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Preparation of protein subunits from alfalfa mosaic virus under mild conditions
236
SHORT COMMUNICATIONS
Preparation of protein subunits from alfalfa mosaic virus under mild conditions It has been shown in several instances that viruses can be degraded into nucleic acid and protein. This process has been studied most extensively in the case of tobacco mosaic virus where degradation at high pH, low pH, high temperature, or with detergents yields protein in the form of uniform particles x (i.e., subunits) of molecular weight about I7 ooo. Protein subunits have also been obtained from turnip yellow mosaic virus ~, tomato bushy stunt virus s and wild cucumber mosaic virus 4 and from the'bacterial viruses T2 (ref. 5) and 9XI74 (ref. 6). It is the purpose of this note to describe experiments which show that alfalfa mosaic virus can be degraded under conditions considerably milder than those mentioned above to yield protein subunits and nucleic acid. It will be shown that alfalfa mosaic virus is degraded in i M NaC1 at neutral pH. Alfalfa mosaic virus is a small plant virus whose infectivity is lost rapidly except at low ionic strength and in a narrow pH range ~. FRISCH-NIGGEMEYER AND STEERE8 have reported that optimum storage conditions are o.oo5 M phosphate buffer at pH 8. It seems clear from our present experiments that the loss in infectivity is due to loss of structural integrity of the virus. Alfalfa mosaic virus was prepared according to the method of BANCROYr AND KaESBERG9. Such preparations contain, in addition to the virus, the several viruslike nucleoprotein particles described by these authors. The protein portions of the virus and the virus-like particles are believed to be identical in composition. 5 ml of a solution of the alfalfa mosaic virus preparation at a concentration of 26.2 mg/ml in o.oi M phosphate buffer at pH 7 were combined with an equal volume of 2 M NaC1 and the resultant mixture was kept at 45 ° for 20 rain. During this time the solution became turbid. It was cooled and then immediately centrifuged at low speed. The supernatant solution contained most of the nucleic acid and/or its degradation products as judged by ultraviolet absorption measurements. Almost all the ultraviolet absorbancy at 26o m# of the original preparation remained in the supernatant solution and also the shape of the ultraviolet absorption curve was identical with that of pure RNA of alfalfa mosaic virus. The pellet contained the protein. It was washed with I M NaC1 and was centrifuged again. The resulting pellet was dissolved in o.oI M phosphate buffer (pH 7), containing 0.o05 M sodium dodecyl sulfate. The solution was dialyzed for 24 h against distilled water and then a precipitate was formed by the addition of 0.66 vol. of sat. ammonium sulfate solution. The precipitate was resuspended in phosphate buffer containing 0.05 M dodecyl sulfate and then dialyzed for 24 h against phosphate buffer containing 0.oo 5 M dodecyl sulfate When the dodecyl sulfate is omitted from the above buffers, the protein solutions are not stable. At first the solutions are clear, but after several days in the cold, turbidity develops and precipitation occurs. The protein obtained by means of the above procedure exhibits a single component in the analytical centrifuge with a sedimentation coefficient of 3 S. When the protein is prepared in the absence of sodium dodecyl sulfate there occurs in addition material which sediments slightly more rapidly.There is no evidence whatever of undegraded virus. The ultraviolet spectrum is typical of that expected for a protein. Its absorption maximum is at 279 m/~ and the ratio of the absorbancy at 260 m/~ to Biochim. Biophys. Acta, 55 (~96z) 236-237
SHORT COMMUNICATIONS
237
that at 2:80 m/~ is 0.69. The low value of the 260/280 ratio shows that the preparation is substantially free of RNA. (The 26o/28o ratio for pure alfalfa mosaic virus RNA was determined to be 2.06. The 26o/28o ratio for pure alfalfa mosaic virus protein was calculated from its tyrosine, tryptophan and phenylalanine content to be about 0.60. The absorbancy of alfalfa mosaic virus RNA at 260 m# is about 4° times that of alfalfa mosaic virus protein. From these data it is readily calculable that the RNA content of the preparation is less than 0.5 %). The molecular weight of the protein has been measured by the approach to equilibrium method described by EHRENBERG1°. The value of the molecular weight obtained shows a small but definite dependence on the time of centrifugation. This indicates that the material is not entirely homogeneous. The molecular weight determination yields 38 ooo early in the experiment; this is the weight-average molecular weight of the preparation. After extensive sedimentation the molecular-weight determination tends toward a value of 34 ooo; this is the molecular weight of the smallest particles in the preparation. The preparation is serologically active against antiserum prepared from undegraded alfalfa mosaic virus indicating that the tertiary structure of the protein ihas not been disrupted extensively. Thus we conclude that low-molecularweight, apparently undenatured protein subunits can be prepared from alfalfa mosaic virus by the use of these mild chemical procedures. Evidently the protein subunits are held together in the virus particles by very weak forces. Even milder procedures induce some breakdown of alfalfa mosaic virus. For example, raising the NaC1 concentration from 0-0. 3 M at room temperature causes substantial turbidity. Analytical centrifugation shows that the amount of undegraded virus has decreased and that there is a slowly moving boundary corresponding to nucleic ~%cidreleased from degraded virus particles.
Department o/ Biochemistry, Univer,~ity o/ Wisconsin, Madison, Wisc. (U.S.A.) t t s t s • T a | xe
JOHN J. KELLEY PAUL KAESBERG
H. G. 't~VITTMANN,Experientia, 15 (1959) 174. j . I. HARRIS AND J. HINDLEY, J. Molec. Biol., 3 (1961) I I 7. R. T. I:[ERSH AND H. K. SCHACHMAN,Virology, 6 (I958) 234. H. YAMAZAKI AND P. KAESBERG Biochim, Biophys. Acta, 53 (1961) 173. H. VAN VUNAKIS, W. BAKER AND R. BROWN, Virology, 5 (1958) 327 • R. L. $,INSHEIMRR, Federation Proc., 2o (1961) 661. A. F. l~oss, Phytopathology, 31 (1941) 394. W. FRISCH-NIGGEMEYER AND R. L. STEERR, Virology, 14 (1961) 83. J. a . BANCROFT AND P. KAESBERG, Biochim. Biophys. Acta, 39 (196o) 519 • A. EHS:ENBERG, Acta Chem. Scand., i i (1957) 1257.
Received May 23rd, 1961
Biochim. Biopkys. Acta, 55 (I962) 236-237
SHORT COMMUNICATIONS
Preparation of protein subunits from alfalfa mosaic virus under mild conditions It has been shown in several instances that viruses can be degraded into nucleic acid and protein. This process has been studied most extensively in the case of tobacco mosaic virus where degradation at high pH, low pH, high temperature, or with detergents yields protein in the form of uniform particles x (i.e., subunits) of molecular weight about I7 ooo. Protein subunits have also been obtained from turnip yellow mosaic virus ~, tomato bushy stunt virus s and wild cucumber mosaic virus 4 and from the'bacterial viruses T2 (ref. 5) and 9XI74 (ref. 6). It is the purpose of this note to describe experiments which show that alfalfa mosaic virus can be degraded under conditions considerably milder than those mentioned above to yield protein subunits and nucleic acid. It will be shown that alfalfa mosaic virus is degraded in i M NaC1 at neutral pH. Alfalfa mosaic virus is a small plant virus whose infectivity is lost rapidly except at low ionic strength and in a narrow pH range ~. FRISCH-NIGGEMEYER AND STEERE8 have reported that optimum storage conditions are o.oo5 M phosphate buffer at pH 8. It seems clear from our present experiments that the loss in infectivity is due to loss of structural integrity of the virus. Alfalfa mosaic virus was prepared according to the method of BANCROYr AND KaESBERG9. Such preparations contain, in addition to the virus, the several viruslike nucleoprotein particles described by these authors. The protein portions of the virus and the virus-like particles are believed to be identical in composition. 5 ml of a solution of the alfalfa mosaic virus preparation at a concentration of 26.2 mg/ml in o.oi M phosphate buffer at pH 7 were combined with an equal volume of 2 M NaC1 and the resultant mixture was kept at 45 ° for 20 rain. During this time the solution became turbid. It was cooled and then immediately centrifuged at low speed. The supernatant solution contained most of the nucleic acid and/or its degradation products as judged by ultraviolet absorption measurements. Almost all the ultraviolet absorbancy at 26o m# of the original preparation remained in the supernatant solution and also the shape of the ultraviolet absorption curve was identical with that of pure RNA of alfalfa mosaic virus. The pellet contained the protein. It was washed with I M NaC1 and was centrifuged again. The resulting pellet was dissolved in o.oI M phosphate buffer (pH 7), containing 0.o05 M sodium dodecyl sulfate. The solution was dialyzed for 24 h against distilled water and then a precipitate was formed by the addition of 0.66 vol. of sat. ammonium sulfate solution. The precipitate was resuspended in phosphate buffer containing 0.05 M dodecyl sulfate and then dialyzed for 24 h against phosphate buffer containing 0.oo 5 M dodecyl sulfate When the dodecyl sulfate is omitted from the above buffers, the protein solutions are not stable. At first the solutions are clear, but after several days in the cold, turbidity develops and precipitation occurs. The protein obtained by means of the above procedure exhibits a single component in the analytical centrifuge with a sedimentation coefficient of 3 S. When the protein is prepared in the absence of sodium dodecyl sulfate there occurs in addition material which sediments slightly more rapidly.There is no evidence whatever of undegraded virus. The ultraviolet spectrum is typical of that expected for a protein. Its absorption maximum is at 279 m/~ and the ratio of the absorbancy at 260 m/~ to Biochim. Biophys. Acta, 55 (~96z) 236-237
SHORT COMMUNICATIONS
237
that at 2:80 m/~ is 0.69. The low value of the 260/280 ratio shows that the preparation is substantially free of RNA. (The 26o/28o ratio for pure alfalfa mosaic virus RNA was determined to be 2.06. The 26o/28o ratio for pure alfalfa mosaic virus protein was calculated from its tyrosine, tryptophan and phenylalanine content to be about 0.60. The absorbancy of alfalfa mosaic virus RNA at 260 m# is about 4° times that of alfalfa mosaic virus protein. From these data it is readily calculable that the RNA content of the preparation is less than 0.5 %). The molecular weight of the protein has been measured by the approach to equilibrium method described by EHRENBERG1°. The value of the molecular weight obtained shows a small but definite dependence on the time of centrifugation. This indicates that the material is not entirely homogeneous. The molecular weight determination yields 38 ooo early in the experiment; this is the weight-average molecular weight of the preparation. After extensive sedimentation the molecular-weight determination tends toward a value of 34 ooo; this is the molecular weight of the smallest particles in the preparation. The preparation is serologically active against antiserum prepared from undegraded alfalfa mosaic virus indicating that the tertiary structure of the protein ihas not been disrupted extensively. Thus we conclude that low-molecularweight, apparently undenatured protein subunits can be prepared from alfalfa mosaic virus by the use of these mild chemical procedures. Evidently the protein subunits are held together in the virus particles by very weak forces. Even milder procedures induce some breakdown of alfalfa mosaic virus. For example, raising the NaC1 concentration from 0-0. 3 M at room temperature causes substantial turbidity. Analytical centrifugation shows that the amount of undegraded virus has decreased and that there is a slowly moving boundary corresponding to nucleic ~%cidreleased from degraded virus particles.
Department o/ Biochemistry, Univer,~ity o/ Wisconsin, Madison, Wisc. (U.S.A.) t t s t s • T a | xe
JOHN J. KELLEY PAUL KAESBERG
H. G. 't~VITTMANN,Experientia, 15 (1959) 174. j . I. HARRIS AND J. HINDLEY, J. Molec. Biol., 3 (1961) I I 7. R. T. I:[ERSH AND H. K. SCHACHMAN,Virology, 6 (I958) 234. H. YAMAZAKI AND P. KAESBERG Biochim, Biophys. Acta, 53 (1961) 173. H. VAN VUNAKIS, W. BAKER AND R. BROWN, Virology, 5 (1958) 327 • R. L. $,INSHEIMRR, Federation Proc., 2o (1961) 661. A. F. l~oss, Phytopathology, 31 (1941) 394. W. FRISCH-NIGGEMEYER AND R. L. STEERR, Virology, 14 (1961) 83. J. a . BANCROFT AND P. KAESBERG, Biochim. Biophys. Acta, 39 (196o) 519 • A. EHS:ENBERG, Acta Chem. Scand., i i (1957) 1257.
Received May 23rd, 1961
Biochim. Biopkys. Acta, 55 (I962) 236-237