Circular Dichroism Studies of the Structure of DNA Complex with Berberine

Abstract

The binding of the benzodioxolo-benzoquinolizine alkaloid, berberine chloride to natural and synthetic DNAs has been studied by intrinsic and extrinsic circular dichroic measurements. Binding of berberine causes changes in the circular dichroism spectrum of DNA as shown by the increase of molar ellipticity of the 270nm band, but with very little change of the 240nm band. The molar ellipticity at the saturation depends strongly on the base composition of DNA and also on salt concentration, but always larger for the AT rich DNA than the GC rich DNA The features in the circular dichroic spectral changes of berberine-synthetic DNA complexes were similar to that of native DNA but depends on the sequence of base pairs.

On binding to DNA and polynucleotides, the alkaloid becomes optically active. The extrinsic circular dichroism developed in the visible absorption region (300–500nm) for the berberine-DNA complexes shows two broad spectral bands in the regions 425–440nm and 340–360nm with the maximum varying depending on base composition and sequence of DNA While the 425nm band shows less variation on the binding ratio, the 360nm band is remarkably dependent on the DNA/alkaloid ratio. The generation of the alkaloid associated extrinsic circular dichroic bands is not dependent on the base composition or sequence of base pairs, but the nature and magnitude of the bands are very much dependent on these two factors and also on the salt concentration. The interpretation of the results with respect to the modes of the alkaloid binding to DNA are presented.     

Circular dichroism studies of the structure of DNA complex with berberine.

The binding of the benzodioxolo-benzoquinolizine alkaloid, berberine chloride to natural and synthetic DNAs has been studied by intrinsic and extrinsic circular dichroic measurements. Binding of berberine causes changes in the circular dichroism spectrum of DNA as shown by the increase of molar ellipticity of the 270nm band, but with very little change of the 240nm band. The molar ellipticity at the saturation depends strongly on the base composition of DNA and also on salt concentration, but always larger for the AT rich DNA than the GC rich DNA. The features in the circular dichroic spectral changes of berberine-synthetic DNA complexes were similar to that of native DNA, but depends on the sequence of base pairs. On binding to DNA and polynucleotides, the alkaloid becomes optically active. The extrinsic circular dichroism developed in the visible absorption region (300-500nm) for the berberine-DNA complexes shows two broad spectral bands in the regions 425-440nm and 340-360nm with the maximum varying depending on base composition and sequence of DNA. While the 425nm band shows less variation on the binding ratio, the 360nm band is remarkably dependent on the DNA/alkaloid ratio. The generation of the alkaloid associated extrinsic circular dichroic bands is not dependent on the base composition or sequence of base pairs, but the nature and magnitude of the bands are very much dependent on these two factors and also on the salt concentration. The interpretation of the results with respect to the modes of the alkaloid binding to DNA are presented.     

Optical properties of hyaluronic acid. Ultraviolet circular dichroism and optical rotatory dispersion

The molar optical rotation at 220 nm and ellipticity values at 210 nm of both sodium hyaluronate and hyaluronic acid are greatly enhanced in comparison to the values for the monomeric units and oligosaccharides indicating a degree of preferred order. With increasing hydrogen ion concentration, there is no appreciable change in the 210 nm circular dichroic band, but the second circular dichroic band below pH 4 changes abruptly to the positive side and reaches a maximum value at pH 2·5. This positive circular dichroic band of hyaluronic acid is temperature and concentration dependent. The major change in sign and position of the second circular dichroic band of hyaluronic acid below pH 4 is attributed to the conformational change of a single polysaccharide chain or to a chain-chain interaction. The results indicate that increase in concentration or decrease in temperature and in the ionization of carboxyl group promotes the formation of ordered cross-link regions. The conformational changes found in solution have been interpreted as an order-disorder transition in the crosslink regions based on the interconversion of random coil and double helix.     

Retention of helical structure of pBR322 covalently closed circular duplex DNA at high alkaline pH

Denaturation of covalently closed circular duplex replicative form (RF) I at high pH yields a form with high sedimentation coefficient even after neutralization. This form allowed less ethidium bromide to be intercalated but yielded a circular dichroic spectrum which had reduced magnitude of both positive circular dichroism at 273 nm and negative circular dichroism at 245 nm. The circular dichroic spectrum of this form is similar to that of RFV DNA. Gel electrophoretic analysis of this DNA revealed that, although part is retained in the groove, another part appeared as a faster-moving band, which we designated as RF I_d. This faster-moving form is cleaved by the restriction endonuclease BamHI at a single site giving a single RF III, comigrating with the RF III obtained from RF I by BamHI cleavage. This signifies that the two strands of RF I did not slide over one another during the formation of RF I_d as suggested previously.     

The effect of low ionic strength on the circular dichroic spectrum of chromatin and nucleosomal subunits

Circular dichroism has been used to measure the conformation changes in the DNA of chromatin and chromatin subunits as a function of ionic strength. Transfer of chromatin from 0.15 M to 0.25 mM salt led to an enhancement of the circular dichroic bands at 275 and 285 nm. Removal of histone H1 did not appreciably affect the circular dichroic spectrum when measured in 0.15 M salt, but in 0.25 mM salt H1 depletion led to a marked increase in the ellipticity. Conformation changes due to low ionic strength were also observed with a 145- and a 172-bp chromatin subunit. A linear combination of the ellipticities of the DNA of the two domains in chromatin, namely core and linker, was successful for measurements at 0.15 M salt, but large unexplained discrepancies appeared with the data from measurements in 0.25 mM salt.     

Circular dichroism of native whole SV40 chromatin.

Circular dichroism properties of SV40 virions, isolated minichromosomes from virions, and SV40 Form I (supercoiled) DNA were studied in a buffer of low ionic strength. The isolated minichromosomes are compact as judged by sedimentation and electron microscopy. The molar ellipticity at 284 nm of the virion, which may be regarded as a minichromosome in its native state, is about 1500 deg cm²/dmol phosphate; this value is in the same range as that reported for core particles (1300–2000) isolated from different sources. When the viral capsid is removed, there is a small increase in the molar ellipticity to about 2000. However, both of these values are much lower than that found for SV40 supercoiled DNA (about 8200). The results strongly suggest that the linker DNA of the native whole chromatin contributes in a similar fashion to the circular dichroic ellipticity as the core DNA.     

Circular dichroism of corticotropin, fragment 1-24, and model compounds. An assessment of the contributions of the peptide chromophore and armoatic residues.

The far-ultraviolet circular dichroic spectrum of the 39-residue peptide hormone porcine corticotropin and the biologically active fragment corticotropin 1–24 is negative from 250 nm to 195 nm in water, but in 6M guanidinium chloride a positive band appears at about 225 nm. The temperature and guanidinium chloride dependence of this spectral transition indicates the absence of any stable ordered secondary structure in corticotropin and the spectrum is seen to be in only partial agreement with results using the model peptide chromophore, Ala-Ala-Ala. Using oligopeptides containing aromatic amino acid residues sandwiched between glycyl residues, it is shown that the shape and intensity of the corticotropin 225 nm positive band which appears in 6M guanidinium chloride is in agreement with the far-ultraviolet transitions of the aromatic chromophores in the hormone. Curve resolution of the near-ultraviolet circular dichroic spectrum of corticotropin and comparison of the rotational strengths of the phenylalanyl and tyrosyl bands reveals no evidence for increased rotational freedom in 6M guanidinium hydrochloride. Spectral changes are observed, however, in the transitions arising from the single tryptophan. This study suggests that corticotropin in aqueous solution may serve as a better model for the circular dichroic spectrum of the aperiodic regions in globular proteins than either synthetic homopolypeptides or reference proteins for which spectral and X-ray diffraction data are available.     

Visual Pigments of Goldfish Cones : Spectral Properties and Dichroism

Freshly isolated retinal photoreceptors of goldfish were studied microspectrophotometrically. Absolute absorptance spectra obtained from dark-adapted cone outer segments reaffirm the existence of three spectrally distinct cone types with absorption maxima at 455 ± 3,530 ± 3, and 625 ± 5 nm. These types were found often recognizable by gross cellular morphology. Side-illuminated cone outer segments were dichroic. The measured dichroic ratio for the main absorption band of each type was 2–3:1. Rapidly bleached cells revealed spectral and dichroic transitions in regions near 400–410, 435–455, and 350–360 nm. These photoproducts decay about fivefold as fast as the intermediates in frog rods. The spectral maxima of photoproducts, combined with other evidence, indicate that retinene₂ is the chromophore of all three cone pigments. The average specific optical density for goldfish cone outer segments was found to be 0.0124 ± 0.0015/µm. The spectra of the blue-, and green-absorbing cones appeared to match porphyropsin standards with half-band width Δν = 4,832 ± 100 cm^–1. The red-absorbing spectrum was found narrower, having Δν = 3,625 ± 100 cm^–1. The results are consistent with the notion that visual pigment concentration within the outer segments is about the same for frog rods and goldfish cones, but that the blue-, and green-absorbing pigments possess molar extinctions of 30,000 liter/mol cm. The red-absorbing pigment was found to have extinction of 40,000 liter/mol cm, assuming invariance of oscillator strength among the three cone spectra.     

Circular dichroism of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus

Circular dichroism (CD) spectra of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus exhibit three positive ellipticity bands between 240 and 300 nm (250, 283, and 292 nm), two negative bands at 327 and 480 nm, and a low-intensity positive band at 390 nm. The fractions of helix β-form, and unordered form of the enzyme are 8, 38, and 54%, respectively. The circular dichroic bands at 327 and 480 nm and a part of the positive bands at 292 and 390 nm are associated with enzyme activity. Significant changes in absorption and CD spectra of the enzyme were observed when the temperature of the enzyme preparation was increased to 47°C, coinciding with the sharp decrease in enzyme activity observed at this temperature.     

Complexes of DNA with histones f2a2 and f3. Circular dichroism studies.

Two histones from calf thymus, the slightly lysine-rich histone f2a2 and the arginine-rich f3, were combined separately, with homologous DNA. The complexes were reconstituted by means of guanidine hydrochloride gradient dialysis, and their circular dichroic (CD) spectra were examined in 0.14 M NaCl. The CD spectra of f2a2-DNA complexes are characterized by a positive band at 272 nm which is blue-shifted and greatly enhanced relative to the corresponding band for native DNA. This type of CD change was noted previously with f2a1-DNA and f2b-DNA complexes. In contrast, f3 histone causes only minor distortions in the DNA CD spectrum, and their character depends upon the state of the two sulfhydryl groups in f3. When the cysteines are reduced, f3-DNA complexes have a slightly increased positive band with a small blue shift; when oxidized disulfide is the predominant form, this CD band becomes slightly smaller than native DNA value. This laboratory has now examined complexes reconstituted from DNA and all five histones of calf thymus. The sum of the CD spectra of these complexes, although very similar to the CD curve for reconstituted complexes containing whole histone, does not approximate that of chromatin; the consequence of this observation is discussed.     

Gonadotropin and subunit conformation.

Circular dichroic spectra have been obtained and resolved for the gonadotropins, ovine pituitary luteinizing hormone and human chorionic (urinary) gonadotropin, their subunits and glycopeptides. Much of the gonadotropin ellipticity above 250 nm can be attributed to the disulfide chromophore, although there are discernible contributions from tyrosyl and phenylalanyl residues as well. Of the two dissimilar subunits, the β-subunit makes the greatest contribution to the near-ultraviolet circular dichroic spectrum of the gonadotropins. From the position of the 0-0 tyrosyl band, i.e., 286–287 nm, one can ascertain that at least some of the tyrosyl residues of the gonadotropins are located in a hydrophobic environment. A positive circular dichroic extremum at 232.5 nm, present in luteinizing hormone but not in chorionic gonadotropin, can be ascribed to the α-subunit and probably results from tyrosines 21 and/or 30 in luteinizing hormone.An analysis of the circular dichroic difference spectrum above 230 nm, generated by subtracting the sum of the molecular ellipticities of the respective subunits from the molecular ellipticities of each gonadotropin, indicates that the local environment of disulfides and of tyrosyl residues is altered when gonadotropins dissociate. Circular dichroic difference spectra between the two α-subunits and between the two β-subunits indicated major contributions from- tyrosyl residues, presumably arising from tyrosyl substitutions.Between 200 and 230 nm, both gonadotropins exhibit negative circular dichroic extrema. The extremum occurs at 210 nm for luteinizing hormone and at 207.5 nm for chorionic gonadotropin. Each extremum can be described by two negative resolved bands, one at 215 nm and the other between 207 and 208.5 nm. The 215-nm resolved band is assigned to the peptide chromophore in a β-pleated sheet conformation and there is no evidence of α-helicity. The lower-wavelength resolved band is believed to have a significant contribution from the N-acetyl groups of glucosamine, galactosamine, and sialic acid, particularly since the glycopeptide fractions, prepared from each gonadotropin by digestion of the S-carboxymethyl derivatives with Pronase, exhibited a negative circular dichroic extremum at about 207 nm.The extent of β-structure in both gonadotropins is estimated to be about 28% whereas the separated subunits contain less β-structure, e.g., about 21 and 13% for the α- and β-subunits, respectively. The sum of the subunit β-structure, corrected for the respective molecular weight of each subunit, is about 17%. This is substantially less than that of the native hormone, thus indicating that significant conformational changes occur during gonadotropin dissociation to the biologically inactive subunits. Also, part of the gonadotropin β-structure may arise from intermolecular hydrogen bonding involving a pleated sheet arrangement between the subunits.     

Role of nonhistone chromosomal proteins in determining circular dichroism spectra of chromatin.

Complexing of histone proteins, from WI-38 cells with pure DNA from WI-38 cells, causes a marked decrease in the amplitude of the positive ellipticity band and a red shift in circular dichroism spectra in the 250–300 nm region. Total nonhistone chromosomal proteins from WI-38 cells (without histones) cause an analogous effect, but of significantly reduced magnitude. However, the two effects are not additive, because, when DNA is complexed with both histones and nonhistones, the amplitude of the positive ellipticity band has an intermediate value, between the histone-DNA complex and the nonhistone-DNA complex. Removal of certain nonhistone proteins from chromatin of WI-38 cells, by extraction with 0.25–0.35 m NaCl, causes a decrease in the positive circular dichroism band in the 250–300 nm region. Removal of histones and other nonhistone proteins from chromatin by extraction with 0.75 and 1.5 m NaCl causes a strong increase in positive ellipticity. This suggests the existence of modest but definite effects of nonhistone proteins in determining DNA conformation in native chromatin. Taken as a whole, nonhistone chromosomal proteins have a weaker but analogous effect to that of histones, while the nonhistone proteins extractable with 0.25–0.35 m NaCl have an opposite effect.     

Learning how planarization can affect dichroic patterns in polyfluorenes

The circular dichroism spectra of a single chain of polyfluorene was predicted for a p-twisted helix conformation and local planarized polymer sections in the presence and in the absence of thermal vibrations. Under thermal vibrations at 300 K, the planarized section of polyfluorene affords a red-shifted positive dichroic band between 446 and 456 nm, preserving a degree of chirality. The S1 ← S0 excitation energy decreases from 3.29 eV, for the p-twisted helix to 2.77 or 2.71 eV, for planarized sections with one or two coplanar twists, respectively. Thermal vibrations and intramolecular rotations eventually affect the circular dichroism spectrum patterns, where planarized bent conformers induce a positive band towards 450 nm.     

The circular dichroism of synaptosomal membranes: studies of interactions with neuropharmacological agents

The circular dichroic spectrum of synaptosomal membranes was highly reproducible and qualitatively similar to that of erythrocyte membranes. The spectrum exhibited a minimum at 224 nm, a shoulder at 212 nm and a maximum at 195 nm. The mean molar ellipticity at the maximum and minimum was approximately +8000 and –8000 respectively. The protein components were the dominant source of the CD signal. Quantitative estimates showed negligible contributions to the spectrum from cholesterol, phosphatidyl serine and TV-acyl sugars. Phospholine iodide, eserine, decamethonium, tetramethyl ammonium chloride and acetylcholine at concentrations of 10^-3 and 10^-4M did not produce detectable perturbations of the membrane circular dichroism. The circular dichroic spectrum of d-tubocurarine exhibited a maximum at 198 nm and a minimum at 212 nm. Addition of d-tubocurarine to membrane suspensions in the cuvette yielded a complex spectrum representing the result of a simple additive combination of the circular dichroic spectra of d-tubocurarine and the membranes. However, preincubation of the membranes with 3 x 10^-3m d-tubocurarine at 0-4°C for 5-10 min followed by sedimentation, several washes and resuspension resulted in a circular dichroic spectrum which appeared to involve no change in the membrane contribution, but there was a substantial decrease in the molar ellipticity of the d-tubocurarine remaining with the membranes.     

Circular dichroism of histone-bound regions in chromatin.

Native, NaCl-treated, trypsin-treated, and polylysine-bound nucleohistones were studied in 2.5 × 10^?4 M EDTA, pH 8.0, using circular dichroism (CD) and thermal denaturation. Removal of histone I by 0.6 M NaCl has a much smaller effect on both Δε₂₂₀ and Δε₂₇₈ than the removal of other histones. This indicates that histone I has less helical content and less conformational effect on the DNA in nucleohistone. By extrapolating to 100% binding by histones other than I, the positive CD band near 275 nm is close to zero. Comparison is also made between the effects of binding by the more basic and the less basic halves of histones by trypsin-digestion and polylysine-binding experiments. Trypsin digestion of nucleohistone reduces melting band IV at 82°C much more than melting band III at 72°C. However, the CD changes of Δε₂₇₈ and Δε₂₂₀ induced by trypsin digestion are small, unless melting band III is also reduced by the use of a higher trypsin level. This implies that the less basic halves of histones, which stabilize DNA to 72°C (melting band III), have more helical structure and are more responsible for conformational change in DNA than are the more basic halves, which stabilize DNA to 82°C (melting band IV). Polylysine binding to nucleohistone diminishes melting band III but has no effect on melting band IV. This binding affects only slightly the Δε₂₂₀ of nucleohistone, indicating that polylysine interferes very little with the structure of the less basic halves of bound histones. The implications of these studies with respect to chromatin structure are discussed.     

Role of membrane organization in the Mg+2-mediated regulation of excitation energy transfer in barley chloroplasts

Photosystem II fluorescence of barley chloroplasts has been monitored to understand the role of membrane organization in the cation mediated regulation of excitation energy transfer from photosystem II to photosystem I. Membrane organization has been perturbed by adding 60 mM benzyl alcohol which is known to increase the membrane fluidity and decrease its thickness. An addition of 60 mM benzyl alcohol increases the fluorescence at 683 nm (excitation at 436 mn) by 43% whereas 5 mM Mg⁺² increased the fluorescence by 38%. An addition of 5 mM Mg⁺² to benzyl alcohol treated chloroplasts resulted in only a small increase in the fluorescence (6.5%). Circular dichroic measurements showed that 5 mM Mg⁺² decreased the circular dichroic signals suggesting an alteration in the orientation of the chromophores. However, the effect was insignificant on the benzyl alcohol treated chloroplast membranes. Benzyl alcohol itself had large effect on the circular dichroic signals. Based on these results, it appears that a change in the orientation of photosystem I and photosystem II, rather than their segregation, is responsible for the cation-induced increase in the photosystem II fluorescence.     

On the various types of circular dichroism induced on acridine orange bound to poly(S-carboxymethyl-L-cysteine).

Circular dichroism and absorption spectra are measured on mixed solutions of acridine orange and poly(S-carboxymethyl-L -cysteine) at different pH and P/D mixing ratios. The observed circular dichroism spectra are classified into several types, mainly based on the number and sign of circular dichroic bands in the visible region. Three of them are associated with the absorption spectra characteristic of dimeric dye or higher aggregates of dye. Type I is observed with solutions, of which the pH is acid and P/D is higher than 4, and it has an unsymmetrical pair of positive and negative dichroic bands at 470 and 430 nm. This type is induced on the dye bound to the polymer in the β-conformation. Types II and III are considered to be characteristic of randomly coiled polymers. Type II is exhibited by solutions of P/D higher than 1 at pH 5–7 and has two dichroic bands around the same wavelengths as Type I but with opposite signs and an additional positive band at 560 nm. Type III, shown by solutions of P/D 2–0.6 at pH 6–10.5, has three dichroic bands around the same wavelengths as Type II but with signs opposite to it. The other two types of circular dichroism, induced for the solutions of P/D less than 1 at slightly acid pH, are associated with the absorption spectra of monomeric dye and are observed with disordered or randomly coiled polymer. They have a pair of dichroic bands at 540 and 425 nm, and the signs of these bands are opposite to each other in these two types.     

Circular dichroism of lipoxygenase-1 from soybeans.

The circular dichroism spectra of the three forms of lipoxygenase-1 from soybeans show characteristic differences in the region between 300 and 600 nm. Native lipoxygenase-1 only shows a negative dichroic band around 330 nm. Yellow lipoxygenase-1, obtained by addition of an equimolar amount of 13-F-hydroperoxylinoleic acid to the native enzyme, shows a positive Cotton effect at 425 nm, while the negative band band at 330 nm has increased in intensity. The blue enzyme, representing a complex of yellow enzyme with 13-L-hydroperoxylinoleic acid exhibits a negative dichroic band at 580 nm and positive bands at 410 and 391 nm. The near-ultraviolet CD spectra of the three forms of lipoxygenase are very similar, showing several well resolved positive dichroic bands at 0 degrees C. Using the method of Chen et al. (Chen, Y.-H., Yang, J.T. and Martinez, H.M. (1972) Biochemistry 11, 4120--4131) the contents of alpha-helix, beta- and unordered form of native lipoxygenase-1 were estimated to be 34, 27 and 39% respectively.     

Circular dichroism of aggregated deoxyhemoglobin S and the effect of amino acids.

It is well known that deoxyhemoglobin S (deoxy Hb S) aggregates at 37 °C and that it disaggregates at 1–5 °C. In this study solutions of pure Hb S at concentrations of 20–22 g/100 ml exhibit a normal circular dichroic spectrum in the range 250–650 nm at the temperature 1 °C. However, by the proper manipulation of the following parameters: temperatures of 1, 24 and 37 °C as well as the times required to change temperature and periods of maintaining at a certain temperature, five stages with different circular dichroic spectra can be produced. Not only the dichroic spectra of these stages are different but the kinetic behavior and stability of each of these stages are different. The evidence suggests that the mechanism of aggregation is similar to crystallization; that is, it exhibits a period of nucleation followed by growth. The overall kinetics of circular dichroic changes are described. At representative solution conditions the circular dichroic changes have been compared and found to parallel gel formation with pure Hb S. Also, the effect of certain anti-sickling amino acids (Sophianopoulos, A. J., et al. (1974) Clin. Biochem.7, 112–118) on the minimum Hb S concentration at which circular dichroic changes occur has been studied, and arginine chloride and arginine aspartate were found to raise this minimum concentration appreciably.