- Dietmar PorschkeAffiliated withMax Planck Institut für biophysikalische Chemie
The direction and the magnitude of the absorbance change induced by the field pulses indicates the orientation of the light-absorbing chromophor with respect to the long axis of the DNA,
The time constant(s) of the molecular rotation process indicate(s) the hydrodynamic dimensions of the complex,
The electric parameters of the complexes are usually not a target of investigations on drug—DNA complexes and, thus, are not discussed in this contribution Among the books (1–4) published on the method, the one of Fredericq and Houssler (1) is still the most advisable one for an introduction, although the examples are not up to date.
The direction and the magnitude of the absorbance change induced by the field pulses indicates the orientation of the light-absorbing chromophor with respect to the long axis of the DNA,
The time constant(s) of the molecular rotation process mdicate(s) the hydrodynamic dimensions of the complex,
The electric parameters of the complexes are usually not a target of investigations on drug-DNA complexes and, thus, are not discussed in this contribution
The molecular alignment by electric field pulses requires an electric anisotropy: In the case of DNA, a large dipole moment is induced along the long axis, leading to a high degree of orientation of the molecules with their long axes parallel to the field vector already at relatively low electric field strengths A second requirement is the existence of an optical anisotropy in the case of DNA the absorbance of UV light is highly anisotropic, because the bases are stacked perpendicular to the long axis of the molecules. Thus, the alignment of DNA molecules by electric field pulses can be easily followed by measurements of the absorbance of polarized light
2.1 The Dichroism Amplitude
The degree of molecular orientation and, thus, the magnitude of electric dichroism increases with the electric field strength E. Complete orientation in the direction of the electric field may be expected only in the limit of infinitely high E The dependence of ΔA/A on E is determined by the type and the magnitude of the electric dipole moment. The degree of orientation at a given field strength increases with the magnitude of the dipole moment. At low E-values the electric dichroism ΔA/A increases with E2. The complete dependence of ΔA/A on the field strength E is described by the “orientation function” Φ according to ΔA/A = Φ ΔA/A ∞ (3) where ΔA/A ∞ is the limit value of the electric dichroism at infinite field strength. In the case of induced dipoles, the orientation function is given by: (4) where γ = (αE 2)/(2 kT), α is the polanzability, k the Boltzmann constant, and T the absolute temperature. In the case of permanent dipoles the orientation function is given by. (5) where β = µp. EkT and µp is the permanent dipole moment.
When the electric dichroism of drug-DNA complexes is measured at wavelengths around 260 nm, where both the drug and the DNA contribute to the absorbance, the dichroism contains contributions from both components. Separation of these contributions may be difficult. However, most drugs absorb light at longer wavelengths than DNA. Thus, the electric dichroism of the drugs may be measured selectively in the long wavelength range and may be used to determine the orientation of the drug in the complex.
2.2 The Dichroism Decay Time Constant
When an electric field pulse is terminated, the molecules, which have been aligned under the influence of the electric field, turn back to their random distribution by rotational diffusion. The process of rotational diffusion is very strongly dependent on the molecular size the time required for the transition from the aligned to the random state increases with the cube of the length of rigid rodlike molecules Thus, the dichroism decay time constant, which reflects the rotational diffusion process, is a very sensitive indicator of the length.
3 Experimental Setup
3.2 Pulse Generator
Most of the electric dichroism data described in the literature have been obtained by use of commercial pulse generators These devices generate pulses with amplitudes up to a few kilovolts; the rise and the decay times of the pulses are in the range around 20 us. Generators of this type are distributed by, e.g., Velonex, Santa Clara, CA
3.3 The Measuring Cell
3.4 Spectrophotometric Detection
Standard commercial spectrophotometers are not sufficient for measurements of the electric dichroism, because their time resolution is usually limited to the range between s and ms. However, parts for the assembly of a fast spectroscopic detection system are available from various sources. For optimal signal-to-noise ratios, the light used for the measurements should be of high intensity. Light sources of high intensity are arc lamps, which are offered in many different variations. Monochromators, polarizers, and detector heads with appropriate power supplies are also offered in various forms by different companies.
Because the electric dichroism is induced by field pulses in the kilovolt range, whereas the absorbance changes after photoelectric conversion are usually in the millivolt range, the detection system has to be protected efficiently against perturbations by induction effects For this purpose the photomultipher is shielded in a cover made from µ-metal, protecting mainly against magnetic perturbations, and the multiplier head including the amplifier is mounted in another cover made from metal, protecting mainly against electric induction effects. The connection between the photomultipher head and the transient data storage unit has to be shielded as well.
3.5 Transient Data Storage and Data processing
Owing to the progress in electronics, transient digital storage of expenmental data in the time range of microseconds and below is not a problem anymore The transiently stored experimental data can be easily transferred to PCs, which are sufficient for evaluation of the data. Usually, the software required for the evaluation is prepared individually. (However, standard software for evaluation of exponentials may be obtained free from: Stephen Provencher, Max Planck Institut fur biophysikahsche Chemie, Am Fassberg 11, D-37077 Gottingen, Germany.)
3.6 Automatic Data Acquisition
Under many conditions, the electric dichroism of DNA is large enough, such that single shots are sufficient for data analysis. However, it is often useful to extend measurements to conditions of, e.g., low DNA concentration or low electric field strengths, where an increase of the signal to noise ratio by averaging of many transients is desirable. For this purpose, automatic acquisition of data is very useful. An instrument for automatic measurements of the electric dichroism may be constructed relatively easily.
Standard PCs are sufficient as control units for timing of the field pulses, activating, and reading of the transient data storage and averaging. For construction of an efficient automatic instrument, perturbations should be avoided by galvanic separation of the different units using optoelectronic coupling as much as possible. Application of many field pulses of a given polarity will lead to electrophoretic motion toward one electrode and, thus, the polarity of subsequent pulses should be changed. Another potential source of problems are photochemical reactions. These reactions may be avoided by using a shutter, which is opened only during recording of transients.
4 Experimental Procedures
4.1 Preparation of Samples
The electric dichroism is usually measured at low salt concentrations in order to keep the electric conductivity of the samples as low as possible. At low conductivities the temperature increase of the samples caused by Joule heating during the pulses remains low; furthermore, the decay of the field strength resulting from current flow is also minimized; finally electric polarizabihties of polyelectrolytes are usually maximal at low salt concentrations. In most cases the requirement of low salt concentrations does not impose restrictions. DNA double helices, for example, are quite stable down to low salt concentrations. In most cases the binding of drugs to DNA is stabilized at low salt concentrations, because of an increase of electrostatic interactions.
Because the electric dichroism is very much dependent on the ionic strength, it is very important to do the experiments at a well-defined salt concentration. Of course, the pH of the solutions also must be well-defined Because of the high charge density of DNA, ions of different types are bound with high affinity The most effective procedure for removal of different ions, including other contaminations of low molecular weight, is dialysis. Because multivalent ions bind very strongly to DNA, in particular at low salt concentration, removal of these ions like Mg2+ or Ca2+ requires extensive dialysis against buffers containing EDTA. It is recommended either to exclude bivalent ions by dialysis and addition of a sufficient concentration of EDTA, or to add a well-defined concentration of bivalent ions, e.g., 100 µMMg2+ According to this procedure it is easily possible to get a sufficiently well-defined ionic milieu, which is important, because small variable residual bivalent ion concentrations may strongly affect electro-optical results. Among the different buffers available, cacodylate proved to be useful (pH range 5.3–7.3), because of the small temperature dependence of its pK and because of the strong suppression of bacterial growth.
Some of the technical precautions to be used for unperturbed measurements have been mentioned already above and are not repeated here For any measurement of the electric dichroism, first the sample should be at a well-defined temperature. An accurate control of the temperature is essential for evaluation of the time constants: the viscosity of aqueous solutions is very much dependent on the temperature and, thus, comparison of data and any quantitative evaluation require strict control of the temperature. Usually, the electric field pulse is adjusted to a length, which is sufficient to drive the dichroism to its stationary level. Evaluation of data by any orientation function requires stationary values of the electric dichroism. It is useful to collect data over a broad range of electric field strengths, as broad as possible under the given experimental conditions, which are defined by the pulse generator and the signal-to-noise ratio
Usually it is sufficient to determine the electric dichroism by measurements with light that is polarized parallel to the field vector. However, it is important to check whether the condition defined by Eq. 1 is satisfied. Thus, some transients should also be obtained with light polarized perpendicular to the field vector. Deviations from Eq. 1 may indicate either problems of the optical setup, e.g., strain in the windows of the measuring cell, or some field induced reaction, e.g., dissociation of a ligand or some change of the DNA structure. A further examination is possible by measurements at the “magic angle,” corresponding to an angle of 54.7° of the plane of polarized light with respect to the field vector (8). Under these conditions dichroism effects should disappear and, thus, any remaining field induced changes of the light intensity should be caused by reaction effects
The electric dichroism may be characterized at any wavelength that may be convenient for the measurements. One of the criteria for the selection of an appropriate wavelength is the availability of sufficient light intensity. The signal-to-noise ratio increases with the square root of the light intensity. The highest light intensities are provided at the mercury lines of xenon/mercury high pressure arc lamps. Mercury emission lines are at 248.2, 265 2, 280.4, 289.4, 302.2, 313.2, 334.2, 366.3, 404.7, 435.8, and 546 1 nm; thus useful lines are available for most applications A second cntenum is the position in the absorbance band. Usually (π* ↓ π)-transitions are polarized in the plane of aromatic chromophors, but there are also (π* ↓ ∌)-transitions that are usually polarized in perpendicular direction to the aromatic plane. Thus, the wavelength used for measurements should be selected carefully.
As usual the signal-to-noise ratio may be increased by averaging of transients. Because the signal-to-noise ratio value increases with the square root of the number of transients, an increase of signal-to-noise ratio by a factor of 5, for example, requires 25 transients In order to check for any potential damage of the solutions after a series of measurements, the first shot should be repeated at the end, and the results should be compared. Another useful control is a comparison of absorbance spectra before and after the measurements of the electric dichroism
4.3 Evaluation of Data
4.3.1 Stationary Dichroism
Using the isotropic absorbance A measured at the same wavelength, the electric dichroism is given by These values are determined at different electric field strengths E, where E is the voltage of the applied pulse divided by the electrode distance.
Obviously, the most convenient systems are those, where the dichroism decay can be represented by single exponentials. The observation of more than a single relaxation process may indicate a special, nonsymmetric shape, internal flexibility, or heterogeneity of the sample under investigation The last possibility may be easily checked, e.g., by gel electrophoresis. A decision between the first two possibilities is usually more difficult. It should be mentioned that the theory predicts five exponentials for the dichroism decay of rigid particles without symmetry (9). However, usually most of these exponentials are associated with undetectably low amplitudes The number of clearly detectable processes obtained in many simulations in the author’s laboratory on rigid macromolecules of very different shape did not exceed two. Evidence for the case of internal flexibility may be obtained from a dependence of the observed amplitudes on the electric field strength, because flexible molecules may be stretched under the influence of high electric field pulses.
DNA double helices are known to behave like rigid rods up to chain lengths in the range around 100 bp. The decay of the electric dichroism of rigid rods is represented by single exponentials The observation of single exponential decays of the electric dichroism for much longer DNA chains reported in the literature appears to be partly because of experimental conditions (limited electric field strength) and partly because of a limited experimental accuracy.
4.3.3 Approximate Procedures
Although a complete characterization of the parameters of a system under investigation is always desirable, it often happens that this is rather difficult or even impossible for various reasons. Under such conditions, approximate procedures may be very useful. For the case of drug-DNA complexes such an approximate procedure has been used by Colson et al. (10) for the determination of the orientation of drugs relative to that of the DNA base pairs.
During application of the electric field pulse, the DNA-ethidium complexes in the solution are aligned to a stationary state, which is characterized by a clear increase of the light transmission, corresponding to a negative electric dichroism (cf. Eq. 8). For an interpretation of this observation, information derived from independent experiments on DNA is used: It is known that DNA double helices are aligned by electric field pulses with their helix axis parallel to the direction of the electric field. In addition, we use the fact that absorption of light in the main absorbance band of aromatic compounds like ethidium is polarized in the aromatic plane. In summary, this means that the aromatic planes of the ethidium molecules bound to the DNA must be oriented preferentially in perpendicular direction to the axis.
The analysis of the experimental data may be driven into more detail. For example, the degree of ethidium binding to the DNA should be considered. According to a binding constant obtained for a similar buffer (15), ≈98% of the ethidium molecules are bound to the DNA. If the accuracy of all the experimental data is sufficiently high and all corrections are taken into account, it is possible to determine the fraction of intercalated ethidium molecules precisely, and also get the relatively small fraction of ethidium molecules attached to the outside of the helix
6 Related Experimental Techniques
6.1 Electric Birefringence
The electric birefringence (1–4) is very similar to the electric dichroism. The only difference is the optical parameter used for detection of field-induced orientation. In the case of the birefringence, orientation of the molecules is detected by measurements of the anisotropy of the refraction, whereas the anisotropy of the absorbance is used in the case of the dichroism Measurements of the birefringence can be very sensitive; some authors even conclude that the birefringence is more sensitive than the dichroism; obviously the sensitivity depends very much on the technical details of the instrument used for the measurements and, thus, general statements are hardly justified. A clear advantage of the dichroism is the fact that its interpretation in terms of molecular structure is more simple and straightforward
6.2 Linear Dichroism Induced by Flow Velocity Gradients
Macromolecules may be aligned by flow velocity gradients and this alignment may be studied by measurements of the linear dichroism (16). Various forms of this technique have been used. An advantage of the method is the fact that it may be used at any salt concentration. However, applications are restricted to relatively long polymers. Furthermore, the flow dichroism cannot be used to get information about rotational diffusion in the time range below milliseconds.
6.3 Fluorescence Detected Dichroism
Drugs containing an aromatic component often emit fluorescence, which may be used for a selective measurement of the dichroism (17). Various experimental procedures are possible. One of them is use of polarized light for excitation, as usual in measurements of the dichroism, and detection of the dichroism by collection the fluorescence light under magic angle conditions, i.e., behind polarizers orientated at an angle of 54.7° with respect to the field vector (18). Use of magic angle conditions simplifies the evaluation to the standard procedure, because under these conditions the measured fluorescence intensity is dependent on the molecular orientation only because of the angular dependence of the excitation process, whereas the light intensity resulting from emission itself is independent of the molecular orientation.