Induced Circular Dichroism Bands Research Articles

Effect of Benzo[c]phenanthridine Alkaloids on Reverse Transcriptase and Their Binding Property to Nucleic Acids

Twelve benzo[c]phenanthridine alkaloids, naturally occurring and synthetic model compounds, were examined for their anti-reverse transcriptase activity. Significant inhibitory effects on AMV reverse transcriptase were found with avicine and 6-methylnitidine as well as with nitidine and fagaronine, whose inhibitory effects on this enzyme have been reported by Sethi et al. In the CD spectra, the mixtures of these four alkaloids with either (rA)n · (dT)12-18 or (rA)n · (dT)n showed induced CD bands at longer wavelengths, suggesting that they interacted with the nucleic acids.

Circular differential scattering and circular differential absorption of DNA-protein condensates and of dyes bound to DNA-protein condensates.

DNA-protein condensates that give positive and negative psi-type circular dichroism (CD) spectra (psi condensates) bind intercalative and nonintercalative dyes. CD depends both on circular differential scattering and on circular differential absorption; scattering-corrected CD measurements are approximations to circular differential absorption. The circular differential scattering and scattering-corrected CD patterns observed in the DNA absorption band of psi condensates are mimicked in the induced CD band of intercalators bound to psi condensates. The induced scattering-corrected CD and circular differential scattering patterns of the groove-binding dye Hoechst 33342 bound to psi condensates are the inverse of the patterns seen with intercalative dyes, whereas the groove-binding dye manganese(III) meso-tetrakis(4-N-methylpyridyl)porphine [MnIIITMpyP-4] shows no significant induced CD patterns. The large circular differential scattering and scattering-corrected CD bands are interpreted as resulting from long-range chiral packing, rather than near-neighbor short-range interactions. Dyes intercalated into the DNA of the psi condensates have the same type of long-range chiral packing as the DNA bases. Therefore, the psi-type CD spectra seen in the UV spectra originating from the long-range packing of the DNA bases are also observed in the visible spectra when dyes are intercalated in the DNA of the psi condensates. Our interpretation comes from the observation that the induced circular differential scattering and circular differential absorption of the dye bound to the psi condensates depend only upon the sign of the circular differential absorption and the pattern of the circular differential scattering of the psi condensates without bound dye.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA-binding characteristics of a synthetic analogue of distamycin

The interaction between a synthetic analogue (structure shown in fig.1) of distamycin, and DNA has been studied with a view to understanding the conformational and chemical basis of the sequence specific binding of distamycin with DNA. The complex formation between the trimer and DNA is apparent from the red shift in the UV spectrum and appearance of the induced CD band in 300nm–350nm region. The relevant data suggest: (i) the binding is A-T base specific though the specificity is not as pronounced as in distamycin (1,2) and (ii) it occurs via the minor groove of DNA. The partial loss in A-T base specificity may be due to the replacement of N-methyl pyrrole by benzene or the increase in curvature of the backbone of the ligand as a result of this replacement.

Amino acid residues complexed with eosin 5-isothiocyanate in band 3 protein of the human erythrocyte

The amino-acid residues of band 3 protein taking part in the ionic interaction with an anion-transport inhibitor, eosin 5-isothiocyanate (EITC), were determined by pH titration. The plots of the absorbance of EITC-ghost system against pH reveal five equilibria, at pH 3.7, 6.4, 8.0, 11.0 and 13.1. Since the three equilibria, 3.7, 8.0 and 11.0, are representative of the EITC molecule, the others, 6.4 and 13.1, may be due to the interaction of EITC molecules with histidine and arginine residues, respectively. The same experiment using a reconstituted system of band 3-lipid vesicles gave results in good agreement with the EITC-ghost system. The intensity of the induced CD band at 530 nm of EITC molecules bound to ghosts was decreased by preincubation with arginine-specific reagents, phenylglyoxal and 1,2-cyclohexanedione, or histidine-specific reagents, diethylpyrocarbonate (DEPC) and p-diazobenzene sulfonate. The repression effects by these chemical modifiers were evaluated by measuring the concentrations which elicit 50% reduction. The histidine-specific reagents repressed the CD of EITC more effectively than the arginine-specific reagents. Furthermore, it was found that DEPC effectively inhibited the sulfate efflux from intact erythrocytes. These results suggest that the histidine residues participate in the anion-transport system of human red cells.

Characterization of the anion transport channel protein in human erythrocytes. Induced circular dichroism of inhibitors bound to the anion transport channel

The induced circular dichroism (CD) of erythrocyte ghosts with anion-transport inhibitors has been studied. A ghost-EITC (eosin 5-isothiocyanate) system shows an induced CD spectrum at the wavelength region corresponding to the absorption bands of EITC. Also a ghost-EMI (eosin 5-maleimide) system shows induced CD, but has bands of opposite sign to the EITC system. From the change of the CD intensity, the number of EITC molecules bound to one erythrocyte was estimated to be about 1.4·10 6, being close to the number of band 3 copies per ghost. The CD spectra of EITC and EMI systems show that a configurational structure of the moiety anchoring the EMI molecule is the reverse to that of EITC. The preferred conformation of bound EITC may be twisted in a right-handed sense. From the signs of the induced CD bands in ghost-stilbene disulfonate systems, the chirality of twisted stilbene derivatives seems to be a left-handed sense, as is the case for the EMI derivative. The CD spectra of EITC in the presence of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonate) shows that the binding site of EITC may not be identical with that of DIDS. The results observed in this study reflect the ternary arrangement of the functional amino groups in anion recognition sites.

Interaction of trifluoperazine with porcine calmodulin 19F NMR and induced CD spectral studies

We have used 19F NMR and CD spectra to study interactions of TFP with CaM under various conditions where no aggregation of TFP occurs. It was found that TFP is bound to CaM in the absence of Ca 2+ with K a >4 × 10 4 M in terms of 19F NMR of TFP and that KCl markedly influences the interaction of TFP with CaM. It was also found that TFP causes quite strong induced CD bands corresponding with the absorption bands of the aromatic ring of TFP when TFP is bound to Ca 2+ -CaM. The induction of the CD bands was time-dependent and was composed of fast and slow phases.

Induced circular dichroism in cholesteric and twisted smectic mesophases

AbstractA new twisted smectic mesophase formed in Tween 80 (1) (monooleate of the ether of sorbitan with 4 mol of polyethyleneglycol)‐water system was found. The induced circular dichroism (CD) spectra of achiral molecules dissolved in this twisted smectic mesophase are compared with those in the thermotropic cholesteric mesophase. Pyrene and fluorene and their derivatives, acridine, phenazine, and phenothiazine were used as achiral molecules (solute molecules). It was found that the signs and shape of the induced CD bands observed in the twisted smectic mesophase were dependent on the solute concentration. The magnitude of the induced CD bands was dependent on the symmetry of the solute molecules and the value of the geometrical ratio of short‐axis to long‐axis of the solute molecules. Furthermore, it was demonstrated that the relative polarization directions of the solute molecules could be determined by means of the induced CD in the Tween 80‐water system using a simple modified C2‐symmetry rule.

Electric dichroism and sedimentation velocity studies of DNA-Hg(II) and DNA-Ag(I) complexes

The cause of the induced CD bands of DNA-Hg 2+ and DNA-Ag + complexes has been interpreted as a condensed ψ state (Walter, A. and Luck, G. (1977) Nucl. Acids Res. 4, 539–550). Electric dichroism and sedimentation velocity experiments indicate that there is no tertiary structure formation of DNA-heavy-metal-ion complexes to support a ψ state. The increase of the sedimentation coefficient of DNA-heavy-metal-ion complexes can be accounted for by an increase in molecular weight and a decrease in the partial specific volume upon the binding of Hg 2+ and Ag +. Electric dichroism measurements show that the new optical bands produced by the binding of Hg 2+ to DNA and the mode I binding of Ag + to DNA result in transition moments polarized in the plane of the bases. Binding mode II of the DNA-Ag + complex at 265 nm gives an optical perturbation where the transition moment has an out-of-plane contribution. The CD studies reported in the following paper give further insight into the binding sites and binding modes of these two metal ions.

Structure of anti-fluorescein combining sites in rabbit and sheep: induced circular dichroism of bound haptens

Based on a comparison of the spectroscopic properties of several 6-hydroxyxanthen-3-ones with different substituents at position 9, we have concluded that the induced circular dichroism (CD) of fluorescein bound in the combining sites of rabbit and sheep anti-fluorescein antibodies reflects primarily the mciro-environment surrounding the hydroxyxanthenone moiety of the hapten. Measurements of the directions of the transition fipole moments of the two major absorption bands above 300 nm of fluorescein, together with a consideration of the signs of the induced CD bands, suggest that the observed optical activity is due, at least partly, to dynamic coupling between one or more aromatic chromophores in the combining site and the transitions in the hapten. The temporal response of anti-fluorescein antibody synthesis in a hyperimmunized sheep in terms of structure of the combining sites was found to exhibit some similarities and some differences compared to that observed in rabbits. The sheep produced two of the three types observed in rabbits (type II and type III: Gollogly & Cathou, 1974). Type II was synthesized after initial immunization and after a booster injection. After a second boost injection (“third period”) and also after several further boosts, types II and III were found together in all antibody preparations examined by CD titrations; the two types exhibit very different induced CD spectra of bound lgiand and different affinities for hapten. Further evidence for antibody site structure heterogeneity was provided by fluorescence titrations of fluorescein fluorescence.

Circular dichroism of hapten-antibody complexes: Studies of tryptophanyl residue involvement in the antibody combining sites of MOPC-315 protein heavy and light chain subunits, reconstituted MOPC-315 protein and its Fv fragment

Circular dichroic spectra of the native MOPC-315 mouse IgA myeloma protein and the protein reformed from its isolated heavy and light chains, the amino-terminal variable region fragment (Fv-315) and the isolated subunits provide information about conformational features of the isolated chains, changes which occur upon subunit association and about local protein-protein interactions. A pair of negative peaks in the near-ultraviolet (at 285 and 293 nm) is present in both the MOPC-315 and Fv-315 CD spectra, indicating that the structure responsible for the bands is located in the variable region of the intact immunoglobulin. These CD bands are absent from the CD spectra of the isolated heavy and light chain subunits but are regained in the process of subunit reassociation which results in the formation of the intact antibody combining site. The near-u.v. circular dichroism of a model cyclic tryptophanyl dipeptide provides evidence that two or more closely juxtaposed tryptophanyl residues may be responsible for the protein aromatic chromophore transitions at 285 and 293 nm. Binding of chromophoric haptens (Dnp- and Tnp-aminocaproate) to the native and reformed MOPC-315 protein and to its Fv fragment generates an induced circular dichroism with unique features: (1) an increase of the negative CD at both 285 and 293 nm; (2) a perturbation (blue-shift) of the tryptophanyl band at 293 nm; and (3) an apparent reciprocal relationship between positive induced CD bands of haptenic chromophore (at 370–382 nm) and the large negative induced CD band in the aromatic residue absorption region of the protein (near 300 nm) where haptens display absorption minima. These spectral features imply that the induced CD arises from electronic interactions between the aromatic moiety of the bound hapten and tryptophan(s) in the protein. This pattern of induced CD furnishes additional evidence that the paired CD bands at 285 and 293 nm in the spectra of the intact immunoglobulin and its Fv fragment arise from closely situated tryptophans and indicates that these residues lie in or near the intact antibody combining site. Induced CD bands generated by binding Tnp-aminocaproate to the isolated light chains also exhibit reciprocal relations and may be due to electronic interactions between the aromatic moiety of the bound hapten and tryptophan(s) in the protein subunit. Data available from primary amino acid sequence and affinity-label studies are consistent with the suggestion that variable region tryptophans from different subunits are close neighbora in or near the intact antibody combining site. The negative 293 and 285 nm CD bands of the MOPC-315 and Fv-315 proteins may therefore represent short range electronic interactions occurring within an antibody combining site between chromophoric residues contributed respectively by separate polypeptide chains.

Sequential Appearance of Three Different Anti-Fluorescein Combining Sites in Hyperimmunized Rabbits: Characterization by Circular Dichroism and Binding Studies

Abstract Fluorescein (F1) bound in the combining site of anti-F1 antibodies exhibits extrinsic optical activity which is a function of the fine structure of the site. We have utilized this phenomenon to study structural changes in antibody combining sites as a function of time and repeated boosting. Four rabbits were immunized with F1-keyhole limpet hemocyanin (KLH) and successively boosted whenever the antibody production declined. Antibodies were purified from bleedings taken once or twice a week and characterized by the extrinsic circular dichroism (CD) of their hapten-antibody complexes. After the third boost, three characteristically different CD spectra were observed, each corresponding to a different combining site configuration. Usually each individual bleed contained only one of the three antibody types and the particular type observed depended on the time after boosting: type I was present for the first 3 to 6 weeks of the antibody response, type II was present for a further 2 to 3 weeks and, finally, as the antibody production declined, only type III antibodies were found. This sequential response was repeated after each succeeding boost. Each of the antibody types was functionally homogeneous with respect to its extrinsic CD properties. In contrast, binding data showed that they were somewhat heterogeneous. The induced CD bands of bound F1 originate in the xanthenone ring whereas the binding of F1 depends on the interactions of both the xanthenone and phenylcarboxylate moieties with the site. Therefore, these results suggested that the anti-F1 combining site consists of two distinct subsites. The functional homogeneity of each antibody type may be due to a common tertiary structure of the xanthenone-binding subsite whereas the heterogeneity indicated by the binding data may be due to heterogeneity within the phenylcarboxylate-binding subsite. Possible mechanisms for the sequential appearance of these antibody types are also discussed.

INDUCED CIRCULAR DICHROISM OF RACEMIC METHYLCYCLOHEXANONES INCLUDED IN β-CYCLODEXTRIN

Abstract When cyclohexanone and its methyl derivatives were included in the cavities of cyclodextrins, the induced circular dichroism (CD) bands were observed in the absorption region of n-π* transition of the carbonyl chromophore. The sign of induced CD band of 2-methylcyclohexanone included in β-cyclodextrin was same as that of resolved R-isomer. From these observations the mode of inclusion and structure of β-cyclodextrin complex with methylcyclohexanone were discussed.

Induced cotton effects of hyaluronic acid-acridine orange complex and conformation of the polymer

Hyaluronic acid-acridine orange complexes in water show a symmetric doublet CD band centered at 455 nm. The exciton-like CD band indicates a preferred left-handed chirality for this polymer in solution, which correlates with the proposed left-handed double helical structure of hyaluronic acid in oriented film. The sign and general shape of the induced CD band with hyaluronic acid (dye concentration 2.5×10 −5M) is the same throughout the range of polymer-dye ratio studied. The single CD band which appears at a 10 −4M concentration of dye has been attributed to dye-dye interaction. Hyaluronic acid, preheated to 95°C for one hour, followed by rapid cooling, does not show any induced CD band.