Characterization of the binding interactions between EvaGreen dye and dsDNA.

Understanding the dsDNA·EG binding interaction is important because the EvaGreen (EG) dye is increasingly used in real-time quantitative polymerase chain reaction, high resolution melting analysis, and routine quantification of DNA. In this work, a binding isotherm for the interactions of EG with duplex DNA (poly-dA17·poly-dT17) has been determined from the absorption and fluorescence spectra of the EG and dsDNA·EG complex. The isotherm has a sigmoidal shape and can be modeled with the Hill equation, indicating positive cooperativity for the binding interaction. A Scatchard plot of the binding data yields a concave-down curve in agreement with the Hill analysis of the binding isotherm for a positive cooperative binding interaction. Analysis of the Scatchard plot with the modified McGhee and von Hippel model for a finite one-dimensional homogeneous lattice and nonspecific binding of ligands to duplex DNA yields the intrinsic binding constant, the number of lattice sites occluded by a bound ligand, and the cooperativity parameter of 3.6 × 105 M-1, 4.0, and 8.1, respectively. The occluded site size of 4 indicates that moieties of the EG intercalate into the adjacent base pairs of the duplex DNA with a gap of 1 intercalation site between EG binding sites, as expected for a bifunctional molecule. Interestingly, at high [EG]/[base pair], the intercalation is disrupted. A model is proposed based on the fluorescence spectrum where the formation of anti-parallel stacked chains of EGs bound externally to the duplex DNA occur at these high ratios.

UV Raman spectroscopy of hydrocarbons.

In this paper, the UV Raman spectra of a large number of saturated and alkyl-substituted monocyclic, bicyclic and polycyclic aromatic hydrocarbons are obtained at 220 and 233 nm excitation wavelengths. Also included are nitrogen- and sulphur-containing hydrocarbons. The spectra obtained are fluorescence free, even for such highly fluorescent compounds as perylene, consistent with earlier reports of UV Raman spectra of hydrocarbons. The hydrocarbon UV Raman spectra exhibit greatly improved signal-to-noise ratio when in the neat liquid or solution state compared with the neat solid state, suggesting that some surface degradation occurs under the conditions used here. Assignments are given for most of the bands and clear marker bands for the different classes of hydrocarbons are readily observable, although their relative intensities vary greatly. These results are discussed in the context of structure and symmetry to develop a consistent, molecular-based model of vibrational group frequencies.

Raman spectroscopy as a discovery tool in carbohydrate chemistry.

Raman spectra of nine anomerically stable monosaccharides have been obtained in aqueous solution in the 700-1700 cm(-1) spectral range. Good-quality spectra are obtained of solutions with concentrations as low as 10 mM and volumes as small as 15 microL. Interestingly, the Raman spectra appear to be exquisitely sensitive to the configuration of the carbon centers; unique spectra are obtained of all nine monosaccharides. The unique Raman spectral fingerprint observed for each monosaccharide, and for each anomer of each monosaccharide, suggests that Raman spectroscopy may be a useful technique for the identification and characterization of biologically relevant oligosaccharides. To test this idea, Raman spectra of three unknown disaccharides were obtained in a single-blind study. Identification of the individual monosaccharide components and their anomeric configuration was completely successful. All of these results suggest that development of Raman spectroscopy as a fast, sensitive discovery tool in glycobiology and carbohydrate chemistry is straightforward.

Identification of the quinone cofactor in mammalian semicarbazide-sensitive amine oxidase.

Mammalian semicarbazide-sensitive amine oxidase (SSAO) enzymes have been classified as EC 1.4.3.6 [amine:oxygen oxidoreductase (deaminating)(copper-containing)]. However, both the identity of the quinone cofactor and the presence of copper remain unconfirmed, and SSAO has proved impossible to purify to homogeneity in sufficient yield to permit cofactor identification. To circumvent this problem, we have partially purified SSAO enzymes from bovine and porcine aortae and have established, with a redox-cycling assay, that no other quinoproteins were present in enzyme preparations. Enzymes were then derivatized with (p-nitrophenyl)hydrazine (p-NPH), which forms a covalent yellow complex with the quinone cofactor. Visible absorbance spectra of derivatized bovine and porcine enzymes (respective lambdamax values 456 and 476 nm at neutral pH, shifting to 580 and 584 nm in 2 M KOH) were consistent with the presence of (2,4,5-trihydroxyphenyl)alanine quinone (TPQ) as cofactor. Resonance Raman spectra were essentially identical to that for pea seedling amine oxidase, a known TPQ-containing enzyme. Extensive digestion of SSAO enzymes, and of porcine kidney diamine oxidase, with pronase E yielded species with identical chromophoric properties characteristic of the dipeptide, TPQ(p-NPH)-Asp. Thermolytic digestion of porcine SSAO gave two cofactor-containing peptides that contained a TPQ consensus sequence, Asn-X-Asp-Tyr-Tyr, where X is a blank cycle corresponding to TPQ. N-terminal sequencing of whole enzymes revealed a membrane-spanning region typical of an extracellular type II glycoprotein. These results confirm the presence of TPQ in mammalian membrane-bound SSAO ectoenzymes.

Structure of the retinal chromophore in 7,9-dicis-rhodopsin.

Bovine rhodopsin was bleached and regenerated with 7,9-dicis-retinal to form 7,9-dicis-rhodopsin, which was purified on a concanavalin A affinity column. The absorption maximum of the 7,9-dicis pigment is 453 nm, giving an opsin shift of 1600 cm-1 compared to 2500 cm-1 for 11-cis-rhodopsin and 2400 cm-1 for 9-cis-rhodopsin. Rapid-flow resonance Raman spectra have been obtained of 7,9-dicis-rhodopsin in H2O and D2O at room temperature. The shift of the 1654-cm-1 C = N stretch to 1627 cm-1 in D2O demonstrates that the Schiff base nitrogen is protonated. The absence of any shift in the 1201-cm-1 mode, which is assigned as the C14-C15 stretch, or of any other C-C stretching modes in D2O indicates that the Schiff base C = N configuration is trans (anti). Assuming that the cyclohexenyl ring binds with the same orientation in 7,9-dicis-, 9-cis-, and 11-cis-rhodopsins, the presence of two cis bonds requires that the N-H bond of the 7,9-dicis chromophore points in the opposite direction from that in the 9-cis or 11-cis pigment. However, the Schiff base C = NH+ stretching frequency and its D2O shift in 7,9-dicis-rhodopsin are very similar to those in 11-cis- and 9-cis-rhodopsin, indicating that the Schiff base electrostatic/hydrogen-bonding environments are effectively the same. The C = N trans (anti) Schiff base geometry of 7,9-dicis-rhodopsin and the insensitivity of its Schiff base vibrational properties to orientation are rationalized by examining the binding site specificity with molecular modeling.

Why are blue visual pigments blue? A resonance Raman microprobe study.

A resonance Raman microscope has been developed to study the structure of the retinal prosthetic group in the visual pigments of individual photoreceptor cells. Raman vibrational spectra are obtained by focusing the probe laser on intact photoreceptors frozen on a 77 K cold stage. To elucidate the mechanism of wavelength regulation in blue visual pigments, we have used this apparatus to study the structure of the chromophore in the 440-nm absorbing pigment found in "green rods" of the toad (Bufo marinus). The 9-cis isorhodopsin form of the green rod pigment exhibits a 1662-cm-1 C = NH+ Schiff base stretching mode that shifts to 1636 cm-1 in deuterium-substituted H2O. This demonstrates that the Schiff base linkage to the protein is protonated. Protonation of the Schiff base is sufficient to explain the 440-nm absorption maximum of this pigment without invoking any additional protein-chromophore interactions. The absence of additional perturbations is supported by the observation that the ethylenic band and the perturbation-sensitive C-10-C-11 and C-14-C-15 stretching modes have the same frequency as those of the 9-cis protonated retinal Schiff base in solution. Our demonstration that a blue visual pigment contains an unperturbed protonated Schiff base provides experimental evidence that the protein charge perturbation responsible for the opsin shift in the 500-nm absorbing pigments is removed in the opsins of blue pigments, as suggested by the sequence data.

Excited-state structure and isomerization dynamics of the retinal chromophore in rhodopsin from resonance Raman intensities.

Resonance Raman excitation profiles have been measured for the bovine visual pigment rhodopsin using excitation wavelengths ranging from 457.9 to 647.1 nm. A complete Franck-Condon analysis of the absorption spectrum and resonance Raman excitation profiles has been performed using an excited-state, time-dependent wavepacket propagation technique. This has enabled us to determine the change in geometry upon electronic excitation of rhodopsin's 11-cis-retinal protonated Schiff base chromophore along 25 normal coordinates. Intense low-frequency Raman lines are observed at 98, 135, 249, 336, and 461 cm-1 whose intensities provide quantitative, mode-specific information about the excited-state torsional deformations that lead to isomerization. The dominant contribution to the width of the absorption band in rhodopsin results from Franck-Condon progressions in the 1,549 cm-1 ethylenic normal mode. The lack of vibronic structure in the absorption spectrum is shown to be caused by extensive progressions in low-frequency torsional modes and a large homogeneous linewidth (170 cm-1 half-width) together with thermal population of low-frequency modes and inhomogeneous site distribution effects. The resonance Raman cross-sections of rhodopsin are unusually weak because the excited-state wavepacket moves rapidly (approximately 35 fs) and permanently away from the Franck-Condon geometry along skeletal stretching and torsional coordinates.