Electric birefringence study of the purple membrane of Halobacterium halobium

An electric birefringence study was carried out on aqueous suspensions of the purple membrane of Halobacterium halobium. In addition to the characterization of both native and modified membrane samples, the dependence of electric birefringence on pH and ionic strength was also investigated. The results indicate that purple membrane shows electric birefringence at a field strength as low as 200 V/cm. The permanent dipole moment and polarizability ranged from 20,500 debyes and 1.01 × 10^?14 cm³ for a purple membrane concentration of 0.40 mg/mL to 41,000 debyes and 2.05 × 10^?14 cm³ for a concentration of 0.80 mg/mL. It was also found that removal of the retinyl group of bacteriorhodopsin substantially decreases but does not eliminate the electric birefringence of the membrane. The solubilization of the membrane by Triton X-100, however, completely abolishes the electric birefringence. These experiments indicate that there is an interaction between adjacent bacteriorhodopsin molecules within the purple membrane via the retinyl chromophore moiety that builds up the permanent dipole moment. They also suggest that there are two types of response when purple membrane suspensions are placed in an electric field. One is an alignment of the disk-shaped particles with the field. The other is a stacking of the particles following their alignment by the electric field, which is promoted by the induced dipole moment.     

Flexibility of nucleosomal DNA

In this study transient electric birefringence (TEB) has been used to investigate the molecular flexibility of short fragments of DNA. Nucleosomal DNA always exhibits negative birefringence and Kerr behavior was observed up to high field strengths (6 KV/cm). The value of the Kerr constant is 3.5 10^?2 e.s.u.. Birefringence decays were single exponentials and a field dependence of the molecular orientational relaxation time τ was found: it is explained by an inherent flexibility of the DNA molecule. A 20 % decrease in the calculated length was observed with fields applied as low as 2 KV/cm. The results obtained at very low fields establish TEB as a method well suited to calculate accurate values for the length of small fragments of DNA: the τ value of 4.3 μsec corresponds to a DNA length of 660 Å.     

Electric birefringence of restriction enzyme fragments of DNA: optical factor and electric polarizability as a function of molecular weight

The electric birefringence of restriction enzyme fragments of DNA has been investigated as a function of DNA concentration, buffer concentration, and molecular weight, covering a molecular weight range from 80 to 4364 base pairs (bp) (6 × 10⁴–3 × 10⁶ daltons). The specific birefringence of the DNA fragments is independent of DNA concentration below 20 μg DNA/ml, but decreases with increasing buffer concentration, or conductivity, of the solvent. At sufficiently low field strengths, the Kerr law is obeyed for all fragments. The electric field at which the Kerr law ends is inversely proportional to molecular weight. In the Kerr law region the rise of the birefringence is accurately symmetrical with the decay for fragments ≤ 389 bp, indicating an induced dipole orientation mechanism. The optical factor calculated from a 1/E extrapolation of the high field birefringence data is ?0.028, independent of molecular weight; if a 1/E² extrapolation is used, the optical factor is ?0.023. The induced polarizability, calculated from the Kerr constant and the optical factor, is proportional to the square of the length of the DNA fragments, and inversely proportional to temperature. Saturation curves for DNA fragments ≤ 161 bp can be described by theoretical saturation curves for induced dipole orientation. The saturation curves of larger fragments are broadened, because of a polarization term which is approximately linear in E, possibly related to the saturation of the induced dipole in high electric fields. This “saturated induced dipole” is found to be 6400 D, independent of molecular weight. The melting temperature of a 216-bp sample is decreased 6°C in an electric field of 8 kV/cm, because the lower charge density of the coil form of DNA makes it more stable in an electric field than the helix form.     

Stretching and overstretching of DNA in pulsed field gel electrophoresis. I. A quantitative study from the steady state birefringence decay

Using a sensitive birefringence instrument, the birefringence arising from the orientation of the DNA chain during electrophoretic transport has been recorded. This birefringence is shown to proceed both from the alignment (stretching) of the molecule in the direction of the electric field and from the extension of the length of its primitive path (overstretching). The contribution of these two processes can be separated in the decay of the birefringence after the end of the application of the electric field. The fast relaxation of the overstretching occurs first and is demonstrated to be the main contribution to the birefringence. The orientation factor of the remaining stretched state and its decay can be quantitatively understood using the biased reptation model. It provides, in addition, a high value for the tube diameter or gel pore size a (4500 ± 450 Å for a 0.7% agarose gel with a c^?0.6_g dependence in the agarose concentration c_g) and a low value for the effective charge per base pair (0.2e as compared to 0.5e using the condensation hypothesis). The contribution of overstretching to the birefringence is also quantitatively interpreted in term of the change in the mean length l of DNA inside a pore size a. The dynamics of decay of this overstretching is well represented by a stretched exponential with a stretching exponent α = 0.44. The mean decay time decreases slightly with increasing fields and scales with the overall DNA length close to N²₀. © 1993 John Wiley & Sons, Inc.     

Decay processes of electric birefringence in chemically reacting system

Analytical and numerical calculations of the decay processes of the electric birefringence in an isomerizing system have been performed. Two modes of isomerization are considered: in the first mode, the direction of the axis (of the optical anisotropy) of a molecule is conserved during isomerization; in the second mode, it is not. In the first mode, if G_Ais not equal to G_B, and that, if k_A^op_A² is not equal to k_B^op_B² (when the orienting electric field is weak), in which G_A, G_B and p_A, p_b are the optical anisotropy and permanent electric dipole moments, of the molecule in two states A and B of the isomerization, and k_A^o and k_B^o are the rate constants for the transitions A → B and B → A, respectively, and if diffusion rates are very much slower than the chemical rates, the relaxation due to the chemical reaction can be detected in the decay of the electric birefringence. In the second mode, if diffusion rates are somewhat slower than the chemical rates, the relaxation due to the chemical reaction appears in the decay, even though there are no differences in optical anisotropy and electric moments in the two states. When the rotary diffusion coefficients are different in the two states, the decay process becomes almost one-component, bringing the chemical rate about ten times higher to diffusion coefficients in both modes.     

Electric properties of macromolecules. IX. Dipole moment, polarizability, and optical anisotropy factor of collagen in solution from electric birefringence

The electric birefringence of collagen solutions has been measured over a wide range of field strength with the pulse technique. The soluble collagen was from rat tail tendon. The solvent used was dilute acetic acid. Very pronounced saturation of the electric birefringence was observed, permitting calculation of the optical anisotropy factor. The Kerr constant was determined by extrapolation to zero field strength. From the dependence on field strength of the birefringence, the permanent dipole moment and the anisotropy of polarizability were separately determined. The contribution of the former to the Kerr constant was found to be twice as large as that of the latter. The same conclusion was obtained from the initial slope of the rise curves of the birefringence at low fields. The permanent dipole moment was 1.5 × 10⁴ Debye, and the anisotropy of polarizability was about 3 × 10^?15 cm.³. The magnitude of the latter indicates that the ion atmosphere polarization is important. Effects of added salt and thermal denaturation on the electric birefringence were explored.     

Electro-optic scattering studies on deoxyribonucleic acid

Measurements have been made of the intensity of light scattered from aqueous solutions of calf thymus DNA with and without the application of electric fields. For fields approaching 150 V/cm and frequencies below 2.5 KHz, changes (ΔI) of up to 10% in the residual scattered intensity were observed. In agreement with previous dielectric and electric birefringence measurements, a low frequency dispersion of ΔI was observed, from which a rotary diffusion constant (D) of 1200 s^?1 was determined. Interpreting the electric field data in terms of the classical dipolar orientation theory led to values of 2.4 × 10^?25 cm (7.4 × 10^?14 esu) and 4.3 × 10^?25 cm (13 × 10^?14 esu) for the permanent dipole moment and the anisotropy of the electric polarisabilities respectively. Furthermore the permanent dipole moment was along the major molecular axis and the particles orientated in the field as rigid entities. The zero field data indicated a molecular shape which was not rodlike but corresponded to the Kratky-Porod “stiffness” parameter of x = 24 for the wormlike coil model. Although curved, the molecules appeared to orientate in low-intensity electric fields as rigid, but not rodlike molecules. The implications of this on recent discrepancies in D determined by two or more dynamic relaxation methods is briefly discussed.     

Electric birefringence of native DNA in an alternating field

The relaxation of the birefringence of native DNA in solution was investigated in a pulsed sine-wave electric field. Relaxation times were calculated from the degree of damping of the birefringence signal and were studied as a function of the strength and frequency of the applied field, the molecular weight of the DNA, and the viscosity and ionic strength of the solvent. Relaxation times decrease with increasing field strength. For high-molecular weight DNA (>10⁶ daltons), the relaxation times decreased with frequency and increased less than linearly with viscosity. For low-molecular-weight DNA (<6 × 10⁵ daltons), the relaxation times were independent of frequency, increased linearly with viscosity, and varied with the 1.65 ± 0.1 power of the molecular weight. The average birefringence of high-molecular-weight DNA decreased with frequency in 0.001M Na₂ EDTA plus NaOH, pH 7.0, but is much less frequency-dependent if the EDTA concentration is reduced tenfold, while the average birefringence of sonicated DNA increases in both solvents with increasing frequency.     

Transient electric birefringence of agarose gels. II. Reversing electric fields and comparison with other polymer gels

The transient electric birefringence of low electroendosmosis (LE) agarose gels oriented by pulsed unidirectional electric fields was described in detail in Part I [J. Stellwagen and N. C. Stellwagen (1994), Biopolymers, Vol. 34, p. 187]. Here, the birefringence of LE agarose gels in rapidly reversing electric fields, similar in amplitude and duration to those used for field inversion gel electrophoresis, is reported. Symmetric reversing electric fields cause the sign of the birefringence of LE agarose gels, and hence the direction of orientation of the agarose fibers, to Oscillate in phase with the applied electric field. Because of long-lasting memory effects, the alternating sign of the birefringence appears to be due to metastable changes in gel structure induced by the electric field. If the reversing field pulses are equal in amplitude but different in duration, the orientation behavior depends critically on the applied voltage. If E < 7 V/cm, the amplitude of the birefringence gradually decreases with increasing pulse number and becomes unmeasurably small. However, if E > 7 V/cm, the amplitude of the birefringence increase more than 10-fold after ～ 20 pulses have been applied to the gel, suggesting that a cooperative change in gel structure has occurred. Because there is no concomitant change birefringence must be due to an increase in the number of agarose fibers and /or fiber bundles orienting in the lectric field, which in turn indicates a cooperatice breakdown of the noncovalent “junction zones” that corss-link the fibers in to the fgel matrix. The sign of the birefringence of LE agarose gels is always positive after extensive junction zone breakdown, indicating that the agarose fibers and fiber bundles preferentially orient parallel to the lectric field when they are freed from the constraints of the gel matrix. Three other gel-forming polymers, high electroendosmosis (HEEO) agarose (a more highly changed agarose), β-carrageenan (a stereoisomer of agarose), and polyacrylamide (a chemically corss-linked polymer) were alos studied in unidirectional and rapidly reversing electric fields. The birefringence of HEEO agarose backbone chain. The β-carrageenan gels exhibit variable orientation behavior in reversing electric fields, suggesting that its internal gel structure is not as tightly interconnected as that of agaroise gels. Both HEEO agarose and β-carrageenan gels exhibit a large increase in the amplitude of the birefringence with increasing pulse number when asymmetric reversing pulses > 7 V/cm are applied to the gels, suggesting that junction zone breakdown in a common feature of polysaccharide gels. Chemically cross-linked polyacrylamide gels exhibit very small birefringence signals, indicating that very little orientation occurs in pulsed lectric fields. The sign of the birefringence is independent of the polarity of the lectric field, as expected from the Kerr law, and normal orientation behavior is observed in reversing electric fields. Hence, the anomalous change in sign of the birefringence observed for agarose gels in reversing electric fields must be due to the metastable junction zones in the agarose gel matrix, which allow gel fiber rearrangements to occur. © 1994 John Wiley & Sons, Inc.     

Nonlinear electric properties of DNA solutions.

DNA solutions are shown to present a nonlinear electric behavior. This property is measured through the third harmonic current intensity, which appears when the solution is placed in a sinusoidal electric field of moderately high strength (about 100 V·cm^?1). The influence of different parameters has been examined: fundamental frequency, field strength, concentration, molecular weight, and conformation. By progressive sonication, it is shown that the harmonic current is linearly proportional to the DNA molecular weight, but under an M_r of approximately 10⁶, the nonlinear electrical property decreases sharply and its spectrum shows a drastic change. It is thought that the harmonic current is not related to an orientational phenomenon; an explanation based on the electrical deformation of the molecule is suggested.     

A Birefringence Relaxation Determination of Rotational Diffusion of Magnetotactic Bacteria

The orientational relaxation of the magnetotactic bacterium Aquaspirillum magnetotacticum is observed by the decay of the optical birefringence upon switching off an aligning magnetic field. The data yield a rotational diffusion constant D_r [unk] 0.13 s^-1 and information about cell sizes that is consistent with optical microscopy data.     

Electric birefringence of eukaryotic ribosomes

Eukaryotic 60S ribosomal subunits were studied by transient electric birefringence. Conformations of subunits in active and inactive states upon changes in magnesium concentration were compared by electric birefringence and orientational relaxation time measurements. Active subunits exhibit a positive birefringence and a relaxation time of the order of 8 microseconds. In the presence of EDTA, inactive subunits show no birefringence. When Mg2+ is reverted in the cold to its initial level, the electro-optical properties of the subunits are partially restored although the particles remain biologically inactive.     

Transient electric birefringence of T-even bacteriophages. I. T4B in the absence of tryptophan and fiberless T4 particles

Measurements of the electric birefringence of suspensions of T4B in the absence of tryptophan and of fiberless T4 particles show that both kinds of particles are hydrodynamically equivalent. Their rotational diffusion coefficients corrected to 25°C and water viscosity (D_25,w) are 280 ± 9 sec^?1 and 295 ± 10 sec^?1, respectively. These corrected rotational diffusion coefficients are almost independent of buffer concentration and temperature. The sedimentation coefficient (s_20,w) of T4 B is equal to 1023 ± 12 S, a value which is likewise independent of buffer concentration. By analysis of the field strength dependence of the steady-state birefringence and by reversing pulse experiments it could be shown that the orientation in an electric field is largely due to a permanent dipole moment. This dipole moment is somewhat dependent on buffer concentration and amounts to about 24,000 debye for T4B and 95,000 debye for fiberless T4. An approximate calculation shows that the difference in dipole moment may be ascribed to positive charges on the fiber tip (at least ten per fiber), to negative charges along the fiber or (and) positive charges on the fiberless particle at those places where the fibers are attached in normal particles.     

Electric birefringence of bacterial flagellar protein filaments: evidence for field-induced interactions.

The time course for the build-up and decay of birefringence induced by a rectangular voltage pulse was measured on solutions of flagellar filaments from Salmonella equi-abortus (strain SJ25). These filaments are tubular polymers of protein (degree of polymerization ≈ 10³) constituted by non-covalent linkage of flagellin monomers of molecular weight 4 × 10⁴. The effect on electro-optical properties of solutions of filaments due to variations in temperature, concentration and mean length of protein filaments, and the duration and intensity of the applied electric field is described. Analysis of the field intensity dependence of the birefringence and comparison of the build-up and decay processes indicate that orientation in the field is due primarily to the existence of a permanent dipole moment in the filaments. At 18 °C the following values were obtained for a solution of filaments with mean length and standard deviation of 0.39 and 0.30 μm: specific Kerr constant (K_sp) = 6.14 × 10⁻³ electrostatic units; optical anisotropy factor (g₁ — g₂) = 5.66 × 10⁻³; dipole moment (μ) = 1.01 × 10⁵ Debye units; and mean relaxation time ($\text{}\text{̄}$gt) = 9.20 ms. At temperatures below 20 °C there is a marked increase in the optical anisotropy factor of the filaments which may be due to a change in their flexibility. The large values of K_sp obtained indicate the highly responsive nature of these filaments to an electric field. The birefringence decay curves were decomposed by computer into a specified number of exponential terms from which both the mean length and the size distribution of these polydisperse filaments were calculated. The results obtained were in substantial agreement with the values of these parameters observed by electron microscopy. A cumulative field effect dependent on field intensity and filament concentration was observed. Repeated pulsing of electric field, above threshold values of field intensity and filament concentration, produced decreases in the birefringence near 60% of its initial value. The effect was reversible with a time constant greater than two minutes. No appreciable change in the relaxation time for decay of birefringence was observed on multiple pulsing of these solutions. These results are interpreted consistently to arise from the sidewise aggregation of filaments induced by electrical impulses of sufficient intensity and duration. These properties appear relevant to bacterial motility: variations in electric potential along the membrane of the bacterium might serve first to orient these organelles and then to induce their coalescence to “bundles” of filaments. The latter structures are commonly observed in vivo. In this way the activity of flagella might be co-ordinated.     

The effects of cooperativity, finite chain length, and field-induced changes of the conformational equilibrium on the electric birefringence of polypeptides in the range of the helix-coil transition.

An electric birefringence apparatus with an He–Ne laser is described, which permits a high sensitivity of measurement for field strengths up to 20 kV/cm and pulse durations between 10^?5 to 1 sec. It is applied to poly(γ-benzyl-L -glutamate) and poly(β-benzyl-L -aspartate) in a nonaqueous solvent mixture, especially under the conditions of the helix-coil equilibrium. A theoretical treatment of the problem shows that the usual neglect of end effects is only justified for comparatively long chains. On the basis of a computational procedure taking into account the finite chain lengths, the experimental data for 15 kV/cm reveal an obvious enhancement of the coil-to-helix transition caused by the electric field. This effect is quantitatively confirmed by a pertinent theory. The cooperative parameter σ may be best determined from the initial linear increase of the bire-fringence versus the square of the field strength where the conformational equilibrium is not affected.     

Structural aspects and orientation mechanism of mitochondrial F1 adenosinetriphosphatase. Evidence for a negative electric birefringence due to a permanent moment perpendicular to the long axes of the particle

The electric birefringence technique was used to investigate the steady-state birefringence, the orientational relaxation time, and the orientation mechanism of pig heart mitochondrial F1 adenosine-5'-triphosphatase (F1-ATPase). The electrooptical properties of this enzyme in solution were studied as functions of pH, protein concentration, and applied electric field. The F1-ATPase exhibits a surprising negative electric birefringence with a specific Kerr constant of -1.5 X 10(-3) esu cgs. The field-independent relaxation time was found to be 0.65 +/- 0.05 microseconds, corresponding to a rotational diffusion constant of 2.55 X 10(5) s-1. The overall size and shape of F1-ATPase have been calculated from both translational and rotational diffusion constants. The enzyme may be assumed to be an oblate ellipsoid of revolution with dimensions of about 170 X 170 X 70 A. The orientation mechanism of F1-ATPase was analyzed by fitting experimental birefringence rising curves with theoretical rising functions. The ratio of the permanent to induced dipole moment is found to be very high; therefore, the birefringence of F1-ATPase is due to a strong permanent dipole moment in a direction perpendicular to the long axes of the particle. These particular electric properties can be explained by the oligomeric structure of the protein and seem likely to play a role in its mechanism of functioning.     

Electric Birefringence of Dilute Agarose Solutions

Abstract

The technique of transient electric birefringence was used to investigate the orientation of agarose solutions in pulsed electric fields. If the agarose was dissolved in deionized water, the sign of the birefringence was positive when the electric field was small, indicating that the agarose molecules were orienting parallel to the electric field lines. The decay of the birefringence was rapid, consistent with the orientation of individual agarose helices. The amplitude of the birefringence, but not the birefringence decay times, increased as the agarose solution aged, suggesting that the helices formed slowly from the sol state. Increasing the amplitude or duration of the pulsed electric field caused additional negative, and then positive, birefringence signals to appear, characterized by much slower rise and decay times, consistent with the formation of aggregates. The slowest decay times ranged from 7.5–9.0 s, suggesting that the aggregates were several microns in size. When agarose was dissolved in dilute Tris buffer instead of deionized water, the fast positive birefringence signal was not observed, suggesting that individual helices were not present in solutions containing dilute buffer.     

Transient electric birefringence of agarose gels. I. Unidirectional electric fields

The orientation of agarose gels in pulsed electric fields has been studied by the technique of transient electric birefringence. The unidirectional electric fields ranged from 2 to 20 V/cm in amplitude and 1 to 100 s in duration, values within the range typically used for pulsed field gel electrophoresis (PFGE). Agarose gels varying in concentration from 0.3 to 2.0% agarose were studied. The sign of the birefringence varied randomly from one gel to another, as described previously [J. Stellwagen & N. C. Stellwagen (1989), Nucleic Acids Research, Vol. 17, 1537–1548]. The sign and amplitude of the birefringence also varied randomly at different locations within each gel, indicating that agarose gels contain multiple subdomains that orient independently in the electric field. Three or four relaxation times of alternating sign were observed during the decay of the birefringence. The various relaxation times, which range from 1 to ～ 120 s, can be attributed to hierarchies of aggregates that orient in different directions in the applied electric field. The orienting domains range up to ～ 22 μm in size, depending on the pulsing conditions. The absolute amplitude of the birefringence of the agarose gels increased approximately as the square of the electric field strength. The measured Ker constants are ～ 5 orders of magnitude larger than those observed when short, high-voltage pulses are applied to agarose gels. The increase in the Kerr constants in the low-voltage regime parallels the increase in the relaxation times in low-voltage electric fields. Birefringence saturation saturation curves in both the low- and high-voltage regimes can be fitted by theoretical curves for permanent dipole orientation. The apparent permanent dipole moment increase approximately as the 1.6 power of fiber length, consistent with the presence of overlapping agarose helices in the large fiber bundles orienting in low-voltage electric fields, the optical factor is approximately independent of fiber length. Therefore, the marked increase in the Kerr constants observed in the low-voltage regime is due to the large increase in the electrical orientation factor, which is due in turn to the increased length of the fiber bundles and domains orienting in low-voltage electric fields. Since the size of the fiber bundles and domains approximates the size of the DNA molecules being separated by PFGE, the orientation of the agarose matrix in the applied electric field may facilitate the migration of large DNA molecules during PFGE. © 1994 John Wiley & Sons, Inc.     

Light Scattering Investigation of Electric Field Alignment of Phospholipid Tubules

The polymerizable diacetylenic phospholipid 1,2-bis(10,12,tricosadiynoyl)-sn-glycero-3-phosphocholine (DC₂₃PC) forms straight hollow cylinders in water. Using an ac electric field it was possible to achieve significant orientational alignment of the tubules parallel to the field direction, and from light scattering results deduce an effective dielectric susceptibility anisotropy Δχ_E. Moreover, we suggest that the alignment arises from an orientational anisotropy of the total electrostatic enthalpy for a dielectric tubule in an electric field, rather than an inherent polarizability anisotropy of the constituent DC₂₃PC molecules, as was the case with magnetic field alignment.     

Reversing-pulse electric birefringence of poly(γ-benzyl-L-glutamate). I. Transient behavior of fractionated samples in the low electric field region

Reversing-pulse electric birefringence (RPEB) was measured for the first time for four fractionated poly(γ-benzyl-L -glutamate), [Glu(OBzl)]_n, samples in N,N-dimethylformamide (DMF) at 20°C and at 535 nm. The RPEB signal showed a deep minimum for each sample on reversal of an applied electric field. The profiles of the reverse-transient signal were analyzed by taking into account the polydispersity for the continuous distribution of molecular lengths. The best set of three quantities (l_w, l_w/l_n, (β_w)²/2_γw), which determine a signal profile, was evaluated for each sample. By combining the experimental data of intrinsic viscosity and RPEB, the diameter of a cylinder, which is assumed for the [Glu(OBzl)]_n helix, was found to be 17 Å. The value (β_w)²/2_γw, which is related to the ratio of weight-average permanent dipole moment μ_w, and electric polarizability anisotropy Δα_w, was found to be in the range of 20–45. This indicates that the former contributes predominantly to electric field orientation, but the latter also should not be ignored. With the three parameters from the reverse portion, the rise and decay curves were regenerated theoretically in excellent agreement with experimental signals.