Transient electric birefringence study of highly dilute agarose solutions

In order to characterize the first step of agarose gelation, highly dilute solutions (2·10^?3 to 0.5 g/L) have been studied by means of the transient electric birefringence technique. The field-free decay curves of the birefringence are well described by a stretched-exponential B(t) ≈ exp(?t/τ)^β; the value of the exponent β is close to 0.5 whatever the agarose concentration. The suspended particles observed by electron microscopy present a rod-like shape with a constant diameter (～50 Å), without any branching; they are polydisperse with a distribution of lengths approximately exponential. The mean length of these fibers, deduced from their mean rotational diffusion coefficient, is proportional to the 0.37 power of the agarose concentration in the solution. Furthermore, these particles possess a strong permanent electrical dipole confirming the side-to-side arrangement of helices into bundles; this dipole is roughly proportional to the particle length, indicating a self-similarity in the unidirectional growth of the agarose fibers, even when approaching the gelling concentration.     

Electric birefringence in concentrated solutions of tobacco mosaic virus.

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. It was found that if the applied electric field is sufficiently high, the steady-state birefringence becomes becomes positive even at 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.     

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.     

Electric birefringence of myosin subfragments.

Electric birefringence measurements have been made on aqueous solutions of myosin subfragments, heavy meromyosin, subfragments 1 and 2 (S-1 and S-2). All of these showed positive electric birefringence. Heavy meromyosin and S-2 showed a large intrinsic Kerr constant. From the analysis of the build up and decay process of the birefringence, the contribution of the slow induced dipole moment was concluded in heavy meromyosin and S-2, although the existence of the permanent dipole moment was not completely excluded. The decay process of the birefringence of heavy meromyosin was found to consist of two components; the fast one of which had a relaxation time of the same order as that of S-1. This is probably due to the presence of a flexible hinge in heavy meromyosin.     

Improvements in procedures for electrophoresis in dilute agarose gels

Procedures have been developed for performing electrophoresis in agarose gels with agarose concentrations as low as 0.035%. Using these procedures, agarose gel electrophoresis of the following has been performed: (a) bacteriophage T7 missing its tail fibers; no detectable sieving of this spherical particle (radius = 30.1 nm) occurred below 0.075% agarose, (b) duplex DNAs with molecular weights between 26.5 × 10⁶ and 110 × 10⁶.     

Electric birefringence studies of rabbit skeletal tropomyosin

The electric birefringence of rabbit skeletal tropomyosin and its nonpolymerizable derivative was studied as a function of protein concentration, pulse length, and electric field strength. Analyses of the zero field birefringence decay curves show that nonpolymerizable tropomyosin, which has had on average six C-terminal residues removed, and tropomyosin are both well approximated by rigid cylinders in solution at low salt concentrations at 20°C. The measured relaxation times for the monomers of polymerizable and nonpolymerizable tropomyosin are 1.5 ± 0.4 μs and 1.30 ± 0.2 μs, respectively, in good agreement with the values calculated from the known dimensions. For tropomyosin the electrical pulse induces the formation of linear dimers. Orientation occurs primarily by a permanent dipole mechanism. Permanent and induced dipole moments were calculated from reversing field experiments and from the saturation of the birefringence. Removal of the six C-terminal residues decreases the measured permanent dipole moment by 9.5%, from 6300 to 5700 Debyes, which is in good agreement with the 7% decrease calculated for permanent dipole contributions arising from the peptide dipoles and from the asymmetric distribution of the formal charges. This change is due primarily to the removal of Asp 280.     

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.     

Study of large DNA fragments in agarose gels by transient electric birefringence

The pulsed-field gel electrophoresis (PFG) is a newly developing technique used in the fractionation of large DNA fragments. Advances in PFG demand a better understanding in the corresponding mechanisms of DNA dynamics in the gel network. Detailed experiments are needed to verify and to extend existing theoretical predictions as well as to find optimum conditions for efficient separation of large DNA fragments. In the present study, deformation of large DNA fragments (40-70 kilobase pairs) imbedded in agarose gels were investigated by using the transient electric birefringence (TEB) technique under both singular polarity and bipolarity electric pulses at low applied electric field strengths (E less than or equal to 5 V/cm). The steady-state optical retardation (delta s) of DNA molecules is linearly proportional to E2. At a given E, the amplitude of optical retardation [delta(t)] increases monotonically with the pulse width (PW) and then reaches a plateau value [delta(t = 0) = delta s] where t = 0 denotes the time when the applied field is turned off or reversed. The field-free decay time (tau-a few minutes) is several orders of magnitudes slower than that from previous TEB observations using high electric field strengths (E-kV/cm) and short pulse widths (PW-ms). The degree of deformation (stretching and orientation) and the time of restoration to the equilibrium conformation of overall DNA chains have been related to delta and tau. In field inversion measurements, exponentially rising and linearly falling of birefringence signals in the presence of forward/inverse applied fields were observed. The rising and falling of birefringence signals were reproducible under a sequence of alternating pulses. Comparison of our results with literature findings and discussions with theories are presented.     

Fractionation of high molecular-weight duplex DNA molecules on dilute agarose gels

Fractionation of high molecular weight duplex DNAs was studied by electrophoresis on 0.05 to 1% agarose gels. Only the most diluted gels allowed the separation of T4 and T7 phages DNAs. The method makes use of progressive melting of the gel columns and may be applied to composite acrylamide agarose gels.     

Transient electric birefringence studies of xanthan solutions

Transient electric birefringence studies have been performed on heat denatured xanthan in 4 m urea. The induced birefringence was positive, the Kerr law was obeyed at low field amplitudes and the birefringence saturated at high fields. The orientation mechanism appears to be mainly induced dipolar in character and the magnitude of the induced dipole moment can be explained on the basis of counterion polarization. The molecules behave as independent rods of mean length 0.65 μm with no evidence for ‘hindered rotation’ in moderately concentrated solutions. The molecular rigidity is attributed to extension of the polyanion due to charge charge repulsions or steric hindrance due to the side chains.     

Electrical birefringence study of monodisperse collagen solutions

Acid-soluble collagen solution in 0.1 M acetic acid are studied by electrical birefringence. Specific birefringence is independant of concentration (for c < 100 mg/l.) and follows Kerr's law at low fields. Birefringence decays present a single relaxation time, and reversing pulse experiments show a very low contribution of induced moment compared to permanent dipole orientation. This result is also confirmed by birefringence rise measurements.     

Electric birefringence studies on polyglutamic acid.

The electric birefringence for the aqueous solution of poly-L -glutamic acid (PGA) in the helical form was studied. PGA samples were fractionated by gel column chromatography. PGA showed a positive electric birefringence. The permanent dipole moment of the PGA molecule was suggested to be largely suppressed. The measurements of the intrinsic Kerr constants for various molecular lengths showed that the electric anisotropy (polarizability) of PGA is proportional to the 1.5 power of the length. The electric birefrigence measurement was also carried out in the helix–coil transition region. The Kerr constant of PGA was largely reduced on going from the helical form to the coiled form.     

Flow birefringence of xanthan and other polysaccharide solutions

A xanthan sample with molecular weight M = 2.2 x 10(6) was investigated in three solvents: bidistilled water, 0.2 M aqueous NaCl and cadoxen by flow birefringence and viscometry methods in dilute solutions. It was shown that the optical shear rate coefficients of xanthan in aqueous and cadoxen media differ by two orders of magnitude. An estimation of xanthan optical anisotropy in different conformational states has been made and compared with values for other polysaccharides: dextran, pullulan, cellulose and chitosan. The process of denaturation and the flow birefringence of renaturated xanthan in aqueous solutions (after heat treatment at 121 degrees C) have also been studied.     

Convection-free boundary sedimentation of dilute protein solutions

Use of the density gradient sedimentation velocity technique appears to be essential for the accurate determination of the mean sedimentation coefficients of dilute protein solutions. When performed on an analytical ultracentrifuge equipped with a photoelectric-scanning-absorption optical system, the density gradient sedimentation velocity technique has been shown to be particularly useful in studying the subunit association-dissociation equilibria of multisubunit enzyme systems. The time factor has been shown to be a major advantage of the density gradient sedimentation velocity technique, as opposed to the sedimentation equilibrium technique, in studying the subunit association-dissociation equilibria of multisubunit enzymes such as rabbit muscle apo-glyceraldehyde-3-phosphate dehydrogenase, which is very unstable in dilute solution.