Optical Properties of the Hexon of Adenovirus
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Eur. J. Biochem. 29,537-541 (1972)
Optical Properties of the Hexon of Adenovirus
Loren A. DAY, Richard M. FRANKLIN, Ulf PETTERSSON, and Lennart PHILIPSON The Public Health Research Institute of the City of New York,
and Microbiologiska Institutionen, Wallenberglaboratoriet, Uppsala Universitet
(Received June 14, 1972)
The ultraviolet absorbance, circular dichroism (CD) and optical rotatory dispersion of the hexons of adenovirus type 2 have been measured and analyzed in terms of secondary structural features. A curve-fitting method for the CD spectra from 190 nm to 240 nm indicates 17O/, a-helical, 2601, t9, and 5601, random conformations ; the classical parameter b, indicates from 15 to 20/, a-helical conformation. The far ultraviolet absorbance indicates considerable hypochromicity in comparison to the absorbance expected from the chromophore composition of the protein, which indicates very high a-helical content. We have applied corrections for absorbance flattening in the analysis of all the spectra and demonstrate the relatively large magnitude of such corrections in the regions of intense absorbance for this protein complex of molecular weight around 400000. In spite of such corrections, the discrepancy in apparent a-helical contents indicated by ultraviolet hypochromism and by optical rotatory methods persists.
This study was undertaken to obtain additional structural information on the hexon of adenovirus type 2, an icosahedral animal virus cootaining DNA. The hexons form the bulk of the outer capsid and are located a t the local six-fold symmetry axes. The hexon ha0 been and is being studied by X-ray crystallography and electron microscopy [I -51. In this study we have used ultraviolet absorbance, circular dichroism (CD) and optical rotatory dis- persion (ORD). Since these methods are still under- going testing and development, it is important to apply them to proteins, the structures of which are known or will eventually be known. In the analysis we have applied correction factors for the absorbance flattening [6-81 of the spectra.
MATERIALS AND METHODS Preparation and Handling of Hexon Protein
Hexons were obtained from infected cells accord- ing to the method of Pettersson et al. [l] with the modifications described by Franklin et al. . The samples used for the present study had been stored at 4 C in 0.01 M Tris-acetate, O.Olo/o sodium azide, pH 8. The samples were dialyzed exhaustively against 0.01 M sodium phosphate buffer pH 7.0 for measure- ments in the dispersed state. In this buffer, the hexons are present as monomeric capsomers of molecular weight 320000 to 360000 .
Abbreviations. CD, circular dichroism; ORD, optical rotatory dispersion.
Instrumental Methods The absorption coefficient for hexons in 0.01 M
sodium phosphate, pH 7 was obtained by deter- mining the refractive increment at room temperature of a hexon solution which had been dialyzed for two days, the last 6 h of which were at room temper- ature. A Brice-Phoenix differential refractometer, calibrated against standard KC1 solutions, was used. From the measured refractive increment, the pro- tein concentration was obtained with an assumed value of dn/dc at 546 nm of 0.185 & 0.005 ml/g (cf. compilation by Timasheff ). The protein con- centration so determined was considered accurate
Absorbance measurements were made with a Cary 14 recording spectrophotometer. Wavelength calibration in the visible and near ultraviolet regions was carried out with a mercury spectral line source. Oxygen band spectra were used for wave- length calibration below 200 nm. For absorbance measurements in the low ultraviolet region, special care was taken in the cleaning and handling of the cuvettes and solutions. Absorbance amplitudes of test samples recorded by our Cary 14 were the same as those given by a Zeiss PMQII single-beam spectrophotometer at 260 nm and at 365 nm, within the legibility of the amplitude scales of the two instruments. To ascertain that stray-light levels were within acceptable limits for measurements in the low ultraviolet range with our Cary 14, we placed a wire mesh screen which had a measured trans-
538 Optical Properties of the Hexon of Adenovirus Em. J. Biochem.
mittance of very nearly loo/, (absorbance = 1) throughout the range from 210 nm down to 185 nm in the reference beam of the instrument and a 1-cm cuvette containing isopropanol in the sample beam, and then recorded the apparent absorbance of the isopropanol down to 185 nm. The slide wire registered its maximum reading of 2 absorbance units through- out the range, hence the stray light was considered to be less than 0.lo/,. The importance of this type of test and other aspects of such measurements has been discussed by Gratzer [lo]. The slit widths used for recording the spectra of the samples did not exceed those used in the calibration procedure. Base lines were obtained before and after the absorbance scans.
Circular dichroism and optical rotatory disper- sion measurements were done with Cary 14 and Cmy 60 instruments. Wavelengths were calibrated with the mercury spectral line source and were known to within the legibility of the wavelength indicators, approximately 0.2 or 0.3 nm. National Bureau of Standards sucrose was used as the rotation standard, and based on this and on the calibration data of Cassim and Yang [ll] for d-camphor-sulfonic acid, the correct ellipticity amplitudes relative to our rotation amplitudes were established to within 2O/,. Optical cells of 1-mm and 10-mm path length were used and the total absorbance of the measured samples, including buffer absorbance, did not ex- ceed 1.2 units.
Computational Methods Corrections for light-scattering contributions
to the near ultraviolet absorption spectrum were obtained as usual by plotting the log absorbance us log wavelength in the nonabsorbing range from 400 nm down to 320 nm and making a linear extra- polation into the absorbing range. The extrapolations were extended into the range below 200nm, and the apparent light-scattering contributions indicated were less than 2O/, of the maximum low ultraviolet absorbance amplitudes measured. However, no corrections in this range were applied.
Corrections for flattening of spectral amplitudes were applied for the hexon in the 0.01 M phosphate buffer . The observed amplitudes are related to the corrected amplitudes as (obs) = QAA;')~ (corrected), [Fl(obs) = QB [PI (corrected) and [e](obs) = Q B [ ~ ] (corrected). The correction factors QA and QB, for absorbance and optical activity, respectively, were computed for a cylinder of radius 4.4nm and height 11 nm, the size and shape of the hexon as determined by low-angle X-ray scattering (Lind- quist, I., personal communication) according to the method of Day and Hoppensteadt , which is a direct extension of the methods of Duysens  and or Gordon and Holzwarth  to the case of randomly
oriented cylinders. The values of QA and QB calcu- lated for the hexon as a function of absorbance A?)m were found to fit the empirical relations QA = exp (- 1.27 x lo-* A:@$m) QB = exp (- 2.57 x Aiabm). Very nearly the same relations would have been obtained from the results of Duy- sens  and Gordon and Holzwarth  had we approximated this particular cylinder as a sphere of equivalent volume . Absorption coefficients per unit length of path within the material of the protein necessary for the computation  were obtained as y = In ( Io/I) per nanometer = 2.303 x where the partial specific volume B = 0.727 . No corrections have been applied for various distortions to the spectra introduced by light scattering [18,19], but their possible effects will be discussed below.
Transformation of the observed and corrected circular dichroism to optical rotatory dispersion was carried out according to the Kronig-Kramers relationship 
[p1 (A)] = $ J [ O (A')] [A'/(A2 - X2)] d L' by approximating the integral as a sum over the CD spectrum a t intervals of A' of 0.5nm. The summation was done by computer.
RESULTS Absorbance Xpectrum of Adenovirus Hexom
The complete absorbance spectrum in 0.01 M sodium phosphate pH 7 is given in Fig.l. The ab- sorption coefficient corrected for light scattering as described above was found to be 279nm = 14.6 & 0.6. From the amino acid composition, the absorption coefficients for N-acetyl methyl esters of tyrosine, tryptophan and phenylanine compiled by Gratzer , and the data of Yanari and Bovey  for spectral shifts of these chromophores in detergent micelles relative to H,O, we compute values of AFPm = 14.8 and 14.3 a t 280 nm for model spectra in detergents and water, respectively, in agreement with the measured value. The measured spectrum clearly shows a shoulder a t about 291 nm characteristic of tryptophan, and appears red- shifted relative to the calculated one, probably the result of localization of the chromophores in hydrophobic regions of the hexon.
An analysis of the far ultraviolet absorbance data according to the method of Rosenheck and Doty  is presented in Table 1. The corrections for side-chain contributions to low ultraviolet absorbance were taken from the data of McDiar- mid . The unusual result is the extreme apparent hypochromicity calculated for the peptide chromo- phores of the protein backbone, even after the experimental absorbance has been increased by
Vo1.29, No.3, 1972 L. A. DAY, R. M. FRANKLIN, U. PETTERSSON, and L. PHILIPSON 539
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Table 1. Analysis of the far-ultraviolet-absorbance spectrzcm of hexons in 0.01 M sodium phosphate pH 7.0
190nm 197nm 205nm
Measured A:'~m QA
Corrected Atopm &(to