Electrical and hydrodynamical properties of polypeptides in solution. I. Poly(L-glutamic acid) in methanol-water mixtures

The electric birefringence of poly(L -glutamic acid) (PLGA) in methanol–water mixtures has been measured by the use of the rectangular pulse technique at 25°C. The permanent dipole moment, the anisotropy of electrical polarizability, and the optical anisotropy factor of PLGA in solution were obtained from the dependence of the steady-state birefringence on the electric field strength. Further, the mean length of PLGA in solution was calculated by a parameter method developed for analyzing the decay curve of electric birefringence. The permanent dipole moment per unit length obtained from these studies was 2.9₆, 2.4₈, 2.3₀, 2.6₆ D/Å in pure methanol, 10, 30, and 50 vol-% water, respectively. The increase of water content caused the decrease of the mean length and broadened the length distribution of PLGA. These results are discussed in relation to the viscosity and the electrical conductivity of PLGA solutions.     

Electric and hydrodynamic properties of polypeptides in solution. IV. A new conformational change of poly(L-glutamic acid) in dimethylsulfoxide–methanol mixtures

The electric birefringence of poly(L -glutamic acid) (PLGA) in dimethylsulfoxide (DMSO)–methanol mixtures has been measured by use of the rectangular pulse technique. The length distribution curve, the mean molecular length, and the mean apparent permanent dipole moment of PLGA in solution have been obtained from the decaycurve and field strength dependence of the steady-state birefringence according to the method developed for analyzing the electric birefringence of a polydisperse system. The length distribution curve exhibits one or two peaks. The length corresponding to a high peak and the mean length of PLGA undergo an abrupt change in the vicinity of 50 to 60 vol % DMSO at 30°C. Moreover, a sharp change of the Moffitt b₀ parameter with the solvent composition is observed. These results provide evidence for the existence of a solvent-induced transition from a helical conformation (presumably α-helix) to another helical conformation with shorter length per amino acid residue. Further, the temperature dependence of the length distribution of PLGA in 50 vol % DMSO suggests the existence of a temperature-induced helix ? helix transition.     

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.     

Electric and hydrodynamic properties of polypeptides in solution. 3. Aggregation of poly(L-glutamic acid) in aqueous methanol solvents studied by electric birefringence

The rise and decay of electric birefringence for poly(L -glutamic acid) (PLGA) in aqueous solvents containing 20 and 10 vol % methanol have been found to be unusual. The decay curves have been analyzed on the assumption that there exist two kinds of particles, namely, one (component I) with a shorter relaxation time exhibiting positive birefringence and the other (component II) with a longer relaxation time exhibiting negative birefringence at low fields. From the field strength dependence of the steadystate birefringence the permanent dipole moment, the anisotropy of electric polarizability, and the saturation value of birefringence have been determined for each component. Furthermore, from the relaxation time the length of component I and the diameter of component II have been computed on the models of cylindrical rod and oblate ellipsoid, respectively. The dipole moment, the anisotropy of electric polarizability, and the relaxation time of component II are much larger than those of component I. Both the anisotropy of electric polarizability and the optical anisotropy factor are positive in sign for component I and negative for component II. It is concluded that component I is the helical PLGA molecule itself and component II is the side-by-side (antiparallel) aggregate composed of many helical PLGA molecules. The optical anisotropy factor of each component has been discussed on the basis of Peterlin-Stuart theory.     

Electric birefringence of poly-L-lysine hydrobromide in methanol-water mixtures and helix-coil transition induced by high electric fields

The electric birefringence of poly-L -lysine hydrobromide in methanol–water mixtures has been measured at 25 °C over a wide range of field strengths by use of the rectangular pulse technique. An abrupt change in the specific Kerr constant was observed between 87 and 90 vol % methanol, corresponding to the solvent-induced helix–coil transition. The specific Kerr constant increased rapidly with dilution in the random coil form, and more slowly in the helical conformation. The field strength dependence of the bire fringence at various concentrations, for both the helical and coil conformations, can be described by a common orientation function, which resembles the theoretical one for the case of permanent dipole moment orientation. This is interpreted in terms of the saturation of ion–atmosphere polarization. The optical anisotropy for the helical conformation was much larger than that for the coil form. Anomalous birefringence signals were observed above a critical field strength (about 5 kV/cm) in 90 vol % methanol. The birefringence passed through a maximum and began to decrease slowly before the pulse terminated, reaching a steady-state value. This steady-state value was closer to that of the coil in the coil in the limit of very high fields. The results indicate that a transition from the charged helix to the charged coil is induced by high electric fields in the transition region. This effect can be explained on the basis of the polarization mechanism proposed by Neumann and Katchalasky.     

Further evidence of the protonation of the C-terminal of the poly-γ-benzyl-L-glutamate helix by acids

The change of the specific Kerr constant upon the addition of several acids to poly-γ-benzyl-L -glutamate solution in ethylene dichloride has been measured by the rectangular pulse method. The addition of a small amount of strong acid (trifluoroacetic acid, trichloroacetic acid, monochloroacetic acid, or hydrogen chloride) caused a rapid decrease of the specific Kerr constant. On the other hand, the effect of weak acids (formic acid, acetic acid, and propionic acid) was small. These facts show that the apparent dipole moment of a helical poly-γ-benzyl-L -glutamate molecule is considerably diminished by the protonation of terminal peptide groups. The electric conductivity of poly-γ-benzyl-L -glutamate solution in ethylene dichloride–dichloroacetic acid mixtures has been measured. It was found that the specific conductivity per unit concentration of poly-γ-benzyl-L -glutamate increased considerably at small fractions of dichloroacetic acid. This shows that poly-γ-benzyl-L -glutamate can react as a base (proton acceptor) with dichloroacetic acid. This result also confirms the previous conclusion.     

Effect of aliphatic alcohols on the conformation of poly (l-ornithine hydrobromide) as studied by square-pulse and reversing-pulse electric birefringence

The electric birefringence and circular dichroism spectra of poly(l-ornithine hydrobromide) have been measured in ethanol/water, 2-propanol/water and tertiary butyl alcohol/water mixtures of various compositions. This charged polypeptide underwent a transition from the coil conformation to the helical conformation at high alcohol content in every case tested. Anomalous birefringence signals, indicative of a field-induced helix-to-coil transition. were observed at high electric fields only in the case of ethanol/water mixtures. The reversing-pulse electric birefringence of this polypeptide has been studied in ethanol/water mixtures and in neutral aqueous solution. Upon rapid reversal of the pulse field, no transient could be observed. This confirms that the electric-field orientation of poly(l-ornithine hydrobromide) results predominantly from the contribution of the counterion-induced dipole moment, regardless of its molecular conformations. It is very probable that the backbone permanent dipole moment of the helical conformation is largely suppressed by the counterion-induced dipole moment in the ionized form.     

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.     

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.     

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.     

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.     

Kerr constants of amides and peptides

Molar Kerr contants _mK have been determined for N-methylacetamide, N-ethyl-acatamide, N-methylpropionamide, N-acetylglycine-N′-methylamide, N-acetyl-L -ala-nine-N′-methylamide, and N-acetyl-L -leucine-N′-methylamide from electric birefringence measurements on dilute solutions in dioxane. The resultes are compared with configurational averatges 〈_mK〉 calculated on the basis of conformational energies. Values of_mK calculated for various fixed confromations on the peptides vary over a wide range depending on the choice of conformation; hece, the configurational averages 〈_mK〉 are sensitive to smalll changes in the conformational enerey. Kerr constants are calculated for random coiled oligoglycine and oligoalanine peptides as functions of chain length. The Kerr constant is an especially acute index of the average conformation.     

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.     

Circular dichroism studies of α-aminoisobutyric acid-containing peptides: Chain length and solvent effects in alternating Aib-L-Ala and Aib-L-Val sequences

The CD spectra of the peptides Boc-X-(Aib-X)_n-OMe (n = 1, 2, 3) and Boc-(Aib-X)₅-OMe, where X = L -Ala or L -Val have been examined in several solvents. The X = Ala and Val peptides behave similarly in all solvents, suggesting that the Aib residues dominate the folding preferences of these peptides. The decapeptides adopt helical conformations in methanol and trifluoroethanol, with characteristic negative CD bands at 222 and 205 nm. In the heptapeptides, similar spectra with reduced intensities are observed. Comparison with nmr studies suggest that estimates of helical content in oligopeptides by CD methods may lead to erroneous conclusions. The pentapeptides yield solvent-dependent spectra indicative of conformational perturbations. Peptide association in dioxane results in an unusual spectrum with a single negative band at 210 nm for the decapeptides. Disaggregation is induced by the addition of methanol or water to dioxane solutions. Aggregation of the heptapeptides is less pronounced in dioxane, suggesting that a critical helix length may be necessary to promote association stabilized by helix dipole–dipole interactions.     

Electric and flow birefringence properties of myosin in aqueous urea and sodium pyrophosphate.

The electric dipole moment of rabbit skeletal myosin was estimated from the electric and flow birefringence properties. Myosin formed small polydisperse aggregates (0.2-1.1 microM in length) with an apparent electric dipole moment of 5,000-20,000 Debye in aqueous urea or sodium pyrophosphate at low ionic strength. Permanent dipole moment contributed substantially to the apparent dipole moment. An anti-parallel association of myosin was suggested from the dependence of the apparent dipole moment on myosin concentration. Some interactions between myosin and C-protein were detected in 1 M urea by flow birefringence and analytical ultracentrifugation studies. The apparent dipole moment of myosin aggregates was less dependent on myosin concentration in the presence of C-protein.     

Electro-optical studies on the electric field-induced helix-to-coil transition of poly(l-ornithine hydrobromide) in methanol/water mixtures

Transient electric birefringence and circular dichroism measurements have been made on poly(L-ornithine hydrobromide) in methanol/water mixtures of various compositions. The specific Kerr constant and the molar ellipticity at 222 nm underwent an abrupt change between 93 and 98% (v/v) methanol at 25°C corresponding to a solvent-induced helix-coil transition. Anomalous birefringence transients were observed at high electric fields between 96 and 98% (v/v) methanol, i.e. on the helix side of the transition region. The double logarithmic plots of the steady-state specific birefringence versus the square of field strength for different solvent compositions could be superimposed on one another by horizontal and vertical shifts, except for the range where anomalous birefringence transient were observed. This behavior served to determine the threshold field strength. The results indicate that a conformational change from the charged helix to the charged coils is induced by high electric fields in this system, as in the cases of poly(L-lysine hydrobromide) and poly(L-αγ-diaminobutyric acid hydrochloride) in methanol/water mixtures.     

Electric dichroism studies: interaction of trifluoroacetic acid with poly (gamma-benzyl L-glutamate)

Electric dichroism studies on poly(γ-benxyl L -glutamate) show that the addition of small amounts (up to 5%) of trifluoroacetic acid causes complete disappearance of dichroism in contrast to the electric birefringence, which drops to an observable plateau at 20% of its initial value. This loss of dichroism cannot be explained simply by a decrease in the effective dipole moment of the benzyl ester side chains, and the nature of this interaction is explored by nuclear magnetic resonance and infrared spectral studies. Theories of the helix-coil transition which require an initial protonation of the helix backbone to form a more flexible rod consisting of helical segments interrupted by regions of random coil are shown to be inadequate to describe the changes in poly(γ-benzyl L -glutamate) effected by small amounts of trifluoroacetic acid. Rather, trifluoroacetic acid in small amounts interacts with the ester carbonyl oxygen in the side chain, either by hydrogen bonding or protonation, before there is evidence of any effect on the backbone or of loss of helix content.     

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.     

Electric field effect on the helix–coil transition of poly(L-α,γ-diaminobutyric acid hydrochloride) in methanol/water mixtures

Transient electric birefringence of poly(L -α,γ-diaminobutyric acid hydrochloride) in methanol/water mixtures has been measured over a wide range of field strengths and solvent compositions and at different polymer concentrations and temperatures. The molar ellipticity at 222 nm and the specific Kerr constant underwent an abrupt change between 75 and 80 vol % methanol at 25°C, accompanied by a solvent-induced helix–coil transition. Anomalous birefringence transients were observed between 78 and 80 vol% methanol above threshold field strengths. The double logarithmic plots of the steady-state specific birefringence versus the square of field strength for different solvent compositions and polymer concentrations could be superimposed by shifting them horizontally along the abscissa and vertically along the ordinate except for the range where anomalous transients were observed. The threshold field strength could be estimated from the point at which a downward deviation occurred. It increased with increasing polymer concentration and with increasing methanol content on the verge of the transition region. The results were interpreted as indicating that a conformational change from the charged helix to the charged coil is induced by high fields in this system, as in the case of poly(L -lysine hydrobromide) in methanol/water mixtures.     

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.