VOL. 15,383-392 (1976)

BIOPOLYMERS

Electric Birefringence in Concentrated Solutions of Tobacco Mosaic Virus HIROSHI ASAI and NOZOMU WATANABE, Department of Physics, Waseda University, Tokyo 160, Japan

Synopsis A tentative and phenomenological analysis of negative electric birefringence, which has often been observed as an anomalous birefringence phenomenon in a concentrated solution of rodlike macromolecules, is presented. Tobacco mosaic virus (TMV) was used as a typical example for the investigation. I t was found that if the applied electric field is sufficiently high, the steady-state birefringence becomes positive even a t a very high concentration of TMV. From this finding and analysis of the time course of birefringence transients, it was suggested that the TMV (common strain, OM type), which originally has no inherent permanent dipole, behaves as if it possesses a permanent dipole perpendicular to its long axis. Supporting evidence was also obtained from birefringence experiments on concentrated solutions of the HR strain of TMV, which has an inherent permanent dipole along its long axis. Other possibilities, for example, the effects of the walls of electrodes or of polymerization of TMV molecules, were excluded.

INTRODUCTION During the past 20 years, the method of pulsed electric birefringence has become increasingly popular as a technique for investigating both the electrical and geometrical parameters of nonspherical macromolecules in solutions.1-6 This method has been applied usually in very dilute solutions. In 1950 O’Konski and Zimm observed that a concentrated solution of tobacco mosaic virus (TMV) has a negative electric birefringen~e.~ Kobayashi et a1.8 also observed a similar negative birefringence phenomenon for F-actin solutions. Anomalous transient birefringence patterns for concentrated collagen solutions were also reported? However at the present there is no good picture of the nature of such negative birefringence. We thought it is important to perform a systematic analysis of birefringence phenomena in concentrated solutions of a rodlike macromolecule, and then to elucidate the nature of the negative birefringence in terms of interactions between the charged anisotropic macromolecules in their concentrated solutions. We employed the common (OM type) and HR strains of TMV as samples. 383

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1976 by John Wiley & Sons, Inc.

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EXPERIMENTAL Apparatus As described in a previous paper,4 usually a rectangular voltage pulse is generated by a transient connection between an electrode in a Kerr cell and a voltage supply output through a vacuum mercury switch. The rise and decay times of the pulse are about 0.3 psec, and the duration time of the pulse is from 5 to 20 msec. Occasionally, a reversing rectangular pulse was used to analyze permanent dipole contributions in the TMV solution. The electronic circuit and performance of the reversing pulse generator will be published elsewhere. Electrodes in the Kerr cell were made with two platinum coated plates. Their separation and length are 0.2 and 2.0 cm, respectively, unless otherwise indicated. An He-Ne gas laser was used as a light source. A XI4 plate was located between the Kerr cell and an analyzing Rochon prism rotated 4’ from the cross position.

Materials The common strain of TMV (OM type) and also the Holmes Rib Grass strain of TMV (HR strain), which has a permanent dipole contribution along its long axis, were used for experiments. The concentrations of TMV were determined spectrophotometrically using an extinction coefficient Ei?,&, = 30 a t 260 nm. Prior to use, a concentrated solution of TMV was dialyzed exhaustively in a very dilute buffer solution, and then diluted with the buffer to the desired extent. The pH of solutions was periodically checked before and after experiments.

RESULTS Birefringence at Low Concentrations of TMV When the concentrations of TMV are lower than 0.03%, all the solutions display positive birefringences, which means that the TMV molecules are oriented to the electric-field direction. A t such concentration ranges, the optical retardations were proportional to the square of the applied electric field and also to the concentration of TMV. A typical example of the relation between the optical retardation and the square of the applied electric field is shown in the insert of Figure 1. The specific Kerr constant K,, of the common strain of TMV in 0.1 mM phosphate buffer was found to be 6.9 X 10-l2 at pH 5.9,6.6 X at pH 6.9, and 8.7 X 10-l2 a t pH 7.5, which are of the same order of magnitude found by O’Konski and Haltner2 (6.9 X 10-l2 a t pH 7.0). The rise and decay times of birefringence patterns in TMV solutions of the common strain were about 0.8 msec, which is somewhat larger than that reported by O’Konski and Haltner2 (0.5 msec). This is due to par-

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10

5

0

-5

0-

A-

-10

b-15 : m n

.-c -2c

-IN - 25

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i i

t

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OO

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10

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Fig. 1. Optical retardation ( 8 ) in steady state and square of electric field. T M V solutions of common strain in 0.1 mM phosphate buffer (pH 6.9) were used: ( 0 )0.05%; ( 0 ) 0.1%;(+) 0.2%; ( 0 )0.3%;( A ) 0.4%; (X) 0.5%; (0) 0.7%; (v)0.8%. The insert demonstrates that the optical retardation is proportional to the square of electric field in 0.0065%T M V solution.

tial longitudinal aggregation of our TMV samples. The average length of the TMV molecule was estimated as 4000 A, which is 17%longer than that estimated by O'Konski and Haltner. Since the rise and decay times in birefringence pattern are the same, the common strain of TMV has no inherent permanent dipole contribution. The absence of a permanent dipole contribution was confirmed also by a nonreversing birefringence pattern when a reversing electric field was applied to a TMV solution. A similar experiment was also performed for the HR strain of TMV. A t concentrations lower than 0.05%, the optical retardation was proportional to the square of the applied electric field and also to the concenat pH 6.5, tration of TMV. The specific Kerr constant was 4.3 X 6.7 x at pH 7.1, and 13.9 X at pH 7.5. The rise and decay times in the birefringence pattern were 0.73 msec and 0.5 msec, respectively, which indicates the presence of a permanent dipole contribution.

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The permanent dipole moment in the HR strain of TMV was estimated to be 1.4 2.0 X lo4 along its long axis.

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Steady-State Birefringence at High Concentrations of TMV The electric birefringence especially in the steady state becomes negative in highly concentrated solutions of the common strain of TMV, as reported similarly by O’Konski and Zimm.7 Such negative birefringence reveals that the rodlike TMV molecules are oriented perpendicular to the direction of the electric field. Such an extraordinary orientation of TMV molecules at high concentration is not due to an artifact caused by the walls of two electrodes. This was confirmed by the following experiments. The electric birefringence patterns, when electrodes separated by 1.3 cm are used, were compared with those using electrodes separated by 0.2 cm. It was found that the transient birefringence patterns for electrodes separated by 1.3 cm were exactly the same as those for the electrodes separated by 0.2 cm a t the same strength of electric field. This fact suggests that the orientation of TMV molecules perpendicular to the direction of the electric field is not due to propagation of any wall effects. We tried also to use Kerr cell whose electrodes are made of metal nets, instead of platinum plates. Again, the steady-state and transient birefringences were completely the same a t the same electric field, regardless of the kinds of electrodes used. The relationships between optical retardations in steady states and squares of electric field for a series of experiments a t various concentrations of the common and HR strains of TMV are summarized in Figures 1 and 2, respectively. It is clearly seen in both figures that even a t a very high concentration of TMV, the steady-state birefringence becomes positive when the applied electric-field strength is increased greatly. A typical example is seen in Figure 2; the steady-state electric birefringence of 0.37% TMV solution of the HR strain reaches a minimum at about 17 Vlcm, zero a t about 26 Vlcm, and then increases positively with field strength. This finding, that the steady-state birefringence becomes positive at an infinitively high electric field regardless of the kinds of TMV used and the concentrations of TMV, is very important for speculating on the possible molecular origin of anomalous birefringence. Note that in Figures 1 and 2 the negative birefringences in the steady-state a t high concentration ranges of TMV are also proportional to the square of the electric field when the electric fields are sufficiently low, and that the critical TMV concentration a t which the birefringence becomes negative at a sufficiently low electric field is higher for the HR strain of TMV than for the common strain of TMV. The pH dependency on electric birefringence of concentrated TMV solutions was investigated. As seen in Figure 3, the electric birefringence in the steady state at an arbitrary field strength becomes more positive

ELECTRIC BIREFRINGENCE OF TMV

I 0

I

I

1

2

4

8 2 2 -4 E (vOlt/crn) X 10

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Fig. 2. Optical retardation (6) in steady state and square of electric field. TMV solutions of HR strain in 0.1 mM phosphate buffer (pH 7.1) were used. ( 0 ) 0.015%; (0) 0.074%; (+) 0.18%; (V)0.37%; (A)0.74%.

4

-2

0

2

4

Fig. 3. Effect of pH on relationship between optical retardation in steady states and square of electric field. The 0.5% TMV solutions of HR strain were used and their pH in 0.15 mM phosphate buffer were adjusted to 6.7 (X), 7.0 ( O ) ,and 7.4 ( 0 ) .

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Fig. 4. Effect of pH on relationship between optical retardation in steady states and square of electric field. The 0.26% TMV solutions of common strain were used and their and 7.5 (0). pH in 0.15 mM phosphate buffer were adjusted to 6.0 (X), 6.9 (e),

with increasing pH of the TMV solutions of the HR strain. For the TMV solutions of common strain, the pH dependency on electric birefringence in the steady state becomes less significant, as seen in Figure 4.

The effect of the addition of neutral salt or buffer on electric birefringence was also investigated for TMV solutions of the common strain a t a constant pH. A typical example is shown in Figure 5. As seen in this figure, the field strength where electric birefringence in the steady state becomes zero decreased with increasing buffer concentration. This means that the addition of salt tends to make the electric birefringence more normal. In the insert of Figure 5, the relationships between optical retardation and the square of field strength for diluted TMV solutions of common strain at various buffer concentrations are also shown for a reference. It is clear from Figures 3-5 that a decline of the electric polarizability of the individual TMV molecule along its long axis always causes its electric birefringence to be more normal.

Birefringence Transient at High Concentration of TMV In Figure 6 we illustrate, as typical examples, the transient patterns of electric birefringences induced by the application of rectangular electric pulses of various field strengths in the common and HR strains of TMV. The transient patterns of birefringence just after the electric field is applied or extinguished are very peculiar, especially a t low and

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Fig. 5. Effect of addition of salt on relationship between optical retardation in steady states and square of electric field. The 0.86%TMV solutions of common strain in various 0.5 mM; (A) 1.0 concentrations of Tris HCl buffer (pH 7.6) were used: ( 0 )0.2 mM; (0) mM; ( X ) 1.5 mM. The insert shows similar experiments for 0.009% TMV solutions. ccmnon strain

-.- .... fleld strength

-0-

HR s t r a l n

+ .__._

Fig. 6. The birefringence patterns of the common and HR strains of TMV when various electric fields of rectangular pulses are applied.

intermediate strengths of the applied electric field. A similar birefringence pattern has been reported by Haschemeyer and TinocolO for a dilute solution of fibrinogen a t pH 4.7-4.9, and interpreted to be due t o a permanent electric dipole perpendicular to the long axis of the fibrinogen molecule. Because of its small size, the electric field applied to the

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fibrinogen solution was very strong. As seen in Figure 6, both the steady-state and transient birefringences become positive when the applied electric field is very high. The decay time in electric birefringence, which is indicative of the rotary diffusion coefficient or of the length of TMV, was not very dependent on the concentration of TMV, or on the electric-field strength. The decay time of electric birefringence in a highly concentrated solution of TMV was a t most twice that of a very dilute solution of TMV. Because the decay time of electric birefringence in a solution of long rodlike macromolecules is proportional to the third power of the length of the rod, the above observation means that the TMV molecules in a highly concentrated solution are not significantly aggregated. Therefore, anomalous birefringence patterns in TMV solutions studied above must be interpreted in terms of electric or steric interaction between long rodlike macromolecules in solution, and not simply in terms of lateral or longitudinal aggregation. It is well known that at a very high concentration of TMV, (e.g., 2 or 3%) the anisotropic phase appears in the TMV so1ution.l' Our experiments were made at a much lower concentration where spontaneous birefringence, which is indicative of the appearance of the anisotropic phase in solution, was not observed.

DISCUSSION Negative birefringence in concentrated solutions of TMV has been mysterious since O'Konski and Zimm7 observed it in 1950. Negative birefringence phenomena seem to appear generally in long rodlike and charged macromolecular solutions. It was found in the present experiments that negative birefringence in the steady state is proportional to the square of the applied electric field in the range of very weak fields and becomes positive in the range of sufficiently high electric fields. It seems that there is no significant lateral or longitudinal aggregation of TMV molecules in a highly concentrated solution and that the possibility of a wall effect of electrodes has been excluded. Therefore, anomalous negative birefringence must be interpreted as the electric or steric interaction between the long rodlike macromolecules in solution. The method of electric birefringence applicable to a low rectangular pulse in a diluted solution of an anisotropic macromolecule has been established theoretically by Benoitl and by Tinoco and Yamaoka.12 Their theories, of course, can not explain the above observations on the relationship between electric birefringence and applied electric field. On the other hand, electric birefringence in the range of high electric fields has been investigated t h e ~ r e t i c a l l y l ~ -and ' ~ experimentally.16J7 From theoretical calculations, it was found that if the macromolecule has a large permanent dipole contribution perpendicular to its long axis,

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the steady-state birefringence is negative in the range of low electric field. A t sufficiently high electric field, the birefringence becomes positive, since the orienting torque by the induced dipole contribution is proportional to the square of the electric field, and that by the permanent dipole contribution is proportional to electric field. Such a relationship between the steady-state birefringence and the applied electric field has been observed experimentally in the cases of fibrinogen'O and bentonitels solutions. However, contrary to the case of concentrated TMV solutions, the above relationship was essentially independent of the concentrations used. This means that both fibrinogen and bentonite have an inherent permanent dipole perpendicular to their long axes. I t is, therefore, concluded by analogous inference with the cases of fibrinogen and bentonite that the TMV molecule of common strain, which has no inherent permanent dipole in its diluted solutions, begins to have an apparent permanent dipole contribution perpendicular to its long axis in a concentrated solution. Such an interpretation for anomalous negative birefringence is supported by the fact that negative birefringence for the HR strain of TMV, which has a large inherent permanent dipole and an induced dipole along its long axis, appears in much higher concentration than that for the common strain of TMV (Figures 1 and 2, about 0.2% for the common strain and 0.4% for the HR strain). The experiments on the effect of the addition of salt and of varying pH also support such an interpretation. Some of the transient and anomalous patterns in electric birefringence just after application or extinction of electric field as shown in Figure 6 must certainly be due to steric interaction between TMV molecules, since the theory of electric birefringence has predicted that the time course of birefringence just after the extinction of low or high electric field must be an exponential decay curve in a monodisperse and dilute solution. Quantitative and even phenomenological analysis of steric interactions between anisotropic macromolecules during their orientations is extremely difficult. It is not known how the appearance of the apparent permanent dipole in concentrated rodlike macromolecular solutions can be related to steric or electric interactions between the macromolecules. The change in sign of electric birefringence is theoretically expected to appear at a very high electric-field region. However, we observed a change in sign of birefringence a t a very low electric field. This problem is not explained well a t the present. It is worth noting that this problem also appeared in the case of bentonite solutions.18 Probably, the effective electric field, which affects the orientation of TMV molecules, may be very high even when an externally applied electric field is very low. Electric potential in the vicinity of TMV molecules is expected to be extremely high in a low ionic solution. The apparent permanent dipole of the TMV molecule in its highly concentrated solution might be created transiently by the application of an electric field or might have been present originally because of an asymmetric ionic at-

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mosphere in the free volume of a TMV molecule. However, an explanation by an electrophoretic effect is certainly impossible, because the electrophoretic effect may occur by an asymmetric ionic atmosphere but can not predict both negative birefringence in a low electric field and positive birefringence in a relatively high electric field. A thermal fluctuation of ionic atmospherelg might statistically cause an apparent permanent dipole in a concentrated macromolecular solution. The authors wish to express their thanks t o Prof. C. A. Knight, University of California, for his kind supply of the HR strain of TMV, and to Dr. Y. Kiho, Institute of Plant Virus Research for his kind supply of the common strain of TMV. Thanks are also due to M. Inoue in the author’s laboratory for helping to construct the Kerr apparatus, and to H. Nishira, 0. Nishikawa for their help with additional experiments.

References Benoit, H. (1951) Ann. Phys. 6,561-609. O’Konski, C. T. & Haltner, A. J. (1957) J. Amer. Chem. SOC.79,5634-5648. Krause, S. & O’Konski, C. T. (1959) J. Amer. Chem. SOC.81,5082-5088. Asai, H. (1961) J. Biochem. (Tokyo)50,182-189. Houssier, C. & Fredericq’, E. (1966) Biochim. Biophys. Acta 120,113-130. Fredericq’, E. & Houssier, C. (1973) Electric Dichroism and Electric and Birefringence, Oxford Univ. Press, Oxford, England. 7. O’Konski, C. T . & Zimm, B. H. (1950) Science 111,113-116. 8. Kobayashi, S., Asai, H. & Oosawa, F. (1964) Biochim. Biophys. Acta. 88.528-540. 9. Kahn, L. & Witnauer, L. P. (1971) Biochim. Biophys. Acta 243,388-397. 10. Haschemeyer, A. & Tinoco, Jr., I. (1962) Biochemistry 1,996-1004. 11. Oster, G. (1950) J . Gen. Physiol. 33,445-473. 12. Tinoco, Jr., I. & Yamaoka, K. (1959) J . Phys. Chem. 63,423-427. 13. O’Konski, C. T., Yoshioka, K. & Orttung, W. (1959) J . Phys. Chem. 63,1558-1565. 14. Shah, M. J. (1963) J . Phys. Chem. 67,2215-2219. 15. Holcomb, D. N. & Tinoco, Jr., I. (1963) J. Phys. Chem. 67,2691-2698. 16. Krause, S. & O’Konski, C. T. (1963) Biopolymers 1,503-515. 17. Riddiford, C. L. & Jennings, B. R. (1963) Biopolymers 5,757-771. 18. Shah, M. J., Thomson, D. C. & Hart, C. M. (1963) J . Phys. Chem. 69,1170-1178. 19. Oosawa, F. (1973) J. Theor. Biol. 39,373-386. 1. 2. 3. 4. 5. 6.

Received November 12,1974 Returned for revision January 7,1975

Electric birefringence in concentrated solutions of tobacco mosaic virus.

VOL. 15,383-392 (1976) BIOPOLYMERS Electric Birefringence in Concentrated Solutions of Tobacco Mosaic Virus HIROSHI ASAI and NOZOMU WATANABE, Depart...
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