José Bontemps

Comparative binding study of the interaction of quinacrine and ethidium bromide with DNA and nucleohistone.

Abstract The degree of binding of quinacrine dihydrochloride and ethidium bromide to DNA and nucleohistone has been determined by direct and indirect methods. The results obtained by the equilibrium dialysis experiments have been analyzed in terms of various theoretical models and have led us to propose for the interaction of the dyes with DNA at low ionic strength a structural scheme where the external binding sites are next to the intercalative ones. The equilibrium dialysis results were used to check those obtained by the indirect methods, i.e. absorption and fluorescence titrations, and to identify the origin of the discrepancies. The comparative study of the binding of these dyes to DNA and nucleohistone has shown that the accessible part of DNA in the nucleoprotein is to some extent different from free DNA itself.

Analysis of dansyl derivatives of di- and polyamines in mouse brain, human serum and duodenal biopsy specimens by high-performance liquid chromatography on a standard reversed-phase column

The concentrations of putrescine, spermine and spermidine were measured in human serum, childrens duodenal biopsy specimens and mouse brain homogenates by high-performance liquid chromatography. The chromatographic analysis was performed on dansyl derivatives of the polyamines using a reverse-phase system with an ion-pairing retention mechanism (heptane sulphonate). Capacity factors were determined at different concentrations of acetonitrile. Simple linear gradients were set up for fast (15 min) or routine (25 min) analysis. Three fluorescence detectors were compared for these determinations and their detection limits determined. The minimum detectable amount of polyamines was 25 fmol compared to 500 fmol with standard detectors. While samples prepared from tissues did not require a high sensitivity, a detector of better performance was needed to assay the polyamines in human serum.

Determination of thiamine and thiamine phosphates in excitable tissues as thiochrome derivatives by reversed-phase high-performance liquid chromatography on octadecyl silica

The analysis of thiamine and thiamine phosphates by high-performance liquid chromatography owes its high sensitivity to the fluorescent derivatives or thiochromes obtained by chemical oxidation in alkaline medium. The possibility of performing precolumn oxidation with potassium ferricyanide instead of using the hazardous cyanogen bromide has been investigated. The derivatization step has been optimized with respect to the following parameters: concentration of alkali and oxidant, presence of methanol and stability of the thiochromes . A gradient separation with 25 mM phosphate buffer (pH 8.4) and methanol as mobile phase components and an octadecyl silica column as stationary phase has been set up. The analytical run takes 14 min with the following elution order: thiochrome triphosphate, thiochrome pyrophosphate, thiochrome monophosphate and thiochrome. The minimum detectable amount is 0.05 pmol. The method was found suitable for the determination of thiamine compounds in excitable tissues such as nerves and electric organs as well as in proteins extracted from membranes of these organs. It may be useful to study the role of thiamine in the electrical activity of these tissues at the molecular level.

High-yield synthesis of a [3H]ethylenediamine ditetrodotoxin derivative

[3H]Tetrodotoxin [( 3H]TTX) and a [3H]ethylenediamine derivative of TTX are the most widely used ligands for the study of the Na channel. The former ligand presents a low specific radioactivity (1 Ci/mmol) while the latter is highly labeled (30 Ci/mmol). However, its two-step synthesis, i.e., mild oxidation followed by coupling of [3H]ethylenediamine, has been described with a low overall yield of 1.7%. In this work, more favorable experimental conditions are defined for the limiting reaction, i.e., the oxidation step, using [14C]testosterone as a model molecule. Applied to the oxidation of tetrodotoxin, this procedure produces yield values of 30-50%, as determined by high-performance liquid chromatography. Moreover, two oxidized TTX molecules appear to be covalently linked to [3H]ethylenediamine, yielding a new labeled tetrodotoxin derivative with a specific radioactivity of 45 Ci/mmol and a dissociation constant of 0.6 nM for electroplax membranes.

Optical and electro-optical properties of the complexes of dibutylproflavine with DNA and nucleohistone

Abstract A number of optical and electro-optical measurements showed that the binding of dibutylproflavine to calf thymus DNA and nucleohistone in 1 mM NaCl could be resolved on the basis of two external and orientated bound species, whose binding parameters have been determined by a spectrophotometric method. The absorption and the electric dichroism properties were found to show up additivity of contributions from the two bound species. The orientation of the long axis of the strongly bound molecules of species I appeared close to the inclination of the DNA grooves, while that of species II differed by about ten degrees. A spatial proximity of the two binding sites was suggested by the dependence of the circular dichroism signals, the fluorescence quantum yield and the emission anisotropy on the degree of binding. The mobility of the first bound dibutylproflavine molecules was similar to that of the intercalated proflavine molecules, but lower in nucleohistone as compared to DNA.

Determination of thiamine and thiamine phosphates as thiochrome derivatives by reversed-phase chromatography on polystyrene packing materials

SummaryThe chromatography of thiochrome and related phosphate esters was studied on two different polystyrene packing materials.The adsorbents were compared with respect to capacity factors, plate heights and resolution factors measured with different mixtures of phosphate buffer (pH 8.5) and methanol.In contrast with derivatized silica, these phases were found to be stable using an alkaline mobile phase and sample. Fast separations were possible with good peak symmetry at all organic modifier concentrations.

Quantitative analysis of fluorescence profiles of chromosomes. Influence of DNA base composition on banding

Abstract Chromosome banding has been analysed in terms of DNA content and base composition distribution along five human chromosomes. Three intercalative dyes (quinacrine, proflavine and ethidium bromide) whose fluorescence quantum yield in the presence of DNAs of different base compositions has been determined, have been used to examine the influence of base composition on the chromosome patterns. Considering that the amount of DNA as determined by the Feulgen reaction is almost constant along the chromosome arms and assuming that base composition is the only factor influencing the fluorescence of these dyes, a distribution of the A-T base pair content along the chromosomes has been calculated from the fluorescence intensity profiles. From the ratio of the intensity profiles obtained with quinacrine and proflavine, patterns showing the variation of the DNA content and of the A-T base pair content could also be obtained independently. The validity of these different approaches is discussed.

High-speed analysis of dansyl derivatives of polyamines

SummaryAnalytical conditions for the high-speed, reversed-phase, liquid chromatographic of a diamine (putrescine) and polyamines (spermine and spermidine) were determined. Various elution modes were employed using the same mobile phase constituents: 20mM sodium heptane sulfonate and 20mM acetic acid as solvent A and, pure acetonitrile as solvent B. Samples were derivatized with dansyl chloride before injection.Under isocratic conditions, the separation of the three polyamines was achieved in 7 min. The use of a linear elution gradient led to the same analytical time but with a better resolution of the putrescine peak from non-polyamine frontal peaks. For these measurements, the sample size was 5μl. This volume was increased to 20μl and the use of a steep gradient combined with the peak-compression technique allowed a fast analysis in 2–3 minutes, which may be compared with a 30 min run time necessary when a conventional column is used.

Binding of [3H]ethylenediamine di-tetrodotoxin to its solubilized receptor from excitable tissues. Binding measurements by rapid gel-filtration and receptor stabilization by phosphatidylcholine.

The molecular study of bioelectrogenesis requires the purification of the membrane proteins involved in the Na-channel electrical activity. This complex biological structure contains various binding sites for different classes of neurotoxins. Labelled forms of the blocking agent, tetrodotoxin, are used to identified and quantified the solubilized membrane proteins during the purification. Such a specific probe was synthetized in our laboratory and this work reports the experimental set-up of the binding technique. A fast-gel-filtration method has been optimized with respect to column design, centrifugation time and speed and, delay between sample application and column centrifugation.

Protein phosphorylation in nerve and electric organ: Isolation and partial characterization of a high affinity system for ATP.

The impedance variation cycle (IVC) of axonal membranes has been described in terms of a dephosphorylation-phosphorylation cycle. It has been experimentally approached by preparing membranes from the walking nerves of crabs and from the main electric organ of Electrophorus electricus . Membrane proteins were solubilized in 1% Lubrol-PX and purified by chromatographic techniques. We have obtained a protein fraction which was phosphorylated by low [?-(32)P]-ATP concentrations, ranging from 5 x 10(?8)M to 10(?7)M. This fraction of high molecular weight proteins has been partly characterized in denaturing conditions revealing the presence of several protein species with different specific phosphorylation activity. At this experimental stage the two types of investigated materials displayed distinct patterns of phosphorylation versus molecular weight. The influence of tetrodotoxin and veratridine, on the phosphorylation process is a rather complex inhibitory effect, now under further investigation.