The influence of formamide on thermal denaturation profiles of DNA and metaphase chromosomes in suspension

Systematic photometric studies are presented to analyze the thermal denaturation behaviour with and without formamide of metaphase chromosome suspensions in comparison to DNA solutions. Temperature dependent hyperchromicity measurements at 256 nm and 313 nm were performed using an appropriately designed computer-controlled photometer device. Due to an upright optical axis, this allowed absorbance measurements with negligible sedimentation effects not only for solutions of pure DNA, but also for particle suspensions of isolated metaphase chromosomes. This device has a temperature resolution of +/- 0.5 degrees C and an optical sensitivity of 10(-3) to 10(-4) optical density. For calf thymus DNA the reduction of the melting point with the increase of formamide in the solution was measured at pH 7.0 and pH 3.2. The good correlation of the theoretical approximation to experimental data indicated the suitability of the apparatus to quantitatively describe DNA conformation changes induced by thermal denaturation. For metaphase chromosome preparations of Chinese hamster culture cells, absorbance changes were measured between 20 degrees C and 95 degrees C with a temperature gradient of 1 degrees C/min. These measurements were performed at pH 7.0 and at pH 3.2. The denaturation profiles (= first derivative of the absorbance curve) resulted in a highly variable peak pattern at 256 nm and 313 nm indicating complex conformation changes. A statistical evaluation of the temperature values of the peak maxima resulted in temperature ranges typical for chromosomal conformation changes during thermal treatment. Especially the range of highest temperature values was independent from pH modifications. For pH 3.2 the influence of formamide on the denaturation behaviour of metaphase chromosome preparations was analyzed. In contrast to pure DNA solutions, a reduction of the "melting point" (i.e. the maximum temperature at which a conformation change takes place) was not found. However, the denaturation behaviour depended on the duration of formamide treatment before the measurement.     

Direct measurement of the interaction between phosphatidylglycerol bilayers in aqueous electrolyte solutions.

Results are presented of force measurements between deposited bilayers of dimyristoylphosphatidyl glycerol (DMPG) at T greater than Tm, and distearoylphosphatidyl glycerol (DSPG) at T less than Tm. Below a bilayer separation of 100 nm, a repulsive double-layer force is measured, which can be explained through the combined screening and binding effect of the counterions in electrolyte solutions of NaCl, HCl, CaCl2, or mixtures of these. The binding of cations to bilayers in the fluid phase (DMPG) appears to be greater than to bilayers in the gel phase (DSPG). At shorter range, below approximately 3 nm, an attractive interaction is measured in solutions containing CaCl2, which was found to be slightly stronger than the theoretically expected van der Waals interaction. No hydration force was observed to exist in solutions containing CaCl2. In NaCl solutions, the measured interbilayer force can completely be accounted for by the electrostatic repulsion, down to a bilayer separation of at least 2 nm, below which no accurate measurements were possible anymore. Parallel measurements on PG monolayers show that the contraction of a DMPG monolayer following addition of CaCl2 is significantly greater than what is predicted from the change in the double-layer free energy alone. This indicates that changes in the lateral interactions between the lipid headgroups probably involve Ca2+-bridge binding and/or a possible dehydration of the lipid headgroups through Ca2+ binding. The results shed new light on both the interbilayer and intrabilayer interactions of PG and identify the possible factors responsible for the morphological behavior of PG aggregates.     

Metachromatic staining and electron dense reaction of glycosaminoglycans by means of Cuprolinic Blue

Summary The cationic phthalocyanin-like dye Cuprolinic Blue, unlike phthalocyanin dyes such as Alcian Blue or Astra Blue, can definitely exhibit a clear metachromatic reaction with appropriate substrates, The application of Cuprolinic Blue to epoxy-embedded semithin sections revealed that mast cell cytoplasmic granules, goblet cell mucin and cartilage matrix stained in violet shades (metachromatic), whereas nuclear chromatin presented a bright blue coloration (orthochromatic). The metachromatic structures showed a high degree of contrast when ultrathin sections treated with Cuprolinic Blue were examined by electron microscopy.Cytophotometric measurements of stained components from the large intestine showed different absorption maxima: at 580 nm for mucin and at 640 nm for nuclei. The spectroscopical analysis revealed a clear-cut metachromatic shift when the dye was in the presence of chondroitin—4-sulphate. The addition of aluminium metal to Cuprolinic Blue solutions resulted in a striking spectral change; under such conditions the dye showed absorption maximum at 530 nm.     

Equilibrium gel permeation: a single-photon counting spectrophotometer for studies of protein interaction.

When a small column or flow cell packed with gel particles is completely saturated with a solution containing molecular species of interest, the average cross-sectional area occupied by the solute (partition cross section) is conveniently and precisely determined by direct optical scanning. For a mixture of interacting solutes this equilibrium gel permeation measurement yields the weight average of the species partition cross sections and the variation of this quantity with solute concentration permits determination of the solute interaction parameters (stoichiometry, equilibrium constants). We have developed a computer-controlled single-photon counting spectrophotometer for these measurements. The instrument exhibits high precision over a wide range of optical density. With counting times in the range of 10-1000 s the standard deviations on optical densities of protein solutions measured at 220 nm are typically 0.0006 at 1 OD, 0.002 at 2 OD, 0.005 at 4 OD. Beer's law tests show that deviations from linearity are less than these precision limits. Partition cross-section measurements for proteins can be made with an accuracy of better than 0.001 and information can be obtained with protein solutions at least as low as 1 mug/ml.     

Catechol biosensing using a nanostructured layer-by-layer film containing Cl-catechol 1,2-dioxygenase

The detection of aromatic compounds from pesticides and industrial wastewater has become of great interest, since these compounds withstand chemical oxidation and biological degradation, accumulating in the environment. In this work, a highly sensitive biosensor for detecting catechol was obtained with the immobilization of Cl-catechol 1,2-dioxygenase (CCD) in nanostructured films. CCD layers were alternated with poly(amidoamine) generation 4 (PAMAM G4) dendrimer using the electrostatic layer-by-layer (LbL) technique. Circular dichroism (CD) measurements indicated that the immobilized CCD preserved the same conformation as in solution. The thickness of the very first CCD layers in the LbL films was estimated at ca. 3.6 nm, as revealed by surface plasmon resonance (SPR). PAMAM/CCD 10-bilayer films were employed in detecting diluted catechol solutions using either an optical or electrical approach. Due to the mild immobilization conditions employed, especially regarding the pH and ionic strength of the dipping solutions, CCD remained active in the films for periods longer than 3 weeks. The optical detection comprised absorption experiments in which the formation of cis-cis muconic acid, resulting from the reaction between CCD and catechol, was monitored by measuring the absorbance at 260 nm after film immersion in catechol solutions. The electrical detection was carried out using LbL films deposited onto gold-interdigitated electrodes immersed in aqueous solutions at different catechol concentrations. Using impedance spectroscopy in a broad frequency range (1Hz-1kHz), we could detect catechol in solutions at concentrations as low as 10(-10) M.     

Slow photoinduced changes in reaction centers of Rhodobacter sphaeroides R-26 by holographic interferometry

Using the on-line holographic interferometry, changes in the refraction index of illuminated solutions containing the Rhodobacter sphaeroides R-26 reaction centers isolated from membranes by detergents of ionic and nonionic nature were registered. Factors affecting the refraction index of these solutions were analyzed. It was shown that the photoinduced changes in this parameter are due to structural changes in the molecular complex of reaction centers. The results of measurements of the kinetics of absorption spectra at lambda = 865 nm for two detergent types are presented, which coincide qualitatively with the results of holographic measurements. The change in the volume of the molecular complex of reaction centers was estimated to be 0.1 divided by 1%.     

The effect of heating by microwave irradiation and by conventional heating on the aldehyde concentration in aqueous glutaraldehyde solutions

Summary The effect of short time heating of aqueous solutions of glutaraldehyde (GA) on relative aldehyde concentration was determined using spectrophotometric analysis. Because free monomeric GA absorbs U. V. light at 280 nm, whereas the alpha, beta polymeric forms absorb at 235 nm, the purity of GA solutions can be expressed as the ratio: A 235 nm/A 280 nm (purification index, P.I.).Heating of 4 ml aliquots of 0.85% distilled aqueous GA solution resulted in an increase of the absorption at 280 nm which is correlated positively with temperature. No increase of absorption at 235 nm was found when solutions were kept at 40°C for several hours. The increase of absorption at 280 nm is caused by a rapid decyclization of hemiacetals producing an increase in free aldehyde concentration.No major differences in absorption were found between the solutions heated by microwave and by conventional heating. However, because microwave irradiation is known to produce an homogeneous rise in temperature, especially in bulky samples, it is expected that the results of fixation procedures will improve by the combined effect of higher temperature and enhanced diffusion rates of the fixating species.     

Action Spectra for Transformation and Fluorescence of Protochlorophyll Holochrome from Bean Leaves

The action spectrum from 232 to 687 nm was determined for the transformation of protochlorophyllide into chlorophyllide a in solutions of protochlorophyll holochrome Fran bean leaves. The whole ultraviolet region is effective. The peaks at 445 and 639 nm have a height ratio of 4.0. Only radiation absorbed in the protochlorophyllide itself is effective in transformation (absorption in aromatic amino acids of the protein and in carotenoids is ineffective). The activation spectra for fluorescence at 643 and 683 nm are measured for the holochrome before and after transformation, as well as the change in absorption spectrum that takes place upon transformation. By combining the various measurements the spectrum of inactive (non-transformable) protochlorophyllide (peak at 440 nm) and the holochromatic chlorophyllide a are derived. In the latter spectrum the peaks at 419 and 435 nm are of about the same height.     

Near-infrared spectra of Scapharca homodimeric hemoglobin: characterization of the deoxy and photodissociated derivatives.

The near-infrared charge transfer band at 760 nm (band III) has been investigated in deoxy and photodissociated dimeric Scapharca hemoglobin. At 300 K, the 10-ns spectrum of the carbonmonoxy derivative photoproduct is shifted by about 6 nm toward longer wavelengths with respect to the deoxy spectrum, both in buffer and in glycerol/buffer solutions. Moreover, the band III peak occurs at about the same wavelength at 300 K and at 10 K for the 10-ns photodissociated derivative, whereas in the deoxy derivative large changes in peak position and linewidth are observed as a function of temperature. These findings suggest that in dimeric Scapharca hemoglobin the photoproduct has not relaxed after 10 ns. The complete time dependence of the relaxation process has been studied both in buffer and in glycerol/buffer solutions at room temperature. The relaxation from the photoproduct to the deoxy species occurs on a microsecond time scale, in line with recent optical absorption and resonance Raman measurements.     

Acid polysaccharide content of frog rod outer segments determined by metachromatic toluidine blue staining

Sulfonation of periodate-oxidized vicinal hydroxyl groups on a polysaccharide backbone allows binding of toluidine blue (aldehyde bisulfite-toluidine blue or ABT staining) with a concurrent metachromatic shift of the dye's absorption peak from 630 nm (monomer) to 580 nm (isolated dimer interaction at vicinal sulfonate groups) or 540 nm (dye polymer interaction). A molar absorptivity of 2.358 +/- 0.134 X 10(4) at 540 nm for polymeric toluidine blue O chloride (TB) aggregates was determined by spectrophotometry of TB bound to hyaluronic acid (HA) and sulfonated glycogen (SG) in water. Microspectrophotometry of ABT stained frog rod outer segments (FROS) showed spectra similar to TB in aqueous HA and SG solutions with absorbances corresponding to 0.063 M dye bound to sugar. Given two dye molecules bound per sugar residue and a rhodopsin concentration of 3.25 mM in FROS, the above indicates 10 stainable sugars per rhodopsin are contained in these cells. Half of these sugars are sensitive to hyaluronidase digestion implying 5 glycosaminoglycan (GAG) repeating units and 5 stainable oligosaccharide sugar residues per rhodopsin in FROS. The GAGs in FROS appear to be primarily HA. Birefringence measurements at 475 nm indicate that this HA and the oligosaccharide of rhodopsin are anisotropically oriented in these cells.     

Photosensitization of singlet oxygen formation by pterins and flavins. Time-resolved studies of oxygen phosphorescence under laser excitation

To elucidate the biochemical roles of singlet molecular oxygen (1(O2)) in the light-dependent reactions photosensitized by biological blue-light photoreceptors, time-resolved measurements of photosensitized 1O2 phosphorescence (1270 nm) were performed in air-saturated aqueous ((D2)O) solutions of pterins (2-amino-4-hydroxy-6,7-dimethylpteridine (DMP) and 2-amino-4-hydroxy-6-tetrahydroxybutyl-(D-arabo)pteridine (TOP)) and flavins (riboflavin and flavin mononucleotide (FMN)) under excitation with nitrogen laser (337.1 nm) pulses. The 1(O2) quantum yields were found to be 0.16, 0.20, 0.50, and 0.50 for DMP, TOP, riboflavin, and FMN, respectively. The data indicate that pterins and flavins are rather efficient photosensitizers of 1(O2) production that might be important for their photobiological functions.     

Characterization of lens alpha-crystallin tryptophan microenvironments by room temperature phosphorescence spectroscopy

Room temperature phosphorescence techniques were used to study the structural and dynamic features of the tryptophan residues in bovine alpha-crystallin. Upon excitation at 290 nm, the characteristic signature of tryptophan phosphorescence was observed with an emission maximum at 442 +/- 2 nm. The phosphorescence intensity decay was biphasic with lifetimes of 5.4 ms (71%) and 42 ms (29%). Phosphorescence quenching measurements strongly suggest that each component corresponds to one class of tryptophans with the more buried residues having the longer emission lifetime. Three small-molecule quenchers were surveyed, and in order of increasing quenching efficiency: iodide less than nitrite less than acrylamide. A heavy-atom effect was observed in iodide solutions, and an upper limit of 5% was placed on the quantum yield of triplet formation in iodide-free solutions, while the phosphorescence quantum yield was estimated to be approximately 3.2 x 10(-4). The temperature dependence of the phosphorescence lifetime was measured between 5 and 40 degrees C. Arrhenius plots exhibited discontinuities at 26 and 29 degrees C for the short- and long-lived components, respectively, corresponding to abrupt transitions in segmental flexibility. Denaturation studies revealed conformational transitions between 1 and 2 M guanidine hydrochloride, and 4 and 6 M urea. Long-lived phosphorescence lifetimes of 3 and 7 ms were measured in 6 M guanidine hydrochloride and 8 M urea, respectively, suggesting that some structural features are preserved even at very high concentrations of denaturant. Our studies demonstrate the sensitivity of room temperature phosphorescence spectroscopy to the structure of alpha-crystallin, and the applicability of this technique for monitoring conformational changes in lens crystallin proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Static water structure detected by heat capacity measurements on aqueous solutions of a triple-helical polysaccharide schizophyllan

Heat capacity measurements were made on aqueous solutions of a triple-helical polysaccharide schizophyllan by precision adiabatic calorimetry over a wide range of concentrations 30.45-90.93 wt % at temperatures between 5 and 315 K. The heat capacity curves obtained were divided into four groups depending on the weight fraction of schizophyllan w regions I-IV. In region I, triple-helices with the sheath of bound water, structured water, and loosely structured water forming layers around the helix core are embedded in free water. In region II, there is no free water, and loosely structured water decreases until it vanishes, but structured water stays constant with increasing w. In region III, bound water remains unaffected, but structured water decreases with increasing w by overlapping each other. Finally, in region IV, only schizophyllan and bound water exist, the latter decreasing upon increasing w. The maximum thickness of each layer is 0.18(3) nm for bound water, 0.13(4) nm for structured water, and 0.23(6) nm for loosely structured water, and these layers of water are at the enthalpy levels of 53%, 93.7%, and nearly 100%, respectively, between ice (0%) and free water (100%).     

Acid polysaccharide content of frog rod outer segments determined by metachromatic toluidine blue staining

Summary Sulfonation of periodate-oxidized vicinal hydroxyl groups on a polysaccharide backbone allows binding of toluidine blue (aldehyde bisulfite-toluidine blue or ABT staining) with a concurrent metachromatic shift of the dye's absorption peak from 630 nm (monomer) to 580 nm (isolated dimer interaction at vicinal sulfonate groups) or 540 nm (dye polymer interaction). A molar absorptivity of 2.358±0.134×10⁴ at 540 nm for polymeric toluidine blue O chloride (TB) aggregates was determined by spectrophotometry of TB bound to hyaluronic acid (HA) and sulfonated glycogen (SG) in water. Microspectrophotometry of ABT stained frog rod outer segments (FROS) showed spectra similar to TB in aqueous HA and SG solutions with absorbances corresponding to 0.063 M dye bound to sugar. Given two dye molecules bound per sugar residue and a rhodopsin concentration of 3.25 mM in FROS, the above indicates 10 stainable sugars per rhodopsin are contained in these cells. Half of these sugars are sensitive to hyaluronidase digestion implying 5 glycosaminoglycan (GAG) repeating units and 5 stainable oligosaccharide sugar residues per rhodopsin in FROS. The GAGs in FROS appear to be primarily HA. Birefringence measurements at 475 nm indicate that this HA and the oligosaccharide of rhodopsin are anisotropically oriented in these cells.Supported by NIH grants EY00012, EY07035 and EY01583     

Ageladine A, a pyrrole-imidazole alkaloid from marine sponges, is a pH sensitive membrane permeable dye

The alkaloid ageladine A, a pyrrole-imidazole alkaloid isolated from marine Agelas sponges shows fluorescence in the blue-green range during excitation with UV light with the highest absorption at 370 nm. The fluorescence of this alkaloid is pH dependent. Highest fluorescence is observed at pH 4, lowest at pH 9 with the largest fluorescence changes between pH 6 and 7. Ageladine A is brominated, which facilitates membrane permeation and therefore allows for easy staining of living cells and even whole transparent animal staining. To calculate the exact pH in solutions, cells, and tissues, the actual concentration of the alkaloid has to be known. A ratiometric measurement at the commonly used excitation wavelengths at 340/380 nm allows pH measurements in living tissues with an attenuated influence of the ageladine A concentration on calculated values. The fluorescence changes report small intracellular pH changes induced by extracellular acidification and alkalization as well as intracellular alkalization induced by ammonium chloride.     

Vacuum ultraviolet circular dichroism of fibronectin. Dominant tyrosine effects

The spectroscopic authenticity of a very intense negative band at about 183 nm reported previously from conventional circular dichroism (c.d.) studies of bovine plasma fibronectin has now been confirmed by vacuum ultraviolet c.d. measurements on two prototype spectrometers, one using a conventional light source and the other using synchrotron radiation. Closely similar spectra were obtained from both instruments, and from both solid films and solutions. The spectra show no obvious parentage in the known c.d. of the peptide backbone, but have marked similarities to the c.d. of N-acetyltyrosineamide, both in the strong band at 183 nm and in a characteristic positive band at 230 nm, It is concluded that the c.d. of fibronectin is dominated by contributions from tyrosine side-chains and that, as suggested previously, these may provide a sensitive probe for molecular organization and interactions.     

Flame atomic emission spectrometric determination of aluminium in a bovine blood plasma matrix

A flame atomic emission spectrometric method, is described for the determination of aluminium in bovine blood plasma matrices. Plasma samples are wet-digested and solutions are aspirated into a conventional nitrous oxide-acetylene flame. Analyte emission is monitored at 396.15 nm with corrections for background emission being obtained from measurements several tenths nm on both sides of the aluminium line. The mean recovery of 0.3–5 μg/ml aluminium added to model solutions containing 500–5000 μg Na/ml, 50–1000 μg Ca/ml, 2000–5000 μg K/ml, or simulated plasma digests containing Na, K, and Ca was 100,6% (SD = 10.9, df = 60); the mean recovery of 0.3, 0.5, and 1.0 μg/ml aluminium added to blood plasma before digestion was 94.3% (SD = 9.8, df = 33) indicating no serious interferences. For standard solutions, the detection limit (signal: peak-to-peak noise = 1) was 0.02 μg/ml by flame emission, and 0.12 μg/ml by atomic absorption measurements with the same instrument. A sample taken through the analytical procedure, gave a detection limit of 0.05 μg/ml suggesting the submicrogram per milliliter region as the lower practical limit of the method.     

Spectral characterization of the effect of viscosity on Fura-2 fluorescence: excitation wavelength optimization abolishes the viscosity artifact.

A systematic study of the spectral characteristics of the viscosity artifact in Fura-2 based [Ca2+] measurements reveals that, by selecting excitation wavelengths approximately 10 nm longer than those routinely employed and modestly reducing excitation bandpasses, the magnitude of the artifact can be reduced to experimentally undetectable levels without greatly impairing [Ca2+] measurements. The feasibility of this approach was confirmed on a ratio imaging microscope; the magnitude of the artifact observed in dextran-conjugated Fura-2 solutions prepared in water or in 50% sucrose was not statistically significant using an excitation wavelength pair of 361/389 nm, whereas at 350/380 nm [Ca2+] was underestimated by 34% in the higher viscosity solution. Thus, provided potential pitfalls are taken into account, a simple change in imaging protocol can avoid the viscosity artifact without recourse to correction factors. This approach may be employed either routinely, or else merely to test whether apparent [Ca2+]i differences observed at more conventional wavelengths arise from the viscosity artifact.     

Gap junctions. Structural changes after uncoupling procedures

The freeze-fracture appearance of rat stomach and liver gap junctions changes after uncoupling procedures such as inhibition of the metabolism of perfusion with hypertonic sucrose. In control stomach, either fixed immediately or kept for 1 h in a well-oxygenated Tyrode's solution at 37 degrees C, most gap junctions between mucous cells contain particles irregularly packed at an average center-to-center spacing of 10.3-10.5 nm. After 1-h treatment with 2,4-dinitrophenol (DNP), at the same temperature and oxygenation, most particles aggregate hexagonally at an average spacing of approximately 8.5 nm. Similar changes are seen in hypoxic specimens. In control liver, fixed by perfusion, most junctional particles are irregularly packed at an average center-to-center spacing of approximately 10 mm. Small areas of fairly regular hexagonal packing are occasionally seen, where the average particle spacing is 9.2-9.5 nm. In hypoxic liver, the junctional particles form regular hexagonal packings in which the average center-to-center particle spacing is approximately 8.5 nm. In liver perfused with hypertonic sucrose-calcium solutions, following EDTA solutions, most junctions are pulled apart. The separated junctional membranes, expected to be highly impermeable, contain particles regularly and tightly packed as in hypoxic or DNP-treated junctions. Preliminary measurements indicate also a possible change in particle diameter, from approximately 8.6 nm (control) to approximately 7.7 nm (treated). The structural changes are similar to those previously reported in crayfish and may reflect conformational changes in particle subunits resulting in functional uncoupling.     

Excitation energy transfer in the light-harvesting chlorophyll a/b.protein

The "light-harvesting chlorophyll a/b.protein" described by Thornber has been prepared electrophoretically from spinach chloroplasts. The optical properties relevant to energy transfer have been measured in the red region (i.e. 600-700 nm). Measurements of the absorption spectrum, fluorescence excitation spectrum and excitation dependence of the fluorescence emission spectrum of this protein confirm that energy transfer from chlorophyll b to chlorophyll a is highly efficient, as is the case in concentrated chlorophyll solutions and in vivo. The excitiation dependence of the fluorescence polarization shows a minimum polarization of 1.9% at 650 nm which is the absorption maximum of chlorophyll b in the protein and rises steadily to a maximum value of 13.8% at 695 nm, the red edge of the chlorophyll a absorption band. Analysis of these measurements shows that at least two unresolved components must be responsible for the chlorophyll a absorption maximum. Comparison of polarization measurements with those observed in vivo shows that most of the depolarization observed in vivo can take place within a single protein. Circular dichroism measurements show a double structure in the chlorophyll b absorption band which suggest an exciton splitting not resolved in absorption. Analysis of these data yields information about the relative orientation of the So leads to S1 transition moments of the chlorophyll molecules within the protein.