Base protonation facilitates B-Z interconversions of poly(dG-dC) X poly(dG-dC)

Comparative studies on the salt titration and the related kinetics for poly(dG-dC) X poly(dG-dC) in pH 7.0 and 3.8 solutions clearly suggest that base protonation facilitates the kinetics of B-Z interconversion although the midpoint for such a transition in acidic solution (2.0-2.1 M NaCl) is only slightly lower than that of neutral pH. The rates for the salt-induced B to Z and the reverse actinomycin D induced Z to B transitions in pH 3.8 solutions are at least 1 order of magnitude faster than the corresponding pH 7.0 counterparts. The lowering of the B-Z transition barrier is most likely the consequence of duplex destabilization due to protonation as indicated by a striking decrease (approximately 40 degrees C) in melting temperature upon H+ binding in low salt. The thermal denaturation curve for poly(dG-dC) X poly(dG-dC) in a pH 3.8, 2.6 M NaCl solution indicates an extremely cooperative melting at 60.5 degrees C for protonated Z DNA, which is immediately followed by aggregate formation and subsequent hydrolysis to nucleotides at higher temperatures. The corresponding protonated B-form poly(dG-dC) X poly(dG-dC) in 1 M NaCl solution exhibits a melting temperature about 15 degrees C higher, suggesting further duplex destabilization upon Z formation.     

7-Amino-actinomycin D complexes with deoxynucleotides as models for the binding of the drug to DNA

Fluorescence, CD, absorption, and ¹H-nmr studies are reported for complexes of 7-amino-actinomycin D with deoxydinucleotides, deoxytetranucleotides, and poly(dG-dC)· poly(dG-dC). The optical spectra for the 7-amino-actinomycin D complex with pdG-dC, pdG-dC-dG-dC and pdC-dG-dC-dG are similar in shape to the 7-amino-actinomycin D complex with either DNA or poly(dG-dC). The changes in the ¹H chemical shifts of the 7-amino-actinomycin D and the pdG-dC resonances that accompany complex formation show that 7-amino-actinomycin D forms a minature intercalated complex with two pdG-dC molecules. The magnitudes of the induced chemical shifts for the 7-amino-actinomycin D complex formation with pdG-dC are similar to, but slightly different from, the induced chemical shifts which are obtained when actinomycin D forms a minature intercalated complex with two pdG-dC molecules. The pdN-dG dinucleotides (N = C, A, or T) form stacked complexes with 7-amino-actinomycin D. The presence of the 7-amino-group results in a larger dimerization constant (in aqueous solution) for 7-amino-actinomycin D [K_D(6°C) = 4.4 × 10³M^?1], as compared to actinomycin D [K_D(6°C) = 1.7 × 10³M^?1]; the chemical shifts which accompany dimer formation indicate that the chromophores stack in an inverted manner. Intercalation of 7-amino-actinomycin D into minature double helices, as well as into calf thymus DNA, poly(dG-dC)·poly(dG-dC), and poly(dA-dC)·poly(dG-dT), results in an enhancement of the relative fluorescence intensity and a shift in both the absorbance and corrected emission spectra.     

Fluorescence-determined preferential binding of quinacrine to DNA.

Quinacrine complexes with native DNA (Calf thymus, Micrococcus lysodeikticus, Escherichia coli, Bacillus subtilis, and Colstridium perfringens) and synthetic polynucleotides (poly(dA) . poly(dT), poly[d(A-T)] . poly[d(A-T)], poly(dG) . poly(dC) and poly[d(G-C)] . poly[d(G-C)]) has been investigated in solution at 0.1 M NaCl, 0.05 M Tris HCl, 0.001 M EDTA, pH 7.5, at 20 degrees C. Fluorescence excitation spectra of complexes with dye concentration D = 5-30 microM and DNA phosphate concentration P = 400 microM have been examined from 300 to 500 nm, while collecting the emission above 520 nm. The amounts of free and bound quinacrine in the dye-DNA complexes have been determined by means of equilibrium dialysis experiments. Different affinities have been found for the various DNAs and their values have been examined with a model that assumes that the binding constants associated with alternating purine and pyrimidine sequences are larger than those relative to nonalternating ones. Among the alternating nearest neighbor base sequences, the Pyr(3'-5')Pur sequences, i.e., C-G, T-G, C-A and T-A seem to bind quinacrine stronger than the remaining sequences. In particular the three sites, where a G . C base pair is involved, are found to display higher affinities. Good agreement is found with recent calculations on the energetics of intercalation sites in DNA. The analysis of the equilibrium shows also that the strength of the excitation spectrum of bound dye depends strongly upon the ratio of bound quinacrine to DNA. This effect can be attributed to dye-dye energy transfer along DNA.     

Kinetics of exchangeable protons in Z DNA: a UV resonance Raman study.

We have studied the hydrogen-deuterium exchange kinetics of the exchangeable protons of the poly(dG-dC).poly(dG-dC) in the Z form of the polymer, using resonance Raman spectroscopy with 257 nm and 284 nm excitation wavelengths. In our experimental conditions (4.5 M NaCl, phosphate buffer pH7, 2 degrees C) the two amino protons and the imino proton of guanine are exchanged with the same exchange half-time of 13 min, whereas the two amino protons of cytosine are exchanged with the same exchange half-time of 51 min.     

Studies with a fluorescent vital probe for DNA in mammalian cells

Olivomycin is taken up efficiently by HeLa cells and by rat fibroblast cells at 38.5 °C, but not by BHK cells. On irradiation with light of 425 nm wavelength, the nuclei of living cells that have taken up olivomycin fluoresce. When olivomycin complexes with DNA in solution, the emission spectrum broadens and shifts, the excitation wavelength maximum shifts up 15 nm, and the fluorescence polarization increases. In HeLa and fibroblast cells, the fluorescence characteristics indicate that olivomycin is entirely complexed to DNA, and its rotational mobility indicates that it is complexed to DNA in regions where other components of the chromatin offer no steric hindrance.     

Anomalous fluorescence of yeast 3-phosphoglucerate kinase.

The 3-phosphoglycerate kinase (EC 2.7.2.3) of yeast which contains two tryptophyl and eight tyrosyl residues per molecule, displayed an unusualy fluorescence emission spectrum with a maximum at 308 nm when excited at 280 nm. The emission peak shifted to 329 nm when excited at 295 nm. We could confirm that it was due to the efficient quenching of tryptophyl fluorescence as well as to the incomplete energy transfer from tyrosyl to tryptophyl residues. The average fluorescence quantum yield of this protein was 0.076 (excitation at 280 nm) and that of tryptophyl residues was 0.046 (excitation at 295 nm). As the pH of the solution was lowered, the fluorescence intensity of phosphoglycerate kinase at 329 nm dramatically increased between pH 5 and 4, while the position of the peak remained unchanged. When denatured in 4 M guanidine hydrochloride, the protein showed two emission peaks, one at 343 nm and the other at 303 nm.     

Luminescence excitation spectra reveal low-lying excited states in stacked adenine bases

Luminescence emission, polarization, and excitation spectra of polyadenylic acid (poly(A)) have been studied in room-temperature aqueous solution (pH 8). The temperature dependence of the luminescence of poly(A) at two different excitation wavelengths in the range 10-70 degrees C has also been studied and compared with that of adenosine 5'-monophosphate (AMP). It has been found that the luminescence excitation spectrum and the polarization curve of poly(A) solution reveal a low-intensity electronic transition at about 320 nm which is red-shifted by approximately 0.9 eV from the maximum of the absorption spectrum at 260 nm. A model of two luminescent stacked forms is suggested. The difference in the ground state levels of these two stacked forms obtained from the temperature dependencies of the emissions is insignificant ( approximately 1 kcal/mol). This means a lowering of the excited state of the stacked form with the 320 nm/420 nm absorption/emission bands by approximately 0.9 eV as compared to the main form with the 260 nm/400 nm absorption/emission bands. The low-lying excited states suggest the stronger electronic coupling of the bases in a certain stacked form. It is proposed that such clusters of the stacked bases could provide the wire-like conductivity in the short segments of DNA.     

Improved staining method for the simultaneous flow cytofluorometric analysis of DNA content, S-phase fraction, and surface phenotype using single laser instrumentation.

We have developed an improved technique for triple staining that permits the simultaneous flow cytofluorometric analysis of cell surface antigens, bromodeoxyuridine incorporation into DNA, and DNA quantification using 7-amino-actinomycin D. PHA-activated human peripheral blood lymphocytes were incubated with bromodeoxyuridine and stained for cell surface phenotype with phycoerythrin-labeled monoclonal antibodies. Stained cells were fixed serially with 1% paraformaldehyde and 45% ethanol. Fixed cells were sequentially stained with an anti-BrdUrd monoclonal antibody followed by a FITC-conjugated goat anti-mouse antibody and incubated with 7-amino-actinomycin D. Hypotonic buffer was employed for all procedures after fixation. Stained-fixed cells were analyzed by flow cytofluorometry for simultaneous green (525 nm), orange (570 nm), and red (greater than 650 nm) fluorescence. Utilizing this staining technique, we were able to analyze simultaneously cell phenotype, DNA synthesis, and total cellular DNA content with single laser excitation.     

7-Amino-actinomycin D as a specific fluorophore for DNA content analysis by laser flow cytometry

A technique for DNA amount determination by flow cytometry based on the use of 7-amino-actinomycin D (7-amino-AMD), a fluorescent analogue of antibiotic actinomycin has been investigated, and a particular staining procedure has been developed. The procedure includes short fixation in 70% ethanol and staining for 20 min in 10(-5)M solution of 7-amino-AMD at pH7. The results of DNA content measurements are very reproducible. The histograms obtained have a coefficient of variation less than 3%. The absorption maximum of the complex of 7-amino-AMD with DNA is situated in the green spectrum region, making this stain particularly suitable for argon laser flow cytometry.     

Contribution of the resonance Raman spectroscopy to the identification of Z DNA

Poly(dG-dC).poly(dG-dC) at low salt concentration (0.1 M NaCl) and at high salt concentration (4.5 M NaCl) has been studied by Raman resonance spectroscopy using two excitation wavelengths: 257 nm and 295 nm. As resonance enhances the intensity of the lines in a proportion corresponding to the square of the molar absorption coefficient, the intensities of the lines with 295 nm wavelength excitation are enhanced about sevenfold during the B to Z transition. With 257 nm excitation wavelength the 1580 cm-1 line of guanosine is greatly enhanced in the Z form whereas with 295 nm excitation several lines are sensitive to the modifications of the conformation: the guanine band around 650 cm-1 and at 1193 cm-1 and the bands of the cytosines at 780 cm-1, 1242 cm-1 and 1268 cm-1. By comparison with the U.V. resonance Raman spectra of DNA, we conclude that resonance Raman spectroscopy allows one to characterize the B to Z transition from one line with 257 nm excitation wavelength and from three lines with 295 nm excitation. The conjoined study of these four lines should permit to observe a few base pairs being in Z form in a DNA.     

Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein

The green-fluorescent protein (GFP) that functions as a bioluminescence energy transfer acceptor in the jellyfish Aequorea has been renatured with up to 90% yield following acid, base, or guanidine denaturation. Renaturation, following pH neutralization or simple dilution of guanidine, proceeds with a half-recovery time of less than 5 min as measured by the return of visible fluorescence. Residual unrenatured protein has been quantitatively removed by chromatography on Sephadex G-75. The chromatographed, renatured GFP has corrected fluorescence excitation and emission spectra identical with those of the native protein at pH 7.0 (excitation lambda max = 398 nm; emission lambda max = 508 nm) and also at pH 12.2 (excitation lambda max = 476 nm; emission lambda max = 505 nm). With its peak position red-shifted 78 nm at pH 12.2, the Aequorea GFP excitation spectrum more closely resembles the excitation spectra of Renilla (sea pansy) and Phialidium (hydromedusan) GFPs at neutral pH. Visible absorption spectra of the native and renatured Aequorea green-fluorescent proteins at pH 7.0 are also identical, suggesting that the chromophore binding site has returned to its native state. Small differences in far-UV absorption and circular dichroism spectra, however, indicate that the renatured protein has not fully regained its native secondary structure.     

Dual‐color‐emitting green fluorescent protein from the sea cactus Cavernularia obesa and its use as a pH indicator for fluorescence microscopy

We isolated and characterized a green fluorescent protein (GFP) from the sea cactus Cavernularia obesa. This GFP exists as a dimer and has absorption maxima at 388 and 498 nm. Excitation at 388 nm leads to blue fluorescence (456 nm maximum) at pH 5 and below, and green fluorescence (507 nm maximum) at pH 7 and above, and the GFP is remarkably stable at pH 4. Excitation at 498 nm leads to green fluorescence (507 nm maximum) from pH 5 to pH 9. We introduced five amino acid substitutions so that this GFP formed monomers rather than dimers and then used this monomeric form to visualize intracellular pH change during the phagocytosis of living cells by use of fluorescence microscopy. The intracellular pH change is visualized by use of a simple long‐pass emission filter with single‐wavelength excitation, which is technically easier to use than dual‐emission fluorescent proteins that require dual‐wavelength excitation. Copyright © 2013 John Wiley & Sons, Ltd.     

The effect of resonance energy homotransfer on the intrinsic tryptophan fluorescence emission of the bothropstoxin-I dimer.

Bothopstoxin-I (BthTX-I) is a homodimeric Lys49-PLA2 homologue from the venom of Bothrops jararacussu in which a single Trp77 residue is located at the dimer interface. Intrinsic tryptophan fluorescence emission (ITFE) quenching by iodide and acrylamide has confirmed that a dimer to monomer transition occurs on reducing the pH from 7.0 to 5.0. Both the monomer and the dimer showed an excitation wavelength-dependent increase in the fluorescence emission maximum, however the excitation curve of the dimer was blue-shifted with respect to the monomeric form. No differences in the absorption or circular dichroism spectra between pH 5.0 and 7.0 were observed, suggesting that this curve shift is due neither to altered electronic ground states nor to exciton coupling of the Trp residues. We suggest that fluorescence resonance energy homotransfer between Trp77 residues at the BthTX-I dimer interface results in excitation of an acceptor Trp population which demonstrates a red-shifted fluorescence emission.     

Interaction of topotecan,DNA topoisomerase I inhibitor,with double-stranded polydeoxyribonucleotides. 4. Topotecan binds preferably to the GC base pairs of DNA

Interaction of topotecan (TPT) with synthetic double-stranded polydeoxyribonucleotides has been studied in solutions of low ionic strength at pH = 6.8 by linear flow dichroism (LD), circular dichroism (CD), UV-Vis absorption and Raman spectroscopy. The complexes of TPT with poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dC).poly(dG-dT), poly(dA).poly(dT) and previously studied by us complexes of TPT with calf thymus DNA and coliphage T4 DNA have been shown to have negative LD in the long-wavelength absorption band of TPT, whereas the complex of TPT with poly(dA-dT).poly(dA-dT) has positive LD in this absorption band of TPT. Thus, there are two different types of TPT complexes with the polymers. TPT has been established to bind preferably to GC base pairs because its affinity to the polymers of different GC composition decreases in the following order: poly(dG-dC).poly(dG-dC) > poly(dG).poly(dC) > poly(dA-dC).poly(dG-dT) > poly(dA).poly(dT). The presence of DNA has been shown to shift monomer-dimer equilibrium in TPT solutions toward dimer formation. Several duplexes of the synthetic polynucleotides bound together by the bridges of TPT dimers may participate in the formation of the studied type of TPT-polynucleotide complexes. Molecular models of TPT complex with linear and ring supercoiled DNAs and with deoxyguanosine have been considered. TPT (and presumably all camptothecin family) proved to be a representative of a new class of DNA-specific ligands whose biological action is associated with formation of dimeric bridges between two DNA duplexes.     

A Z-DNA binding protein isolated from D. radiodurans

A DNA binding protein isolated from D. radiodurans changes CD-spectrum of Z-form poly(dG-dC) X poly(dG-dC). We have found that a positive band at 268 nm is converted close to that of B-form in the presence of the protein. Concomitantly, a negative band at 295 nm shown by Z-form poly(dG-dC) X poly (dG-dC) was weakened by the protein but not by albumin. Such changes in the CD-spectra were not induced by the protein and by albumin when they were mixed with Z- or B-form poly(dG-me5dC) X poly(dG-me5dC) or with B-form poly(dG-dC) X poly(dG-dC). The protein formed a complex preferentially with Z-form poly(dG-dC) X poly(dG-dC).     

Chain flexibility and hydrodynamics of the B and Z forms of poly(dG-dC).poly(dG-dC).

The solution properties of the B and Z forms of poly(dG-dC).poly(dG-dC) have been measured by static and dynamic laser light scattering. The radius of gyration, persistence length, translational and segmental diffusion coefficients, and the Rouse-Zimm parameters have been evaluated. The persistence length of the Z form determined at 3 M NaCl is about 200 nm compared to 84 and 61 nm respectively for the B forms of poly(dG-dC).poly(dG-dC), and calf thymus DNA, both determined at 0.1 M NaCl. The data on persistence length, diffusion coefficients and the Rouse-Zimm parameters indicate a large increase in the chain stiffness of Z DNA compared to the B form. These results are opposite to the ionic strength effects on random sequence native DNAs, for which the flexibility increases with ionic strength and levels off at about 1 M NaCl.     

Characterization of the fluorescence of the antitumor agent, mitoxantrone

Studies have been conducted on the absorption, fluorescence excitation and fluorescence emission of mitoxantrone, an important antineoplastic agent. Mitoxantrone has been found to fluoresce with excitation maxima at 610 and 660 nm and emission maximum at 685 nm. Further characterizations of the fluorescence were undertaken to study its usefulness in biological studies. Mitoxantrone fluorescence intensity is altered by pH and the emission spectrum is red-shifted by DNA. Furthermore, the fluorescence polarization is enhanced by DNA, confirming the binding of the antitumor agent to DNA. The fluorescence spectra are slightly modified by changes in ionic strength and the addition of albumin. Data establishing the usefulness of fluorescence to measure serum concentrations in the range of 0.0 to 100 nM are presented. Such determinations can distinguish serum mitoxantrone from its non-fluorescent primary metabolite.     

Dimethyl-and monomethyloxyluciferins as analogs of the product of the bioluminescence reaction catalyzed by firefly luciferase

The absorption and fluorescence spectra of dimethyloxyluciferin (DMOL) and monomethyloxyluciferin (MMOL) were studied at pH 3.0-12.0. In the range of pH 3.0-8.0, the fluorescence spectrum of DMOL exhibits a maximum at lambda(em) = 639 nm. At higher pH values an additional emission maximum appears at lambda(em) = 500 nm (wavelength of excitation maximum lambda(ex) = 350 nm), which intensity increases with time. It is shown that this peak corresponds to the product of DMOL decomposition at pH > 8.0. The absorption spectra of MMOL were studied in the range of pH 6.0-9.0. At pH 8.0-9.0, the absorption spectrum of MMOL exhibits one peak at lambda(abs) = 440 nm. At pH 7.3-7.7, an additional band appears with maximum at lambda(abs) = 390 nm. At pH 6.0-7.0 two maxima are observed, at lambda(abs) = 375 and 440 nm. The fluorescence spectra of MMOL (pH 6.0-9.7, lambda(ex) = 440 or 375 nm) exhibit one maximum. It is shown that decomposition of DMOL and MMOL in aqueous solutions results in products of similar structure. DMOL and MMOL are rather stable at the pH optimum of luciferase. It is suggested that they can be used as fluorescent markers for investigation of the active site of the enzyme.     

pH-induced quaternary assembly of Vitreoscilla hemoglobin: The monomer exhibits better peroxidase activity

pH-dependent (pH 6.0–8.0) quaternary structural changes of ferric Vitreoscilla hemoglobin (VHb) have been investigated using dynamic light scattering. The VHb exhibits a monomeric state under neutral conditions at pH 7.0, while the protein forms distinct homodimeric species at pH 6.0 and 8.0, respectively. The dissociation constant obtained using the Bio-Layer Interferometry technology indicates that, at pH 7.0, the monomer–monomer dissociation of VHb is about 6-fold or 5-fold higher (K_D = 6.34 μM) compared with that at slightly acidic pH (K_D = 1.05 μM) or slightly alkaline pH (K_D = 1.22 μM). The pH-dependent absorption spectra demonstrate that the heme microenvironment of VHb is sensitive to the changes of pH value. The maximum absorption band of heme group of VHb shifts from 402 nm to 407 nm when pH changes from 6.0 to 8.0. In addition, the fluorescence emission spectra of VHb, taken at excitation wavelength of 295 nm, suggest that the single Trp122 fluorescence quantum yields in VHb are decreased due to the formation of the homodimeric species. However, the circular dichroism spectra data display that the secondary structures of VHb are little affected by pH transitions. The pH-dependent peroxidase activity of VHb was also investigated in this study. The optimum pH for VHb using 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) as substrate is 7.0, which implies that the monomer state of VHb would exhibit better peroxidase activity than the homodimeric species of VHb at pH 6.0 and 8.0.     

Resolution of electronic absorption bands and nucleotide binding processes in actinomycin D

Complexes of actinomycin D with model dexoxynucleoides have been studied by means of absorption spectroscopy and CD spectroscopy and CD spectroscopy. The CD spectra of the complexes of actinomycin D with 5′-dGMP, pdG-dT, pdT-dG, pdG-dA, and pdA-dG, respectively, all resemble one another. It is presumed that in solution the interactions between the guanine residues and actinomycin D in these complexes are the same as found in the crystalling 1:2 actinomycin D:dG complex [Jain, S. C. & Sobell, H. M. (1972) J. Mol. Biol. 68 , 1–20]. The CD spectrum of the Complexes with pdG-dC differs from of the complexes just mentioned, and resembles those of the complexes formed by actinomycin D with calf-thymus DNA and with poly(dG-dC)-poly(dG-dC). These resulls are consisent with, the nontion that pdG-dC froms a double-staranded intercalated complex with actinomycin D, and that the dG-dC sequence is an important binding site for actinomycin D in polydeoxynucleotides. Titrations of actinomycin D with monodeoxynucleotides were monitored at 380, 425, 440,465, and 480, nm in both absorption and CD modes. Comparisons fo saturation profiles at these wavelengths reveal that the curves obtained at various wavelenths do not superimpose with each other, so that they must reflect different titation processes. In complex formation wiht any given nucleotide, the apparent binding affinity monitored at these wavelengths decreases in the order given above. Based on these resulted and on features noted in the CD spectra of certain complexes, it is concluded that a minimum of theree electronic transitions underlie the visible absorption band of actinomycin D, extending earlier findigs. Comparing the titration proffiles obtained with dAMP and dIMP with the result for these systems in mmr titratins [Krugh, T. R. & Chen, Y. C. (1975) Biochemistry 14 , 4912–4922], it is concluded that one transition, centered at 370 nm, monitors preponderantly effects occurring at the 6 (benzenoid) nucleotide binding site and a second transition, located at 490 nm, is sensitive preferentially to processes occurring at the 4 ( quinoid) binding site. The latter is probably closely asscoiated with 2-amino and /or 3-carbonyl substituents. The third transition, identified with the absorption maxium at 420–440 nm, appears to reflect contributions arising in the entire phenoxazone chromophore. Using these band assignments, it is concluded that the benzenoid site binds nucleotides 1.5–3 times more avidly than does the quinoid site. CD titrations resolve these processes more effectively than do abscrption titrations. Aspects of the structures of these complexes formed in solution are discussed.