Electrically induced fluorescence depolarisation of macromolecules

CHEMICAL PHYSICS LElTERS

Volume 45, number 3

1 February

1977

ELECTRICALLY INDUCED FLUORESCENCE DEPOLARISATION OF MACROMOLECULES B.R. JENNINGS and PJ. RIDLER Physics Department. Bntnel University, Uxbridge. Maddlesex UB8 3PH. UK

Received 16 June 1976 Revised manuscript

received 4 October

1976

Quantifiable charges have been recorded in the polarised fluorescence components as macromolecular solutions were subjected to pulsed electric fields. The advantages of this new molecular characterisation method are discussed. Illustrative

data are presented for DNA tagged with ethidium bromide.

1. Introduction When an electric field is applied to macromolecular solutions, the constituent particles align as their inherent electrical dipoles (both permanent and induced) interact with the applied field. Such alignment should be accompanied by changes in the intensity and state of polarisation of the fluorescence for a solution of mo:ecuIes which have naturally occurring or dehberately attached fluorescent groups, on the condition that the molecular alignment arso induces alignment of the fluorescent groups. Recently the orientation of quinacrine molecules bound to deoxyribonucleic acid (DNA) was studied from the fluorescent changes of these so!utions in suitable flow fields [I J . More recently, Weill and co-workers [2,3 ] developed a theory for electrically induced fluorescent changes. They also attempted measurements using pulsed fields on complexes of DNA with acridine orange and 2-hydroxy4,4’- diamidinostilbene. Unfortunately, their experimental results were not convincing. This seemed to us to be particularly disappointing for three reasons. Firstly, from experience with other electro-optic phenomena, we expected elecrro-optic Ruorescence to be very sensitive because of the high light intensities generally involved in fluorescent phenomena. Secondly, fluorescence is character&d through four polarised components. These are Vv and VH for vertically polarised incident light and Jfv and HH for a horizontally polarised incident beam. The subscripts indicate the

polarisation state of the analysed light. If one could record significant changes in each of these components, the directions of the absorption and emission transition moments and hence of the fluorophores could be estimated relative to the macromolecular geometry. Thirdly, using pulsed electric fields, the rates of decay of the fluorescence intensity changes after the termination of the applied field should indicate the rotary relaxation time (7) of the macromolecule. In this note we report the first significant, quantifiable obser-ations of electrically induced changes in each of the four components of the ff uorescence for macromolecules in solution. Illustrative data are presented for DNA to which the dye ethidium bromide (EB) has been bound. Even in the absence of suitable rigorous theory it is shown that the transient changes in these components can give insight into the nature of the stacking of the fluorescent groups within the DNA helix

2. Theory Equations for the effect have been developed only for rigid rod molecules which orientate in an electric field of intensity E. They are assumed to have either or both a molecular permanent dipole moment (Jo) along the rod axis or an anisotropy (cy3 - ~yt) in the electrical polarisability (CY)associated with the unique (subscript 3) or transverse (subscript 1) rod axes. Furthermore, the

CHEMiCALPHYSICSLETTERS

Volume 45, number 3

theory is only applicable to small degrees of molecular orientation. In such a case, the changes (preftued A) in the polarised components of the fluorescence are given by the interchangeable equations [3], AVH-AI+ V, +2VH =

A&

AHH+2AVH = V, +2V,

=-

+ 2Af.4,

V,+2VH

gcos2JI - cos23/‘)@ .

(1)

Here, the angles 3/ and $ ’ are those between the major rod axis and the absorption transition moment or emIssion transition moment respectively. The orientation function +, as defined originally by O’Konski et al. [4] for the orientation of rigid ellipsoids, has the following form in low intensity electric fields, @ = &[p2/k2T2

+ (a3 - q)lkT]

E2 >

(2)

with k and T the BoItzmann constant and the absolute temperature respectively_ Hence, it can be seen that at low field intensities, a quadratic dependence on E is expected. ln the special case where both the absorption and emission transition moments lie in a plane perpendicular to the rod axis, then the following simpler equation has been developed [2] AVvl VV = -+r [ p2/k2T2

+ (a3 -

q )/kT] E2

(3)

and the factor in the brackets can be evaluated from the relative intensity changes in a single fluorescence component. Finally, by analogy with the transient changes in other electro-optic phenomena for monodisperse macromolecular solutions [5], the decay of the effect foIlowing the termination of the electrical impulse, obeys the rate equation [6] AF lAFo = exp(-t/r)

,

(4)

where AF represents the change in any one fluorescent component at any time Cafter the cessation of the field, at which time t = 0 and AFhas the vahre aF,. A suitable semi-logarithmic plot of the ratio of the left hand side of this equation against t should yield a negative slope from which the molecular relaxation time r is obtained. Curvature of such a plot indicates molecular polydispersity or flexibility.

I Febtuay 1977

3. Experimental and resuks The orientation time of macromoIecuIes in SOIUtion is generally much longer than the Iife times of the absorption and reemission involved in fluorescent processes. Hence, in this work we have used continuous radiation at 488 run wavelength to excite the fluorescence. The apparatus required careful design. It is to be described in detail elsewhere. In principIe, a Iight beam from an argon-ion laser was suitably attenuated and then polarised in either the vertical or horizontal plane using a rotatable pair of Fresnel rhombs. The beam then passed into the solution which was hetd in either a cylindrical or square cell. This had special entrance windows to avoid multiple reflections of the incident laser light. It was also equipped with a pair of horizontal steel electrodes across which the pulse of electric voltage was applied. Accompanying transient changes in two of the polarised components of the fluorescence were recorded simultaneously at right angles to the incident beam direction_ This was achieved by means of two detection unit arms which could be manually rotated coaxially about the cell centre. Each of these held suitably oriented high quality Glan-Taylor polarising prisms, fluorescent filters and fast response, low noise photomultiplier tubes. The other two fluorescence components were measured by rotating the Fresnei rhombs and hence the incident beam polarisation state through 90”, reapplying the electric pulse to the soIution, and recording the two new transient outputs from the photomuhipliers. It was convenient to record the applied field as we11as the transient fluorescent components on an oscilloscope. To illustrate the method and its potential, measurements were made on an aqueous solution of 10-J g ml-’ at neutral pH of calf thymus DNA of 5.0 X 106 relative molecular mass. To this, ethidium bromide had been added to a concentration such that the ratio of dye to DNA phosphorus was 0.05. This was of the order of one dye molecule per 10 base pairs of the nucleic acid. The detection arms were fitted with optical filters which cut off alI radiation below 5% nm wavelength. Rectangular pulsed eIectric fields of up to 4 kV cm-i and up to 800 gs duration were applied to the solutions. A typical set of the changes in the four polarised components of the fluorescence is shown in fig_ 1, along with the profde of the applied

551

V0lume 45, number 3

CHEMICAL

PtIYSICSLEl-t-ERS

1 February 1977

Fig. 1. Transient changes in the polarised components of fluorescence for the ethidium bromide/DNA cornpIe\. In each frame, the upper trace is tile optIcal response and the lower trace is the profile of the applied field of 4 kV cm-‘. Time runs from left to right with 0.2 ms per division. The zero-field mtcnsities were made the same for each component.

field. The clarity, lack of electronic noise and magnitudes of these responses is remarkable. They should be compared with the only other reported attempts to measure them [2,3 ] where the responses were indistinguishable in the noise. We ciaim these data to be the first realistic manifestation of transient electrically induced fluorescence changes in which the relative magnitudes of the four polarised components are readily quantified. The four polarised components differ in their relative magnitudes and this yields important structural information. Had the dye molecules been randomly bound to the DNA, the medium would have been fluorescent but with no significant transient change in its polarised components when the field was applied. Hence the transients of fig. 1 indicate a discrete ordering of the dye molecuIes relative to the nucleic acid helices. The nature of this ordering can be reasoned in the following manner. At any instant, the total ff uorescent intensity is equal to the sum of the components 552

Vv + 2 VH. Both AV, and AV, are negative, hence in the field the fluorescent intensity decreased with molecular alignment. The reduction in these components was progressive with increasing field strength. However, the greater the field strength, the more complete the molecular alignment which, with the geometry of our apparatus, meant that the helices became increasingly parallel to the V direction. The accompanying reduction in overall fluorescent intensity suggested that the absorption transition moment was predominantly in a direction perpendicular to the major helix axis. An extension of this logic implies that, for an absorption truly perpendicular to the electric dipole, at sufficiently high fields, the ratio A Vv/ V, should approach unity. Whereas fig. 2 shows an increase in this ratio with field strength, unity was not obtained. Prior to complete molecular alignment, the increasing field magnitude induces anomalous effects which prevent the realisation of field induced orientation [7,8]. We now consider the emission transition moment.

1 February

CHEMICAL PHYSICS LE-ITERS

Volume 45, number 3

(A&

+ 2AV,)/(V,

-(AHH+2At%J)j(VX,

1977

f 2Hv) = 0.004, +2VH)=O.O06.

Firstly, the agreement between these experimental ratios is encouraging. Secondly, using the experimental value for E and that calculated for a3 -CK~ below, the aforementioned ratios correspond to a vahre of 0.04 for cos2 $ - cos2 IJ ‘. Assuming one of these angles to be 90”, the other must be within 10” of this. This is m concord with the prediction that both the absorption and emission moments are predominantly perpendicuk to the helix axis. As both of these moments are thought to be in the plane of the ethidium bromide moIecuk for visible light [9], a perpendicular interaction of the EB molecules within the DNA helk is indicated_ In such a case eq. (3) can be used to analyse the low field data of fig. 2 where AVv/Vv varies with E2. Assuming that DNA has no significant permanent dipoIe moment [LO] a value of a3 - ol = 2.7 X 1O-31 Fm2 (or 2.4 X LOdt5 cm3)

Fig. 2. Field strength dependence of the relative chmges in the VV polarised component of fluorescence.

At low field strength, where the absorption is still significantly high, AVB/ VB is always greater than AVv/V-v when the negative sign of these changes is considered. Hence, the emission transition moment is associated predominantly with the horizontal plane. This suggests that the emission moment is also predominantly perpendicular to the vertical direction and hence to the helical axes. This reasoning is confirmed by a consideration of the polarised components for horizontal incident light in which ALYB/HB is always greater than AKv/Hv. It would appear therefore that both the absorption and emission transition moments are predominantIy perpendicular to the DNA helical axis. Confirmation of this reasoning is provided by an analysis of the data through eq. (1). It is noted that the three ratio factors on the left hand side of the equation are extremely small. Within the region of the quadratic dependence of the fluorescent component changes on the field strength, these factors were (AVH - AHv)/(Vv

+ 2Hv) = 0.005,

is obtained for the polarisability anisotropy. In addition, the negative sign of the ratio AV,/ VV indicates that a3 S a1 and that the induced dipole moment acts along the major hzliv axis. These results are in concord with those reported in the Literature as cohated and discussed in ref. [IO] . It should be realised however that the dye moiecufes may influence the magnitude of a3 - a1 and hence lead to a different vahre than that obtained for isolated DNA molecules. Finally, molecular information can be obtained from analysis of the decay rates of the fluorescence corn ponent changes. From fig_ 1, it is seen that each component decays at the same rate for a given esperimental field strength. Evaluation of r from a transient decay is illustrated in fig. 3. Departure from linearity for this semi-logarithmic plot indicates sampIe polydispersity whence the graph is composed of two or more exponential contributions and eq. (4) becomes AF = lg (dFo Ii exp(-f/r&

(5)

for n molecular species, each with a rekation time rP The analysis of curved semi-logarithmic plots is commonplace in transient electro-optic studies and so is not discussed here. Suffice it to recall that the dz553

CHEMICAL PHYSICS LE’l-l-ERS

Volume 45, number 3

1 February 1977

of the interpretation of D in terms of the geometry of the DNA molecule.

4. Comments on the method

I 0

T=O

01

02

03 1dxTlME

04

0.5

21ms

06

SEC

Fig. 3. Transient decay analysis. Initial slope corresponds to 7 = 0.21 rns (& = 823 s-‘).Non-linearity indicates more than one relaxing species. The long time asymptote is characteristic of T = 0.44 ms (D = 391 s-l). Within experimental error similar data were obtained from each fluorescent component deQY-

convolution of curves such as fig. 3 does nor relate the initial and final slopes of the curve to the smallest and largest molecules in the sample [ 1 I] . Rather, with rigid molecules, it has been reasoned that the initial slope is equated with the most reliable experimental data which are also associated with discrete averages of the relevant rotary diffusion coefficient D = l/67. Because fluorescence is a manifestation of the optical anisotropy of the molecules, the values for D obtained from the initial slope of curves such as fig. 3 will be identical with those obtained from electric birefringence and dichroism experiments [ 11 ,I2 ] . Hence, at low field strengths and assuming that DNA has only an induced electrical dipole moment, a value of T = 0.21 ms or

8,

= l/67 = 820 (a40) s-*

was obtained for this sample. This is almost identical with the value of T = 0.22 ms obtained from electric birefringence data at the same order of field strength by Miller and Wetmur [ 131 on a sample of calf thymus DNA which also had a relative molecular mass of 5 X 106. Hence, our result is regarded as satisfactory. The reader is referred elsewhere 1141 for a discussion 554

In conclusion, we have been able to observe and record quantifiable changes in the four polarised components of fluorescence for macromolecules, in this case DNA, during the application of pulsed electric fields. In common with the other electro-optic methods which have been developed in recent years, the transient fluorescent method is fast, requires small sample volumes and leads to the evaluation of electrical and geometrical parameters of macromolecules through the direct determination of their permanent dipole moments, electrical polarisabilities and rotary relaxation times. The high light levels involved in fluorescence phenomena result in the field induced changes being easier to detect than in the majority of electrooptic scattering [ 151 or optical rotation [S ] experiments and of comparable ease to those met in electric birefringence and dichroism [lo] measurements. Electric dichroism is a useful method for studying the directions of absorption transition moments for absorbing molecules. The fluorescence method has the added facility in that one can also evaluate the characteristics of the emission moment and hence the packing geometry of inherent fluorescent groups or of tagged dye molecules for the macromolecule. Studies are currently being undertaken on a range of dye-tagged helical polymers and biopolymers. The method has wider potential however. Possible fields of study include (i) the nature of molecular interactions and binding in antibody-antigen reactions, (ii) polymer motion and behavior in electric fields using suitably tagged polymers and (iii) the evaluation of transition moment directions for small fluorescent molecules by binding these with specific directions to suitable macromolecules.

Acknowledgement The Science Research Council is acknowledged for an equipment grant to one of us (B.R.J.) and a postgraduate studentship to the other (P.J.R).

Volume 45, number 3

CHEMICAL PHYSICS LETTERS

References [I ] [2] [ 3] [4]

L.S. Lerman, Biochemistry 49 (1963) 94. G. Weill and C. Hornick, Biopolymers 10 (1971) 2029. G. Weill and J. Sturm, Biopolymers 14 (1975) 2537. CT. O’Konski, K. Yoshioka and W.G. Orttung, J. Phys. Chem. 63 (19.59) 1558. [S] E.D. BaiJy and B.R. Jennings, J. Colloid Interface Sci. 45 (1973) 177. [6] H. Benoit, Ann. Phys. (Paris) 6 (1951) 561. [7] CT. O’Konski and N.C. Stellwagen, Biophys. J. 5 (1965) 607.

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1977

[8] P. Colson, C. Houssier, E. Fredericq and J.A. Bertoletto, Polymer 15 (1974) 396. f9] J.B. Le Pecq and C. Paoletti, J. Bfool.Eliot. 27 (1967) 87. [IO] E. Fredericq and C. Houssier, Electric dichroism and electric birefringence (Clarendon Fress, Oxford, L973). [ 111 3. Schweitzer and B.R. Jennings, Biopoiymers IL (1972) 1077. [ 121 J. Schweitzer and B.R. Jennings, BiopoIymers 12 (1973) 2439. f 131 S.J. Miller and J-G. Wetmur, Biopolymers 13 (1974) 115. [14] S. Takashima, Advan. Chem. Ser. 63 (1967) 232. {lS] B.R. Jennings, in: Li@tt scatter&g from polymer sotutions, ed. M. Huglin (1972) ch. 13.