BIOPOLY MERS
VOL. 16, 573-582 (1977)
Probing DNA Quaternary Ordering with Circular Dichroism Spectroscopy: Studies of Equine Sperm Chromosomal Fibers M. LEONIDE SIPSKI and THOMAS E. WAGNER, Department of Chemistry, Ohio University, Athens, Ohio 45701
Synopsis Studies are described which strongly support a cholesteric liquid crystal-like quaternary structure for the DNA molecules of a biologically native chromosomal preparation from equine sperm cells. Discrete chromosomal fibers released from the head pieces of equine spermatozoon cells were prepared intact and probed for liquid crystalline ordering using reflectance and linear dichroism spectroscopy. Assuming cholesteric liquid crystalline order for the DNA molecules within the chromatin fibers, parameters measured experimentally were used to calculate the circular dichroism (CD) of the fibers. The calculated results compare remarkably well with the experimentally measured CD of the sperm chromosomal fibers and suggest a specific cholesteric liquid crystal-like quanternary structural ordering of DNA molecules in equine sperm chromatin fibers. The potential of CD spectroscopy as a tool for the study of long-range ordering of macromolecules is discussed.
INTRODUCTION DNA condenses from its extended configuration in dilute solution to a compact, relatively dense ($) state in concentrated solutions of neutral or anionic polymers as a result of excluded volume interactions.' This rearrangement of the DNA molecules brings about dramatic alterations in the circular dichroism (CD) spectra.2 The transition involves loss of the conservative spectrum of B-form DNA resulting in a curve which more nearly resembles the negative of the absorption spectrum with a slight shift in the wavelength of the peak. The magnitude of the ellipticity is extraordinarily enhanced. The kinetics of the CD change and the X-ray scattering data of DNA condensed in this way3 suggest that the +-spectra depend on interhelix interactions and reflect a higher order arrangement of the DNA molecules. The structural model proposed by Lerman4 on the basis of fluorescence and polarized microscopy is that of a twisted stack of layers of parallel molecules not unlike that of a cholesteric liquid crystal. DNA in the $-state is of interest, therefore, as an analogue of chromosomal structure and the packing of DNA in viruses. Recently Wagner et al.5 were able to effect the release of the contents of the heads of equine sperm through a disulfide reduction. The CD of the released strands resembles that seen in $-DNA; the curve exhibits a large 573 (c)1977 by
John Wiley & Sons, Inc
574
SIPSKI AND WAGNER
230 240 280 260 270 280 290 300
0
WAVELENGTH (nm) Fig. 1. CD spectra of equine sperm chromosomalmaterial (- - -) observed, (- - -) calculated; and of DNA (-).
negative ellipticity and a slight shift in the wavelength of the peak to 265 nm (Fig. 1). We propose that the structure of the DNA in these fibers is similar to that of $-DNA. We will attempt to show that the features of these CD spectra can be explained qualitatively and roughly quantitatively by invoking a cholesteric liquid crystal model for the arrangement of the molecules in this $-like chromosomal DNA. Cholesteric liquid crystals are noted for their remarkable optical properties including extremely strong rotatory power and selective reflection of circularly polarized light in a narrow region of wavelengths.6 The mesophase has a Cotton effect in the absorbing region of its spectra with the CD band shapes generally resembling those of the absorption bands, but with a "hook," sometimes generating CD of opposite sign, at their long- or short-wavelength edge. These optical characteristics are a direct result of the ordered helical cholesteric structure. Holzwarth et aL73have extended the theory of electromagnetic radiation in liquid crystals6s9to the absorbing case by adding a frequency-dependent complex contribution to the spiraling dielectric tensor of the liquid crystallo allowing calculation of the CD spectra. It was our purpose to determine the measurable input parameters, namely, the linear dichroism and reflectance spectra of the released equine sperm chromatin. These measurements allowed us to determine the orientation of the DNA helix relative to the chromatin fibers and the pitch of the cholesteric arrangement of the molecules. The theory of liquid crystal electromagnetic radiation was then applied to our system allowing calculation of the theoretical CD spectrum in an investigation of the ordered arrangement of the molecules.
DNA QUATERNARY ORDERING
575
MATERIALS A N D METHODS Whole stallion semen (60 ml) was centrifuged a t 5000 rpm for 10 min, washed several times with distilled water to remove all the seminal fluid, and suspended in 100 ml of distilled water. The suspended sperm were treated with ultrasound (Bronwill Biosonic I11 at 60% maximum intensity) for 5 min a t 4OC to dissociate the sperm head pieces from sperm midpieces and tail sections. Centrifugation of the sonicated whole sperm at 8000 rpm for 15 min yielded a mixed pellet of sperm head pieces, midpieces, and tails which were separated by ultracentrifugation a t 28,000 rpm for 70 min in a 2.4 M sucrose suspension. The purified sperm head-piece pellet was washed and resuspended in 2.5 ml of 0.05 M tris buffer pH 8.8 containing 0.005 M EDTA and 0.1 M guanidine HC1 and incubated a t 4OC for several M in 2-merhours. Following incubation the solution was made 5 X captoethanol resulting in the immediate release of chromatin from the suspended head pieces. For the linear dichroism measurements a small amount of the viscous transparent chromatin was transferred to a quartz slide and aligned undirectionally by stroking until good birefringence was seen when viewed between crossed polarizors. The sample was then covered with a quartz coverslip using a plastic spacer to maintain the film thickness at a constant value of 2.54 X cm. The concentration of the film samples varied from 0.130 to 0.168 M . The linear dichroism was measured on the Durrum-Jasco 5-20 Spectropolarimeter modified as mentioned under Instrumentation. The sample was aligned with the stroking direction parallel to the plane of the electric vector of the incident radiation. The resultant curve is a plot of the linear dichroism ( A11 - A I). Rotation of the sample through a 90' turn (alignment of the sample stroking direction perpendicular to the plane of the incident electric vector) produces a plot of A I - A 11. Measurement of the critical wavelength requires that the sample studied be in the plane-texture form, that is, the helical (optical) axes of the cholesteric arrangement are at right angles to the sample surface. Samples for the reflectance measurements were prepared by transferring a small amount of chromatin to a quartz plate and squeezing between two plates until relatively pure spectral colors appeared for incident white light to achieve the plane-texture form. To obtain a sufficient amount of selectively reflected light for the measurement, the sample was illuminated over an area % inches in diameter. This requires a plane-texture uniform over this area. Nonuniformities could be caused by impurities and by deviations of the helical symmetry axis of the crystal from the normal of the sample surface. Since the degree of nonuniformities depends on purity, on unknown interactions at the interfaces, and on thermal history, reproducible results were achieved for a given sample by controlling these factors. The samples were carefully prepared to avoid effects due to surface structures, electrostatic and chemical interactions. The temperature throughout the preparation (and incubation) was maintained a t 4OC until the time of the
576
SIPSKI A N D WAGNER
measurement, a t which time the sample was allowed to warm to room temperature. To assure that the sample was oriented in the plane-texture form, the linear dichroism of samples prepared for reflectance spectroscopy was also measured. In the plane-texture form the helical axes (optical axes) of the cholesteric arrangement are parallel to the incoming radiation, i.e., perpendicular to the plane of the quartz slide. The DNA helices in the molecular layers are then parallel, and thus the bases perpendicular, to the plane of the slide. As the molecules in the layers rotate tracing out the helix, the planes of the DNA bases also rotate to align every 90” with raThe net linear diation incoming either vertically (11) or horizontally (I). dichroism for a plane-textured sample is therefore zero. Although a totally disoriented sample would also be expected to have no net linear dichroism, such samples did not exhibit the selective reflectivity. A sample in which the fibers themselves might have assumed a macroscopic “plane-textured” form would a t some rotational orientation give a net linear dichroism. Samples carefully prepared as above having no linear dichroism and exhibiting the selective reflection of light were taken as being sufficiently oriented in the plane-texture form. Since perfect alignment of the plane texture could not be achieved, the absolute value of the selective reflectivity was not measured. The thickness of the sample film then did not need to be known. The reflectance measurements were made on a Durrum-Jasco 5-20 Spectropolarimeter modified as mentioned under Instrumentation. The sample was placed a t the back opening of the integrating sphere and the relative intensity of reflection measured over the wavelength range of 210-440 nm.
INSTRUMENTATION A Durrum-Jasco 5-20 Spectropolarimeter normally used to measure CD spectra was modified to measure linear dichroism spectra. In the CD mode, the pockel cell modulates plane polarized light into circularly polarized light rotating first left then right. For linear dichroism, the pockel cell was modified to vary between zero retardation (zero voltage) and half-wave retardation (half-wave voltage). The linear polarized light then radiates with first vertical then horizontal polarization. Upon passing through a sample whose stroking direction is aligned vertically, the differential light absorption would be measured as A 11 - A I,the linear dichroism. The linear dichroism mode of our instrument, thus modified, was calibrated using the angularily dependent refraction of a fused quartz plate. A fused quartz plate was mounted in the sample chamber of the instrument on a goniometer head so that the angle of the quartz surface to the incident light beam could be maintained a t discrete fixed angles. The signal from the transmitted beam was measured a t several angles between 0” and 15’ and compared to the transmittance calculated from equations for the refracted beams from light polarized parallel and perpendicular to the tilted plate.
DNA QUATERNARY ORDERING
577
:;(
fraction of light transmitted when beam 4 -COS 4i polarized parallel to tilted quartz plate = ( Z C O S
fraction of light transmitted when beam polarized perpendicular to tilted quartz plate =
)
(COS
4t)
4i) (cos 4t)2
4
4 -cos 4. (cos 4t)
;;(
(2
cos Qt) (cos 4iY
where N1 is the refractive index of air, N2 is the refractive index of quartz (1.500 at 265 nm), q$ is the angle of incidence, and dt is the angle of transmittance. This calibration shows that our instrument is within 5%of the calculated values, an error which is smaller than that introduced by the physical orientation of the chromosomal fibers. For measuring the diffuse reflectance of our samples an integrating sphere reflectance attachment was used with incident circularly polarized light. Radiation reflected from the sample in all directions is intercepted by the sphere and rereflected to the photomultiplier tube for detection. The reflection so measured is reflectance dichroism; any reflected right circularly polarized light is subtracted from the reflected left circularly polarized light. Since for a cholesteric liquid crystal one-half of the incident intensity is reflected with one sense of circular polarization while the other half is transmitted with the opposite sense;ll the sign of the reflectance dichroism indicates the sense of the reflected wave.
CALCULATIONS For wave numbers outside the critical region of reflection, the CD (A[,
- A R )(base 10) is given approximately by8
where Re [6, ( V ) ] is the real part of 6, ( V ) , etc. The data were converted to molar ellipticity through the relation [O] = 3300 [ A A / ( c b ) where ] [O] = degree cm2/dmol, AA = difference absorbance (base lo), b = cm, and c = mol/l. The linear birefringence is symbolized by 60 and to is the mean dielectric constant. Here s denotes sample thickness. The imaginary parts of t(V) were calculated straightforwardly from the measured absorption spectra A ( V ) and A ( ; ) , in base 10. The corresponding real parts of t,(V) and 6, (7) are their Kronig-Kramers transforms and were evaluated numerically. All calculations were performed in the near-uv over the wavelength range of 216-310 nm. The model used to represent the ordered form of DNA can be seen in Figure 2. It closely parallels the structural arrangement of molecules in
578
SIPSKI AND WAGNER
Fig. 2. Conceptual model of the cholesteric liquid crystal-like folding of DNA molecules into a sperm chromosomal fiber.
a cholesteric liquid crystal.12 The sample was assumed to be a perfect cholesteric liquid crystal with a finite thickness measured experimentally. The molecules are arranged in layers being aligned parallel to each other within the layers. The log axes of the molecules in each layer are slightly rotated from those in neighboring layers m d therefore trace out a helix. The directions of circular polarization of a cholesteric liquid crystals are determined by the screw sense of the helix.13 The totally negative ellipticity observed for equine sperm chromosomal fibers corresponds to a helix with a right-handed screw direction. The opposite sense of twist changes the sign of the calculated curve. The orientationof the crystal in a magnetic field would be with the helix axis perpendicular and the crystalline planes parallel to the field. The long axes of the molecules would be at all angles to the field. The absorption data used in the calculations were that of the linear dichroism measured in equine sperm chromatin as described. The thickness
DNA QUATERNARY ORDERING
579
WAVELENGTH (nm) / /
-002I
,/--',
/
\
/
\
-.004-
/
\
/
\
/
\
-005-
/
\-,'
-008-
Fig. 3. Linear dichroism spectra of equine sperm chromosomal material. Curve A ( A 1 A I)(-); Curve B, rotation of sample go", ( A I - A ,I) (- - -).
of the film was 2.5 X cm. The concentration of the film samples varied from 0.130 to 0.168 M . The choice of dielectric constant was made on the basis that the fibers in the sperm head immediately upon release contain little water.14 The mean dielectric constant used, consequently, was that of vacuum-dessicated (%11%water) Na-DNA at 25OC and low frequency, which was 3.1.15 The critical wave number of the reflection band used was that determined in the reflectivity measurements (310 nm). This parameter is important in that it is related to the pitch of the cholesteric arrangement and the optical characteristics of the model are a direct result of the ordered structure. This parameter has a great effect on the magnitude of the spectra and is quite critical to proper evaluation. As a cholesteric arrangement of DNA molecules would represent a well-ordered arrangement, the maximum birefringence commonly observed in DNA fibers was used (0.1O).l6 Varying this parameter from 0.09 to 0.15 produced a corresponding change in the minimum of -4.1 X lo4 to -6.7 X lo4 degcm2/dmol.
RESULTS The linear dichroism spectrum for equine sperm chromatin is given in Figure 3. Curve A for ( A11 - A I)closely resembles the shape of the absorption curve of DNA. The sign of the resultant dichroism AA is positive indicating that the parallel component is being absorbed more strongly than the perpendicular component. A plot of A I - A 11 seen in Curve B (produced by rotation of the sample 90°)yields a dichroism of approximately
580
SIPSKI AND WAGNER
the same magnitude, but opposite in sign, again indicating greater absorption in the parallel direction. The magnitude of the two curves would be expected to be equal given perfect alignment of the molecules with stroking. Native DNA on the other hand has a greater absorption for the electric vector perpendicular to the fiber length.17J8 As the molecules align with their long axes in the direction of shear, DNA absorbs more strongly in a direction normal to the helix axis, in the direction of the planes of the bases. The equine sperm chromatin strands again orient their fiber length in the direction of shear. In order to give a greater absorption in the parallel direction the planes of the bases must be parallel to the direction of the fiber length, that is, the helix axis must be at a 90’ angle to the fiber length. The molecular orientation in the fiber would then be similar to a pleated ribbon. The released strands of equine sperm chromatin must retain some structural arrangement upon release with their helix axis a t right angles to the fiber length. One of the distinguishing features of the cholesteric liquid crystal is the helical arrangement of the molecular planes which can be characterized by a screw direction and a pitch. If white light is incident parallel to the helix axis of the liquid crystal (also the optical axis), it is found that a narrow wavelength band will be reflected while the others are transmitted.6 The reflectance spectrum of equine sperm chromatin (
VOL. 16, 573-582 (1977)
Probing DNA Quaternary Ordering with Circular Dichroism Spectroscopy: Studies of Equine Sperm Chromosomal Fibers M. LEONIDE SIPSKI and THOMAS E. WAGNER, Department of Chemistry, Ohio University, Athens, Ohio 45701
Synopsis Studies are described which strongly support a cholesteric liquid crystal-like quaternary structure for the DNA molecules of a biologically native chromosomal preparation from equine sperm cells. Discrete chromosomal fibers released from the head pieces of equine spermatozoon cells were prepared intact and probed for liquid crystalline ordering using reflectance and linear dichroism spectroscopy. Assuming cholesteric liquid crystalline order for the DNA molecules within the chromatin fibers, parameters measured experimentally were used to calculate the circular dichroism (CD) of the fibers. The calculated results compare remarkably well with the experimentally measured CD of the sperm chromosomal fibers and suggest a specific cholesteric liquid crystal-like quanternary structural ordering of DNA molecules in equine sperm chromatin fibers. The potential of CD spectroscopy as a tool for the study of long-range ordering of macromolecules is discussed.
INTRODUCTION DNA condenses from its extended configuration in dilute solution to a compact, relatively dense ($) state in concentrated solutions of neutral or anionic polymers as a result of excluded volume interactions.' This rearrangement of the DNA molecules brings about dramatic alterations in the circular dichroism (CD) spectra.2 The transition involves loss of the conservative spectrum of B-form DNA resulting in a curve which more nearly resembles the negative of the absorption spectrum with a slight shift in the wavelength of the peak. The magnitude of the ellipticity is extraordinarily enhanced. The kinetics of the CD change and the X-ray scattering data of DNA condensed in this way3 suggest that the +-spectra depend on interhelix interactions and reflect a higher order arrangement of the DNA molecules. The structural model proposed by Lerman4 on the basis of fluorescence and polarized microscopy is that of a twisted stack of layers of parallel molecules not unlike that of a cholesteric liquid crystal. DNA in the $-state is of interest, therefore, as an analogue of chromosomal structure and the packing of DNA in viruses. Recently Wagner et al.5 were able to effect the release of the contents of the heads of equine sperm through a disulfide reduction. The CD of the released strands resembles that seen in $-DNA; the curve exhibits a large 573 (c)1977 by
John Wiley & Sons, Inc
574
SIPSKI AND WAGNER
230 240 280 260 270 280 290 300
0
WAVELENGTH (nm) Fig. 1. CD spectra of equine sperm chromosomalmaterial (- - -) observed, (- - -) calculated; and of DNA (-).
negative ellipticity and a slight shift in the wavelength of the peak to 265 nm (Fig. 1). We propose that the structure of the DNA in these fibers is similar to that of $-DNA. We will attempt to show that the features of these CD spectra can be explained qualitatively and roughly quantitatively by invoking a cholesteric liquid crystal model for the arrangement of the molecules in this $-like chromosomal DNA. Cholesteric liquid crystals are noted for their remarkable optical properties including extremely strong rotatory power and selective reflection of circularly polarized light in a narrow region of wavelengths.6 The mesophase has a Cotton effect in the absorbing region of its spectra with the CD band shapes generally resembling those of the absorption bands, but with a "hook," sometimes generating CD of opposite sign, at their long- or short-wavelength edge. These optical characteristics are a direct result of the ordered helical cholesteric structure. Holzwarth et aL73have extended the theory of electromagnetic radiation in liquid crystals6s9to the absorbing case by adding a frequency-dependent complex contribution to the spiraling dielectric tensor of the liquid crystallo allowing calculation of the CD spectra. It was our purpose to determine the measurable input parameters, namely, the linear dichroism and reflectance spectra of the released equine sperm chromatin. These measurements allowed us to determine the orientation of the DNA helix relative to the chromatin fibers and the pitch of the cholesteric arrangement of the molecules. The theory of liquid crystal electromagnetic radiation was then applied to our system allowing calculation of the theoretical CD spectrum in an investigation of the ordered arrangement of the molecules.
DNA QUATERNARY ORDERING
575
MATERIALS A N D METHODS Whole stallion semen (60 ml) was centrifuged a t 5000 rpm for 10 min, washed several times with distilled water to remove all the seminal fluid, and suspended in 100 ml of distilled water. The suspended sperm were treated with ultrasound (Bronwill Biosonic I11 at 60% maximum intensity) for 5 min a t 4OC to dissociate the sperm head pieces from sperm midpieces and tail sections. Centrifugation of the sonicated whole sperm at 8000 rpm for 15 min yielded a mixed pellet of sperm head pieces, midpieces, and tails which were separated by ultracentrifugation a t 28,000 rpm for 70 min in a 2.4 M sucrose suspension. The purified sperm head-piece pellet was washed and resuspended in 2.5 ml of 0.05 M tris buffer pH 8.8 containing 0.005 M EDTA and 0.1 M guanidine HC1 and incubated a t 4OC for several M in 2-merhours. Following incubation the solution was made 5 X captoethanol resulting in the immediate release of chromatin from the suspended head pieces. For the linear dichroism measurements a small amount of the viscous transparent chromatin was transferred to a quartz slide and aligned undirectionally by stroking until good birefringence was seen when viewed between crossed polarizors. The sample was then covered with a quartz coverslip using a plastic spacer to maintain the film thickness at a constant value of 2.54 X cm. The concentration of the film samples varied from 0.130 to 0.168 M . The linear dichroism was measured on the Durrum-Jasco 5-20 Spectropolarimeter modified as mentioned under Instrumentation. The sample was aligned with the stroking direction parallel to the plane of the electric vector of the incident radiation. The resultant curve is a plot of the linear dichroism ( A11 - A I). Rotation of the sample through a 90' turn (alignment of the sample stroking direction perpendicular to the plane of the incident electric vector) produces a plot of A I - A 11. Measurement of the critical wavelength requires that the sample studied be in the plane-texture form, that is, the helical (optical) axes of the cholesteric arrangement are at right angles to the sample surface. Samples for the reflectance measurements were prepared by transferring a small amount of chromatin to a quartz plate and squeezing between two plates until relatively pure spectral colors appeared for incident white light to achieve the plane-texture form. To obtain a sufficient amount of selectively reflected light for the measurement, the sample was illuminated over an area % inches in diameter. This requires a plane-texture uniform over this area. Nonuniformities could be caused by impurities and by deviations of the helical symmetry axis of the crystal from the normal of the sample surface. Since the degree of nonuniformities depends on purity, on unknown interactions at the interfaces, and on thermal history, reproducible results were achieved for a given sample by controlling these factors. The samples were carefully prepared to avoid effects due to surface structures, electrostatic and chemical interactions. The temperature throughout the preparation (and incubation) was maintained a t 4OC until the time of the
576
SIPSKI A N D WAGNER
measurement, a t which time the sample was allowed to warm to room temperature. To assure that the sample was oriented in the plane-texture form, the linear dichroism of samples prepared for reflectance spectroscopy was also measured. In the plane-texture form the helical axes (optical axes) of the cholesteric arrangement are parallel to the incoming radiation, i.e., perpendicular to the plane of the quartz slide. The DNA helices in the molecular layers are then parallel, and thus the bases perpendicular, to the plane of the slide. As the molecules in the layers rotate tracing out the helix, the planes of the DNA bases also rotate to align every 90” with raThe net linear diation incoming either vertically (11) or horizontally (I). dichroism for a plane-textured sample is therefore zero. Although a totally disoriented sample would also be expected to have no net linear dichroism, such samples did not exhibit the selective reflectivity. A sample in which the fibers themselves might have assumed a macroscopic “plane-textured” form would a t some rotational orientation give a net linear dichroism. Samples carefully prepared as above having no linear dichroism and exhibiting the selective reflection of light were taken as being sufficiently oriented in the plane-texture form. Since perfect alignment of the plane texture could not be achieved, the absolute value of the selective reflectivity was not measured. The thickness of the sample film then did not need to be known. The reflectance measurements were made on a Durrum-Jasco 5-20 Spectropolarimeter modified as mentioned under Instrumentation. The sample was placed a t the back opening of the integrating sphere and the relative intensity of reflection measured over the wavelength range of 210-440 nm.
INSTRUMENTATION A Durrum-Jasco 5-20 Spectropolarimeter normally used to measure CD spectra was modified to measure linear dichroism spectra. In the CD mode, the pockel cell modulates plane polarized light into circularly polarized light rotating first left then right. For linear dichroism, the pockel cell was modified to vary between zero retardation (zero voltage) and half-wave retardation (half-wave voltage). The linear polarized light then radiates with first vertical then horizontal polarization. Upon passing through a sample whose stroking direction is aligned vertically, the differential light absorption would be measured as A 11 - A I,the linear dichroism. The linear dichroism mode of our instrument, thus modified, was calibrated using the angularily dependent refraction of a fused quartz plate. A fused quartz plate was mounted in the sample chamber of the instrument on a goniometer head so that the angle of the quartz surface to the incident light beam could be maintained a t discrete fixed angles. The signal from the transmitted beam was measured a t several angles between 0” and 15’ and compared to the transmittance calculated from equations for the refracted beams from light polarized parallel and perpendicular to the tilted plate.
DNA QUATERNARY ORDERING
577
:;(
fraction of light transmitted when beam 4 -COS 4i polarized parallel to tilted quartz plate = ( Z C O S
fraction of light transmitted when beam polarized perpendicular to tilted quartz plate =
)
(COS
4t)
4i) (cos 4t)2
4
4 -cos 4. (cos 4t)
;;(
(2
cos Qt) (cos 4iY
where N1 is the refractive index of air, N2 is the refractive index of quartz (1.500 at 265 nm), q$ is the angle of incidence, and dt is the angle of transmittance. This calibration shows that our instrument is within 5%of the calculated values, an error which is smaller than that introduced by the physical orientation of the chromosomal fibers. For measuring the diffuse reflectance of our samples an integrating sphere reflectance attachment was used with incident circularly polarized light. Radiation reflected from the sample in all directions is intercepted by the sphere and rereflected to the photomultiplier tube for detection. The reflection so measured is reflectance dichroism; any reflected right circularly polarized light is subtracted from the reflected left circularly polarized light. Since for a cholesteric liquid crystal one-half of the incident intensity is reflected with one sense of circular polarization while the other half is transmitted with the opposite sense;ll the sign of the reflectance dichroism indicates the sense of the reflected wave.
CALCULATIONS For wave numbers outside the critical region of reflection, the CD (A[,
- A R )(base 10) is given approximately by8
where Re [6, ( V ) ] is the real part of 6, ( V ) , etc. The data were converted to molar ellipticity through the relation [O] = 3300 [ A A / ( c b ) where ] [O] = degree cm2/dmol, AA = difference absorbance (base lo), b = cm, and c = mol/l. The linear birefringence is symbolized by 60 and to is the mean dielectric constant. Here s denotes sample thickness. The imaginary parts of t(V) were calculated straightforwardly from the measured absorption spectra A ( V ) and A ( ; ) , in base 10. The corresponding real parts of t,(V) and 6, (7) are their Kronig-Kramers transforms and were evaluated numerically. All calculations were performed in the near-uv over the wavelength range of 216-310 nm. The model used to represent the ordered form of DNA can be seen in Figure 2. It closely parallels the structural arrangement of molecules in
578
SIPSKI AND WAGNER
Fig. 2. Conceptual model of the cholesteric liquid crystal-like folding of DNA molecules into a sperm chromosomal fiber.
a cholesteric liquid crystal.12 The sample was assumed to be a perfect cholesteric liquid crystal with a finite thickness measured experimentally. The molecules are arranged in layers being aligned parallel to each other within the layers. The log axes of the molecules in each layer are slightly rotated from those in neighboring layers m d therefore trace out a helix. The directions of circular polarization of a cholesteric liquid crystals are determined by the screw sense of the helix.13 The totally negative ellipticity observed for equine sperm chromosomal fibers corresponds to a helix with a right-handed screw direction. The opposite sense of twist changes the sign of the calculated curve. The orientationof the crystal in a magnetic field would be with the helix axis perpendicular and the crystalline planes parallel to the field. The long axes of the molecules would be at all angles to the field. The absorption data used in the calculations were that of the linear dichroism measured in equine sperm chromatin as described. The thickness
DNA QUATERNARY ORDERING
579
WAVELENGTH (nm) / /
-002I
,/--',
/
\
/
\
-.004-
/
\
/
\
/
\
-005-
/
\-,'
-008-
Fig. 3. Linear dichroism spectra of equine sperm chromosomal material. Curve A ( A 1 A I)(-); Curve B, rotation of sample go", ( A I - A ,I) (- - -).
of the film was 2.5 X cm. The concentration of the film samples varied from 0.130 to 0.168 M . The choice of dielectric constant was made on the basis that the fibers in the sperm head immediately upon release contain little water.14 The mean dielectric constant used, consequently, was that of vacuum-dessicated (%11%water) Na-DNA at 25OC and low frequency, which was 3.1.15 The critical wave number of the reflection band used was that determined in the reflectivity measurements (310 nm). This parameter is important in that it is related to the pitch of the cholesteric arrangement and the optical characteristics of the model are a direct result of the ordered structure. This parameter has a great effect on the magnitude of the spectra and is quite critical to proper evaluation. As a cholesteric arrangement of DNA molecules would represent a well-ordered arrangement, the maximum birefringence commonly observed in DNA fibers was used (0.1O).l6 Varying this parameter from 0.09 to 0.15 produced a corresponding change in the minimum of -4.1 X lo4 to -6.7 X lo4 degcm2/dmol.
RESULTS The linear dichroism spectrum for equine sperm chromatin is given in Figure 3. Curve A for ( A11 - A I)closely resembles the shape of the absorption curve of DNA. The sign of the resultant dichroism AA is positive indicating that the parallel component is being absorbed more strongly than the perpendicular component. A plot of A I - A 11 seen in Curve B (produced by rotation of the sample 90°)yields a dichroism of approximately
580
SIPSKI AND WAGNER
the same magnitude, but opposite in sign, again indicating greater absorption in the parallel direction. The magnitude of the two curves would be expected to be equal given perfect alignment of the molecules with stroking. Native DNA on the other hand has a greater absorption for the electric vector perpendicular to the fiber length.17J8 As the molecules align with their long axes in the direction of shear, DNA absorbs more strongly in a direction normal to the helix axis, in the direction of the planes of the bases. The equine sperm chromatin strands again orient their fiber length in the direction of shear. In order to give a greater absorption in the parallel direction the planes of the bases must be parallel to the direction of the fiber length, that is, the helix axis must be at a 90’ angle to the fiber length. The molecular orientation in the fiber would then be similar to a pleated ribbon. The released strands of equine sperm chromatin must retain some structural arrangement upon release with their helix axis a t right angles to the fiber length. One of the distinguishing features of the cholesteric liquid crystal is the helical arrangement of the molecular planes which can be characterized by a screw direction and a pitch. If white light is incident parallel to the helix axis of the liquid crystal (also the optical axis), it is found that a narrow wavelength band will be reflected while the others are transmitted.6 The reflectance spectrum of equine sperm chromatin (
