different experiments are given in Table 4. The mean values from these experiments were: M = 355,000 and A = 3.76 x 10ms cm3 mol-l. Clearly no reliable value for r, can be obtained from light scattering data because the wavelength of the light is about 40 times the particle diameter.

MOLECULAR

2O

WEIGHT

OF THE

I73

HEXON

I

I

I

I

0.25

0.5

0.75

I-O

sin2 T+6.6ilo% ’

FIQ. 4. Zimm plot of hexon in 0.01 M-sodium phosphate, of the data

c is the conoentration

pH 7.0. This is a two-dimensional

of the hexon and R(B) is the reduced scattered Kc/R(O)

= (4

+ 2Ac)

(1 + T

z

intensity.

plot

Since

sins:)

the extrapolation: c + 0,B --f 0 gives the inverse of molecular weight, M, on the ordinate, whereas the slope of the extrapolation c + 0, 0 + 0 gives the radius of gyration rg and the second virial coefficient A. c is given in g/l00 ml. (0) Measured points; ( n ) extrapolated points.

(f) Fluctuation

spectroscopy

The second-order correlation function, g2, recorded on punched tape, was processed according to Pusey et al. (1974) with small modifications. Every experiment was first tested by checking whether g2 was of the form g2 (T) = 1 + c . e-2rT, where r = Dq2 and q, the momentum transfer, is q = (47r~&,) sin (012). D is the diffusion constant, c the concentration of macrocomponent, T is the correlation time, and 6 the scattering angle. This check was done by performing a momentum expansion. Essentially 6 In (gs-I)/& is calculated numerically and fitted with a straight line ; when the slope of this fit is smaller than its standard deviation and therefore compatible with zero, the experiment was accepted. Then all weighted least-squares fits involving 5,6 . . . 24 data-points were performed and the three fits with smallest variance were retained. If these three best fits involved not less than 18 data-points, I’ and D were calculated. Figure 5 shows a plot of r verse q2, including errors. The value at each angle is obtained as an average of three experiments. The slope was calculated with a weighted least-squares fit. The result is: D = 3.32 f 0.03 x 10-l cm2 s-l at t = 2O.O”C f 0.05 deg. C and 7 = l-0049 centipoise. D can clearly be taken as the reduced standard diffusion constant D!&+,, i.e. the value reduced to the viscosity of water. Note that D is independent of the concentration of hexons. This was checked separately for four concentrations (Fig. 6). The Stokes’ radius for the hexon is r = KT/6rD7, where T is the absolute temperature and r) the viscosity of the buffer. Its value is 64 f 0.6 A. The molecular weight was calculated using the relation (Svedberg & Pedersen,

174

M. GROTTER

AND

R. M. FRANKLIN

20/ /

15./ -i 2? 5

/

IO-

2

/ /

5i/I /’ OK// 0

,=’ I IO

I 30

I 20

I 40

I 50

I 6C

q2 x 10mg (cmez)

FIG. 6. Plot of r versus pa at different angles of observation. diffusion constant, D = 3*34x 1O-7 cma s-l.

The slope of the fitted curve is the

3.4 ‘w Ng 3.3 -

T .

.

“0 ; 3.2 :: Q 3.1 3.00

I I

I 2

I 3

Concn (mg/ml)

FIG. 6. Diffusion

constant

as a function

of concentration.

1940) M = RT s!&,~/D;,,~ (1 - Cp) where R is the gas constant, T the absolute temperature, p the density of water, and d the partial specific volume. The value is given in Table 4. (g) .Moleculur weight from crystal

density measurement

The density of cross-linked crystals in water was 1.185 f 0.01 g/ml and of native crystals in 0.5 M-citrate buffer (pH 3.2) was 1.234 f 0.01 g/ml. The density of the O-5 M-citrate buffer (pH 3.2) was 1.054 g/ml. Applying the formula M =

(PC -

PA NV

n(1 - Gp,)

’

ignoring the bound water (cf. Matthews, 1974), where 2M is the molecular weight, pc is the crystal density, pS is the density of the solvent, N is Avogadro’s number, V is the volume of the unit cell = 3.368 x lo- l8 cm3 and fi is the partial specific volume of the protein = 0.738 cm3 g-l, we calculated molecular weights of 413,000 for the native crystals and 362,006 for the cross-linked crystals. Different values

MOLECULAR

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175

for the crystal densities were obtained by using different methods of transferring the crystal from its mother liquor to the bromobenzene/xylene mixtures. The differences arise from the varying amounts of water present around the crystal and explain the observed error of about 8 to 10% in molecular weight (Table 4). (h) Polyacrylamide gel electrophoresis These electrophoresis experiments gave a molecular weight of 120,000 to 125,000, for both reduced hexons and for reduced and alkylated (carboxymethylated) hexons. Thus we were not able to confirm earlier reports (Pettersson, 1970) that a subunit of molecular weight 60,060 could be demonstrated after reduction and alkylation. Pronase, however, partially digested the hexon. In the polyacrylamide gel pattern, peaks were obtained at molecular weights 120,000, at about 20,000 and a main peak at 60,000. Thus the polypeptide of molecular weight 60,000 may be a product of limited proteolysis. Chymotrypsin did not have any influence on the hexon. (i) Titration

experiments with organomercurial 5,5’-dithiobis

(2-nitrobenzoic

compounds am?

acid)

2-chloromercuri-4-nitrophenol and 2-chloromercuri-4,6-dinitrophenol undergo large spectral changes when thiols displace a more weakly bound ligand from the mercury

FOO0 c

(b)

FIG. 7. (a) Titration curve of hexon with 2-ohloromercuri-4-nitrophenol (addition of reagent in 2.~1 steps). 0.63 mg hexon corresponding to 6.27 nmol of 120,000 subunit reacted with 31.68 nmol mercury compound (concentration 1.98 mxa in stock solution). 6.0 & 0.2 mercury molecules were bound per 120,000 subunit. (b) Titration curve of hexon with 2-chloromercuri-4.6~dinitrophenol (addition of reagent in 4.~1 steps). 0.66 mg hexon corresponding to 6.6 nmol of 120,000 subunit reacted with 22.4 nmol mercury compound (concentration 1.40 mM in stock solution). 4.07 f 0.1 mercury moleeulen were bound per 120,000 subunit.

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atom (McMurray & Trentham, 1969). Figure 7 (a) and (b) shows difference titration curves. At the titration end-point six molecules of 2-chloromercuri-4nitrophenol and four molecules of 2-chloromercuri-4,6-dinitrophenol had reacted per molecular weight of 120,000. In titration experiments without sodium dodecyl sulphate, 0.135 mg hexon in 0.53 ml reacted with an excess of 5,5’-dithiobis (2 nitrobenzoic acid). The absorption measured at 412 nm in a l-cm cuvette was O-0285 O.D. In titration experiments with dodecyl sulphate 0.135 mg hexon in 0.59 ml reacted with an excess of 5,5’-dithiobis (2-nitrobenzoic acid). The absorbance 5 hours after the addition of dodecyl sulphate, at the end of the reaction, WILSO-154 O.D. The concentration of the thionitrobenzoate ion calculated with E = 1.36 x lo4 mol- l cm-l was 111 pM without dodecyl sulphate and 663 pM with dodecyl sulphate. The concentration of the hexon subunit calculated with 120,000 molecular weight was 113 PM in both experiments. Only one SH-group per 120,000 reacted when protein was in the native state, and six SH-groups per 120,000 reacted after dodecyl sulphate treatment of the protein.

4. Discussion The present work attempts to resolve the current controversy concerning the molecular weight of the hexon and its subunit. In earlier determinations of the molecular weight of hexon a large source of error arose from values assumed for the partial specific volume. A change in the procedure for purifying the protein has given a yield ten times higher than in Pettersson et al. (1967) and five times higher than in Boulanger t Puvion (1973). The higher yield together with the smaller quantity required for density measurements by the mechanical oscillator technique (Kratky et al., 1973), has enabled us to determine the partial specific volume directly. Since the value of B has been determined, it is no longer necessary to give a range of values for the molecular weight. The quantity of protein available also allowed us to make nitrogen and carbon analyses which, with the amino acid composition (Pettersson et al., 1967), gave the concentrations and hence the extinction coetlicient. We found good agreement among the molecular weight determinations using light scattering (355,000), equilibrium sedimentation (355,000), and sedimentation and diffusion (363,500). The molecular weight of 355,000 obtained by Frankhn et al. (1971a) from sedimentation equilibrium recalculated with fi = 0.738 agrees well with our redetermined values, whereas the value from diffusion constant and sedimentation coefficient measurements in Franklin et al. (1971a) is lower (334,000). The value of the diffusion constant calculated from the Stokes’ radius determined by quantitative gel filtration on 6% agarose in Franklin et al. (1971a) differs only 7% from that given here using fluctuation spectroscopy. Gel filtration is therefore a simple and accurate method to obtain an estimation of the diffusion constant. Prom density measurements on crystals, molecular weights of 362,000 from glutaraldehyde cross-linked crystals and of 413,000 from native crystals were obtained. Using the same method Cornick et al. (1973) obtained a value of 310,000 from cross-linked crystals and Franklin et al. (1971a) 356,000 from native crystals, both values calculated using our newly determined fi = O-738. The spread of these values illustrates the limits of this method for molecular weight determination. We prefer to place reliance upon our other physico-chemical methods and to limit our

MOLECULAR

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OF THE

HEXON

157

use of the crystal density determinations to a calculation of the number of asymmetric units per unit cell. Indeed crystal density values for molecular weight determination must be used with extreme caution, particularly in cases where the amount of bound solvent is not known and also when glutaraldehyde-fixed crystals are employed (Matthews, 1974). We calculated the hydration as described in Franklin d al. (1971a) to be 0.98 g water per g protein, which is rather high (cf. Tanford, 1961). From the hexon radms of gyration of 47 A (Jensen et al., 1972), the radius of the dry molecule-assuming a sphere-would be about 60.5 A, whereas the Stokes’ radius (64 f O-6 8) is the radius of the hydrated sphere. The difference in the radii of the hydrated and dry molecules is 4 A, corresponding to more than one layer of water (0.3 g/g; Perutz, 1946) around the molecule, and is consistent with the high value of 0.98 g/g. The subunit size of the hexon has been determined by polyacrylamide gel electrophoresis (Maize1 et al., 1968a,b; Pettersson, 1970) and by equilibrium sedimentation in guanidine hydrochloride (Horwitz et al., 1970). In all cases a molecular weight of 120,000 was found. Pettersson (1970) found, however, from sodium dodecyl sulphate gel electrophoresis of reduced and alkylated hexon a molecular weight of 60,000. Our results, using the same techniques, gave a molecular weight of 120,000. In an attempt to explain Pettersson’s (1970) results we treated the alkylated and reduced hexon with Pronase and found a main band of 60,000, whereas for untreated reduced and alkylated hexon there was only one band at 120,000. Contamination of some hexon samples with proteolytic enzymes may have produced the polypeptide of molecular weight 60,000 as an artifact. Interpretation of 5,5’-dithiobis (2-nitrobenzoic acid) titration experiments, using the polypeptide molecular weight of 120,000 obtained from gel electrophoresis, indicated one binding site per 120,000 or three per 360,000. Further titration experiments with organomercurial compounds were interpreted in terms of integral numbers of bound reagent only when the above molecular weight of 120,000 was assumed. Thus although these titrations do not allow us to determine the total number of sulfhydryl groups, they do provide a further confirmation of our proposed hexon molecular weight. At the same time the titrations showed that mercurial reagents do bind to the hexon, as has also been demonstrated by JGrnvall and co-workers (1974u). Precession photographs (100) of hexon crystals from two of several soaking experiments in organic mercurial reagents show significant intensity differences from those of native crystals and these heavy metal derivatives are now being actively invest,igated as part of our three-dimensional structure analysis of the hexon (M. Griitter, R. M. Franklin & R. Burnett, work in progress). We conclude from our investigations that the hexon has a molecular weight of 360,000 and that it is composed of three identical subunits of 120,000 molecular weight,.

We acknowledge with thanks the help of Dr M. Zulauf and Mr R. Marcoli in fluctuation spectroscopy and light scattering, of Mr A. Lustig, who performed the ultracentri&gation experiments and of Dr H. Wagner from Ciba-Geigy in N and C determination. We thank Mrs Ulls Lips for excellent technical assistance. Professor L. Philipson provided hexon in the early stages of the work and we would like to thank both him and Dr J. Rosenbusch for helpful discussions. This work is part of the doctoral thesis of one of the authors (M. G.). 12

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REFERENCES Acharya, S. A. & Moore, P. B. (1973). J. Mol. Biol. 76, 207-221. Boulanger, P. A. & Puvion, F. (1973). Eur. J. Biochem. 39, 37-42. Cornick, G., Sigler, P. B. & Ginsberg, H. S. (1971). J. Mol. Biol. 57, 397-401. Cornick, G., Sigler, P. B. & Ginsberg, H. S. (1973). J. Mol. Biol. 73, 533-537. Coumou, D. J. (1960). J. CoZZoidSci. 15, 408-417. Crestfield, A. M., Moore, S. & Stein, W. H. (1963). J. BioZ. C&m. 238, 622-627. Day, L. A., Franklin, R. M., Petterson, U. & Philipson, L. (1972). Eur. J. Biochem. 29, 537-541. Dohner, L. & Hudemann, H. (1972). Archivfiir die gesamte Virwforechung, 38, 279-289. Duysens, L. N. M. (1956). Biochim. Biophya. Actu, 19, 1-12. Eagle, H. (1959). Science, 130, 432-437. Ellman, G. L. (1959). Arch. Biochem. Biophys. 82, 70-77. Franklin, R. M., Pettersson, U., Akervall, K., Strandberg, B. & Philipson, L. (1971a). J. Mol. BioZ. 57, 383-395. Franklin, R. M., Harrison, S. C., Pettersson, U., Philipson, L., Brand&, C. I. & Werner, P. E. (1971b). Cold Spring Harbor Symp. Quant. BioZ. 36, 503-510. Henry, C. M. (1972). Ph.D. Thesis, University of Pittsburgh. Horwitz, M. S., Maizel, J. V., Jr & Scharff, M. D. (1970). J. Viral. 6, 569-571. Jensen, B. T., Furugren, B., Lindquist, I. and Philipson, L. (1972). Monatsheftefiir Chenaie, 103, 1730-1736. Jornvall, H., Pettersson, U. & Philipson, L. (1974u) Eur. J. Biochem. in the press. JGrnvall, H., Ohlsson, H. & Philipson, L. (19748). Biochem. Biophys. Re8. Commun. 56, 304-310. Kratky, O., Leopold, H. & Stabinger, H. (1973). Methods in EnzymoZogy, 27, g&110. Macintyre, W. M., Pereira, H. G. & Russell, W. C. (1969). Nature (London), 222,1165-1166. Maize& J. V., Jr, White, D. 0. & Scharff, M. D. (196&z). ‘virology, 36, 115-125. Maizel, J. V., Jr, White, D. 0. & Scharff, M. D. (1968b). Virology, 36, 126-136. Matthews, B. W. (1974). J. Mol. BioZ. 82, 513-526. McMurray, C. H. & Trentham, D. R. (1969). Biochem. J. 115, 913-921. Pereira, H. G., Valentine, R. C. & Russell, W. C. (1968). Nature (London), 219, 946-947. Perutz, M. F. (1946). Trans. Faraday Sot. (Ser. B) 42, 187-197. Pettersson, U. (1970). Ph.D.Thesis, University of Uppsala. Pettersson, U., Philipson, L. C%Hhglund, S. (1967). Virology, 33, 575-590. Pusey, P. N., Koppel, D. E., Schafer, D. W., Camerini-Otero, R. D. & Koenig, S. H. (1974). Biochemistry, 13, 952-960. Schachman, H. K. (1959). Ultracentrijugation in Biochemidry, Academic Press, New York. Svedberg, T. & Pedersen, K. 0. (1940). The UZtracentri,fuge, Oxford University Press, New York and London. Tanford, C. (1961). The Physical Chemistry of Macromolecules, J. Wiley & Sons, New York and London. Timasheff, S. N. (1970). Handbook of Biochemistry (Sober, H. A., ed.), p. C-67, The Chemical Rubber Co., Cleveland. Valentine, R. C. & Pereira, H. G. (1965). J. Mol. BioZ. 13, 13-20. Weber, K. & Osborn, M. (1969). J. BioZ. Chem. 244,4406-4412.