ANALYTICAL
BIOCHEMISTRY
81, 447-449
SHORT Simple
(1977)
COMMUNICATIONS
Determination of Virus Molecular Sedimentation Equilibrium
Weights
by
Sedimentation equilibrium in the analytical ultracentrifuge is a straightforward method of measuring molecular weights. However, for viruses, such low speeds are required that rotor vibration and precession become serious practical problems. Also, partial specific volumes are difficult to determine. Here, we describe two simple techniques to help resolve these problems. Rotor precession can be recognized by a characteristic distortion of Rayleigh interference fringes (1). To correct this, we made a simple lower bearing to steady the axis of rotation in a Beckman Spinco Model E, as shown in Fig. 1. The mercury cup assumbly was replaced by a rubber stopper holding a piece of polytetrafluoroethylene (FTFE), into which a small hole had been drilled to receive the needle in the bottom of the rotor. With the aid of modeling clay, the bearing was correctly positioned to take an imprint of the contact model when the chamber was slowly closed. The RTIC temperature control unit was not used, and the rotor, therefore, came to thermal equilibrium with the chamber walls, which reduced thermal convection. The refrigeration unit was set at a suitable temperature, and the rotor temperature was measured directly after the run. The partial specific volume of a virus available only in tiny amounts cannot be measured by pycnometry or “calculated” from amino acid and nucleic acid compositions. It can be determined from the variation with solvent density, in H,O/D,O mixtures, of sedimentation equilibrium distributions (2) or sedimentation velocities (3). with corrections for hydrogen exchange and viscosity. However, we lacked the wedge-centerpiece cells for interference optics or the multiplexer for ultraviolet absorption optics. necessary to examine several samples spinning together in the same rotor. Therefore, we used two single-sector cells with windows partially blacked out by a felt-tipped marker, as shown in Fig. 2, to observe two samples simultaneously. One other comment may be useful to the nonspecialist. With the advent of small programmable calculators, most of the published methods for converting Rayleigh interference fringe numbers into absolute concen447 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction m any form reserved.
ISSN 0003-2697
448
SHORT
Bottom
COMMUNICATIONS
of
chamber
FIG. 1. Sectional drawing of the anti-precession cup thermistor contact in a Model E ultracentrifuge.
bearing
which
replaces
the mercury
trations are no longer useful. For reasonably homogeneous proteins, we recommend guessing the meniscus concentration and then refining it by trial and error until the curve of apparent molecular weight versus concentration is least curved at the meniscus. However, for most viruses, even at the lowest rotational speed, (800 rpm), the virus concentration at the meniscus falls very near zero, which solves the problem directly. By using the approach described here, in conjunction with a heavy AN-J rotor, we determined the partial specific volume and molecular weight values for a cytoplasmic polyhedrosis virus from Heliothis armigera (4), using less than 0.5 mg of virus, which was recovered undamaged afterward, as judged by electron microscope examination. Its partial specific volume was 0.69, as calculated from the ratio of two sedimentation coefficients in 0 and 75% D,O at pH 7.0, 20°C. and a rotor speed of 12,000 rpm. Its molecular weight, derived from this value and
FIG. images
2. Partial blacking out of two single-sector cells to obtain simultaneous using ultraviolet absorption optics. One cell was only half-filled.
half-cell
SHORT
449
COMMUNICATIONS
‘I O-l 0 C -
-2
1
-3
-4
-5 49
50
51
52
r2tcm2)
FIG. 3. A plot of In (concentration) versus radius2 for a virus equilibrium centrifugation. Three separate interference fringes on the photographic plate were measured. Conditions of the centrifugation were: speed. 800 rpm: temperature. s”C; buffer, 0.03 M sodium phosphate. 0.1 M KCI. pH 7.0: sample volume, 0.4 ml: cell path length. I2 mm; length of run. 3 days.
the sedimentation equilibrium data shown in Fig. 3, was daltons. Our precision was probably limited to about ~20% heterogeneity of the virus. However, we estimate an inherent of perhaps 2 10% obtainable with the type of ultracentrifuge in many biological laboratories.
47.2 x lo6 by slight precision available
ACKNOWLEDGMENT We are most grateful
to Professor
Marc
Van Regenmortel
for his advice.
REFERENCES I. 2. 3. 4.
Durham, A. C. H. (1972) .I. Mol. Biol. 67, 289-305. Edelstein, S. J.. and Schachman, H. K. (1973) Adl,trn. Enz.yno/. 27, 82-98. Bellamy, A. P., Gillies. S. C.. and Harvey. J. D. (1974) J. Viral. 14, 1388-1393 Rubinstein, R.. Stannard. L.. and Poison, A. (1975) Prep. Biochem. 5, 79-90. RNA RUBINSTEIN* ANTHONY C. H.
* Virus Research Unit. Uni\,ersity of‘ Cape Tobvn Medical School t Microbiology Department. lJni\,ersity of Cape Tow,n Rondebosch. Receitved August 23. 1976; accepted April 14. 1977
’ Present Descartes.
address: Institut 67084 Strasbourg.
de Biologie France.
Moltculaire
et Cellulaire
DURHAM?.’
Obsen~ato~, 7925. 7700 South Africa
du C.N.R.S..
IS rue
BIOCHEMISTRY
81, 447-449
SHORT Simple
(1977)
COMMUNICATIONS
Determination of Virus Molecular Sedimentation Equilibrium
Weights
by
Sedimentation equilibrium in the analytical ultracentrifuge is a straightforward method of measuring molecular weights. However, for viruses, such low speeds are required that rotor vibration and precession become serious practical problems. Also, partial specific volumes are difficult to determine. Here, we describe two simple techniques to help resolve these problems. Rotor precession can be recognized by a characteristic distortion of Rayleigh interference fringes (1). To correct this, we made a simple lower bearing to steady the axis of rotation in a Beckman Spinco Model E, as shown in Fig. 1. The mercury cup assumbly was replaced by a rubber stopper holding a piece of polytetrafluoroethylene (FTFE), into which a small hole had been drilled to receive the needle in the bottom of the rotor. With the aid of modeling clay, the bearing was correctly positioned to take an imprint of the contact model when the chamber was slowly closed. The RTIC temperature control unit was not used, and the rotor, therefore, came to thermal equilibrium with the chamber walls, which reduced thermal convection. The refrigeration unit was set at a suitable temperature, and the rotor temperature was measured directly after the run. The partial specific volume of a virus available only in tiny amounts cannot be measured by pycnometry or “calculated” from amino acid and nucleic acid compositions. It can be determined from the variation with solvent density, in H,O/D,O mixtures, of sedimentation equilibrium distributions (2) or sedimentation velocities (3). with corrections for hydrogen exchange and viscosity. However, we lacked the wedge-centerpiece cells for interference optics or the multiplexer for ultraviolet absorption optics. necessary to examine several samples spinning together in the same rotor. Therefore, we used two single-sector cells with windows partially blacked out by a felt-tipped marker, as shown in Fig. 2, to observe two samples simultaneously. One other comment may be useful to the nonspecialist. With the advent of small programmable calculators, most of the published methods for converting Rayleigh interference fringe numbers into absolute concen447 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction m any form reserved.
ISSN 0003-2697
448
SHORT
Bottom
COMMUNICATIONS
of
chamber
FIG. 1. Sectional drawing of the anti-precession cup thermistor contact in a Model E ultracentrifuge.
bearing
which
replaces
the mercury
trations are no longer useful. For reasonably homogeneous proteins, we recommend guessing the meniscus concentration and then refining it by trial and error until the curve of apparent molecular weight versus concentration is least curved at the meniscus. However, for most viruses, even at the lowest rotational speed, (800 rpm), the virus concentration at the meniscus falls very near zero, which solves the problem directly. By using the approach described here, in conjunction with a heavy AN-J rotor, we determined the partial specific volume and molecular weight values for a cytoplasmic polyhedrosis virus from Heliothis armigera (4), using less than 0.5 mg of virus, which was recovered undamaged afterward, as judged by electron microscope examination. Its partial specific volume was 0.69, as calculated from the ratio of two sedimentation coefficients in 0 and 75% D,O at pH 7.0, 20°C. and a rotor speed of 12,000 rpm. Its molecular weight, derived from this value and
FIG. images
2. Partial blacking out of two single-sector cells to obtain simultaneous using ultraviolet absorption optics. One cell was only half-filled.
half-cell
SHORT
449
COMMUNICATIONS
‘I O-l 0 C -
-2
1
-3
-4
-5 49
50
51
52
r2tcm2)
FIG. 3. A plot of In (concentration) versus radius2 for a virus equilibrium centrifugation. Three separate interference fringes on the photographic plate were measured. Conditions of the centrifugation were: speed. 800 rpm: temperature. s”C; buffer, 0.03 M sodium phosphate. 0.1 M KCI. pH 7.0: sample volume, 0.4 ml: cell path length. I2 mm; length of run. 3 days.
the sedimentation equilibrium data shown in Fig. 3, was daltons. Our precision was probably limited to about ~20% heterogeneity of the virus. However, we estimate an inherent of perhaps 2 10% obtainable with the type of ultracentrifuge in many biological laboratories.
47.2 x lo6 by slight precision available
ACKNOWLEDGMENT We are most grateful
to Professor
Marc
Van Regenmortel
for his advice.
REFERENCES I. 2. 3. 4.
Durham, A. C. H. (1972) .I. Mol. Biol. 67, 289-305. Edelstein, S. J.. and Schachman, H. K. (1973) Adl,trn. Enz.yno/. 27, 82-98. Bellamy, A. P., Gillies. S. C.. and Harvey. J. D. (1974) J. Viral. 14, 1388-1393 Rubinstein, R.. Stannard. L.. and Poison, A. (1975) Prep. Biochem. 5, 79-90. RNA RUBINSTEIN* ANTHONY C. H.
* Virus Research Unit. Uni\,ersity of‘ Cape Tobvn Medical School t Microbiology Department. lJni\,ersity of Cape Tow,n Rondebosch. Receitved August 23. 1976; accepted April 14. 1977
’ Present Descartes.
address: Institut 67084 Strasbourg.
de Biologie France.
Moltculaire
et Cellulaire
DURHAM?.’
Obsen~ato~, 7925. 7700 South Africa
du C.N.R.S..
IS rue
