OPTICAL PROPERTIES OF SOLUTIONS OF TOBACCO MOSAIC VIRUS PROTEIN' MAX A. LAUFFER The Rockejeller Institute for Medical Research, Princeton, New Jersey Received July 1 , 1038 INTRODUCTION
Tobacco mosaic virus nucleoprotein was first obtained in crystalline form by chemical means by Stanley in 1935 (13), from the juice of Turkish tobacco plants infected with tobacco mosaic virus. All of the evidence available at present indicates that the active disease-causing agent is this nucleoprotein (14). The results of recent studies indicate that the virus nucleic acid closely resembles yeast nucleic acid, that i t is in combination with protein, and that it is necessary for virus activity (1,9). Because the virus protein has an extremely high molecular weight, a value in the millions (3, 7, 20), it is possible to obtain very superior preparations by differential centrifugation (20). The protein is insoluble at its isoelectric point and in 20 per cent ammonium sulfate solution. It crystallizes in small needle-shaped crystals, visible only with the microscope. Bernal and Fankuchen have studied the x-ray diffraction patterns of the virus protein in many states, and they concluded from their findings that these crystals are in the mesomorphic or paracrystalline state, rather than in the true crystalline state (2). These crystals are, nevertheless, definite solid objects, visible with the microscope, and, inasmuch as crystallinity of any kind is not an infallible criterion of protein purity, the crystallinity of the tobacco mosaic virus protein is probably not much less significant as a criterion of purity than that of other proteins. DOUBLE REFRACTION OF FLOW
Takahashi and Rawlins (16) first showed that the juice from tobacco plants diseased with tobacco mosaic exhibited stream double refraction, and it has since been demonstrated that the solutions of the purified tobacco mosaic virus protein show the phenomenon to a marked degree (1, 8, 17). The question of the interpretation of this double refraction was recently considered in some detail, and it was concluded that the 1 Presented a t the Fifteenth Colloid Symposium, held a t Cambridge, Massachusetts, June 9-11, 1938. 935
936
MAX A . LAUFFER
phenomenon wab due in this case to the orientation of rod-like protein particies in the flowing stream (S). These rod-shaped particles may themselves be either optically isotropic or anisotropic. Wiener (19) has shown from lheoreticai considerations that a system composed of very small isotropic rods arranged parallel to each other in an isotropic medium of different refractive index is optically anisotropic. It brhaves like a uniaxial crystal a i t h the optic axis in the direction of orientation. The folloning equation, which represents the double refraction of such a system, niay be d m r ed from Wiener's considerations (8) : (1)
N e s i l d N o are the indices of refraction of extraordinary and ordinary rays, respectively, h'l and N Zthe refractive indiced of rods and medium
7
/
FIG.1 Pictuie diagram of streaming cell
respectively, and VI and V , their respectire relatile volumes. It is readily apparent'from this equation that, if N1 = M z , one obtains no double refraction. If ( N e - N o )is plotted against N 2 ,a parabola is obtained with a miiiimum a t the point hr2 = N 1 . If rod-shaped particles are oriented in a medium nith a refractive index equal to their own, the system should ahon no double refraction of this sort,-i e , no morphic double refraction. Any double refraction shown by the system under such conditions must be the intrinsic double refraction of the particles themselves. This point was considered by Freundlich (4). Steubel (15) evaluated the intrinsic and morphic double refraction shown by cross-striated muscle by measuring the double refraction of the mu'cles immersed in rarious solvents of different refractive index. This method has been applied to stream double refraction by Signer (12) in his studies on polystyrene solutions. By measuring stream double refraction of tobacco niosaic virus protein in solvents of different refractive indices, such as Trarious glycerol-water mixtures and aniline-glycerol-water mixture$, the effwt of the refractive
937
TOBACCO MOSAIC VIRUS PROTEIN
index of the medium on the double refraction of flow was determined. The apparatus used for this measurement consisted of a streaming cell and a polarizing microscope fitted with a photoelectric tube and a vacuum tube amplifying system, as described previously (8). The change in plate current caused by the flow of the liquid, as measured by galvanometer displacements in millimeters, was taken as a measure of stream double refraction. The streaming cell used in this case differed from that described in the original study. As may be seen in figure l, it consisted of a glass capillary fused to two reservoirs, so arranged that all of the liquids studied could be made to flow a t the same constant pressure. By this means the reduced rete of flow compensates for the increased viscosity of the less fluid solvents, and the mechanical force exerted on the suspended TABLE 1 Double refraction ojJEow of tobacco mosaic virus protein (1.08 mg. per cubic centimeter) in solvents of different compositions COMPOSITION OF SOLVEXT IN VOLUME PER CENT REFRAWWE INDEX
Water
.
1 2 3 4 5 6
7 8 9 10 11
100 80
60 50 30 20 10 2 2 2 2
Glycerol
0 20 40 50 70
80 90 88 68 48 28
Aniline
0 0 0
0 0
0 0 10 30 50 70
1.334 1.362 1.389 1.402 1.430 1.442 1.454 1.477 1.503 1.526 1.55
MILLIMETERS OB GALVANOMETER DEFLECTION
54 42 33 27 23 19 14 6.0 4.5 2.4 0
rods remains constant. It may be seen from table 1 and figure 2 that the double refraction of flow decreases greatly as the refractive index of the medium approaches 1.6, the approximate value of the refractive index of the tobacco mosaic virus protein. This value was calculated from the refractive index of a 1.78 per cent solution of the protein, N250= 1.33715, and that of the solvent, Ni5’ = 1.33405, using the equation presented by Wiener (19),
where N , is the refractive index of the solution and the other symbols have the same meaning as in equation 1.
REFRACTIVE INDEX OF MEDIUM
PIG.2 . The dependence of the stream double refraction cf tobacco mosaic virus proteirl u p y l the refractive index of the solvent. TABLE 2 The efi;;ct of various solvents on the activity of tobacco mosaic virus protein as measured by the half-lc f method on Phaseolus vulgaris _ l l l _ _
1
Composition of Bolvent in volume per cent: Water. . . . . . . . . . . . . . . . . . . . . . . 10 Glycerol. . . . . . . . . . . . . . . . . . . . . . 90 0 Aniline, . . . . . . . . . . . . . . . . . . . . . . Concentration of virus in contact with solvent.. . . . . . . . . . 10 mg. per cc. Time of contact with solvent.. . 7 days Concentration of virus in M/10 phosphate a t time of inoculoe6 g. per cc. lating. ..................... Number of half-leaves inocu38 lated. . . . . . . . . . . . . . . . . . . . . . . Average number of lesions per 111.5 .............. half-leaf Average number of lesions per half-leaf for control a t samc concentration CY), . . . . . . . . . 131.8 9.52 Standard error of X - F(SE;-gl 2.13 (2- y)/SE;-i . . . . . . . . . . . . . .
(x).
2
3
2 88 10
48
n
L
50
10 mg. per cc. 10 min.
.O mg. per cc. 10 min.
10-7 g. per cc.
10-7 g. per cc.
26
26
8.1
5.6
6.8
6.5 0.90 1 .o
0.89
1.5
The control in each case was a sample of virus protein in water from the same batch as that exposed t o the solvent under consideration. It was diluted directly from water solution. (X - B)/SE;-,- is a test for the significance of the difference of the number of lesions per half-leaf shown by sample and corresponding blank. By convention, if this quantity exceeds 2.1, the difference is regarded as being due t o factors other than experimental error. These results indicate that 1 week’s contact with glycerol causes a slight decrease in virus activity, whereas 10 minutes’ contact with the other solvents causes no significant change in virus activity. 938
TOBACCO MOSAIC VIRUS PROTEIN
939
In table 2 are presented the results of activity measurements made on virus protein samples which had been dissolved in the solvents employed in the present study for periods of time great enough to allow double refraction measurements to be made. It seen that no appreciable loss in activity results from this treatment. Since activity in the biological sense is a very sensitive measure of change in the protein molecule, it must be concluded that the reduction in double refraction of flow can not be attributed to denaturation or destruction of the protein by the solvents. This conclusion may be checked more directly for the case of the protein dissolved in glycerol-water solvents. It was found that, when solutions of the virus protein in 90 per cent glycerol were diluted with water, the double refraction per unit concentration of protein increased to the value obtained originally for a solvent of the same composition as that obtained upon dilution. This shows that the effect of the solvent is a reversible one, a necessary condition for an effect due to nothing but the refractive index of the solvent. In view of the facts that the property of stream double refraction shown by the virus protein is lost when the protein is dissolved in a medium whose refractive index is the same as that of the protein and that this effect seems not to be due to any change in the protein, it is cvident that the double refraction of flow of the tobacco mosaic virus protein solutions is due largely, if not entirely, to the effect of the shape of the particles and scarcely, if a t all, t o the intrinsic double refraction of the particles themselves. This fact, as will be seen presently, is of great importance to those who are interested in the nature of plant viruses. THE LIQUID CRYSTALLINE STATE
The liquid crystalline or paracrystalline state is encountered in many biological systems ( 5 ) . As will be seen presently, relatively concentrated solutions of the tobacco mosaic virus protein afford an example of such systems, for they may exist in a state which can be described as twocomponent liquid crystalline. The jelly-like pellets obtained by ultracentrifuging solutions of the virus protein may be regarded as being very concentrated colloidal solutions. Figure 3 is a photomicrograph of a flattened section of the jelly-like material constituting a pellet of the ultracentrifuged tobacco mosaic virus protein, taken with a polarizing microscope. This picture resembles those obtained in a similar manner with substances known to be liquid crystalline or paracrystalline, for example, wetted bromophenanthrenesulfonic acid (11). Furthermore, the x-ray diffraction pattern shown by these pellets may be interpreted as being that of a paracrystalline material (2, 21). It would seem, then, that these jelly-like pellets are true examples of the paracrystalline state. As a result of his studies ~ i t ah great n w b e r of organic compounds, Vorlander (18) has come to regard the ability to exist in the liquid crystalline
940
MAX A. LAUFFER
state as being associated with materials having rod-shaped molcrulcs. This generalization may he carried over from systems of one componrnt to systems of two components, such as tobacco mosaic virus protein in water or bromophenathrenesulfonic arid in water. This paracrystallinity of the tobacco mosaic virus protein pellet, then, must be regarded as constituting additional evidenrc of the rod-like character of the tobarro inosair virus protein
I'I,.
:i
Fm. 3. Photomirrogl.:ipIr of a Rattencd ecctivn of a tobacco mosaic
I.,,.
I
virus pmt,
