Energy of interaction in actinomycin-nucleotide complexes

Complexes of the natural heterocyclic antibiotic actinomycin D (AMD) with its putative carriers: purine and pyrimidine nucleotides, as well as with fragmented DNA and phospholipid liposomes have been studied by high-sensitivity spectrophotometry. The antibiotic is not only adsorbed onto the surface of purine clusters but also is incorporated into them. It is especially readily incorporated into unwound DNA regions. The incorporation is accompanied by a long-wavelength shift of the absorption spectrum. From the magnitude of the shift, the energy of interaction was calculated. In the case of AMD in the complex with caffeine and adenosine, it is 2.4 and 2.7 kcal/mol, and in the complex with guanosine and fragmented DNA it is considerably higher, 3.3 and 3.7 kcal/mol. It is assumed that guanosine, adenosine, caffeine and fragmented DNA may serve as carriers of the antibiotic.     

Stoichiometry of P1 plasmid partition complexes

The P1 plasmid prophage is faithfully partitioned by a high affinity nucleoprotein complex assembled at the centromere-like parS site. This partition complex is composed of P1 ParB and Escherichia coli integration host factor (IHF), bound specifically to parS. We have investigated the assembly of ParB at parS and its stoichiometry of binding. Measured by gel mobility shift assays, ParB and IHF bind tightly to parS and form a specific complex, called I + B1. We observed that as ParB concentration was increased, a second, larger complex (I + B2) formed, followed by the formation of larger complexes, indicating that additional ParB molecules joined the initial complex. Shift Western blotting experiments indicated that the I + B2 complex contained twice as much ParB as the I + B1 complex. Using mixtures of ParB and a larger polyhistidine-tagged version of ParB (His-ParB) in DNA binding assays, we determined that the initial I + B1 complex contains one dimer of ParB. Therefore, one dimer of ParB binds to its recognition sequences that span an IHF-directed bend in parS. Once this complex forms, a second dimer can join the complex, but this assembly requires much higher ParB concentrations.     

Kinetic studies of drug-dinucleotide complexes.

Three classes of kinetic behavior are observed in the complexes of actinomycin or ethidium with deoxydinucleotides. First, the initial dinucleotide binding to form a 1:1 complex is a rapid bimolecular process, whose rate could be measured for combination of actinomycin with d(pTpG) d(pGpT), d(pGpA), d(pGpG) d(pCpGpG), and d(pCpG) andfor combination of ethidium with d(pGpC). Second, with one exception, all reactions in which a second dinucleotide is added to form a 2:1 dinucleotide-drug complex are limited by a first-order step at high concentration. This class includes the combination of actinomycin with all dinucleotides tested except d(pGpC), and the reaction of ethidium with nucleotides of complementary sequence pyrimidine-purine, such as d(pCpG). The final class is the special case of d(pGpC) interacting to form a 2:1 complex with actinomycin. Third-order kinetics is observed, with no evidence for a first-order, rate-limiting step.     

The partition of glycosaminoglycan-quaternary ammonium complexes. I. The effect of phase composition.

The complexes formed between quaternary ammonium cations and polyanionic glycosaminoglycans can be partitioned between partially miscible aqueous inorganic salt and alcohol phases. Small changes in salt concentration can completely shift the complex from one phase to the other. The effect of the phase composition variables: the type of inorganic salt, the type of quaternary ammonium salt, and the alcohol used, were systematically investigated. The sharp transition from solubility in the upper non-aqueous phase to solubility in the lower, aqueous phase was found to be strongly affected by the type of inorganic salt. This transition occurred at higher salt concentrations when NaCl, KCl, or LiCl were used than when CaCl2 or MgCl2 were used. Differences in behavior among glycosaminoglycans were larger for NaCl than for CaCl2. The complex is stabilized to dissociation by salt by increasing hydrophobicity of the non-aqueous phase. However, aggregation of the complex into an insoluble form is also favored by an increasingly hydrophobic environment. The most consistent partition was observed with 1- and 2-butanol. The partition isotherm of chondroitin 4-sulfate was investigated at constant salt concentration. It was found that the partition coefficient varies with the concentration of chondroitin 4-sulfate, although the magnitude of this effect could be diminished by increasing the quaternary ammonium salt concentration.     

Resonance Raman studies of hemerythrin-ligand complexes.

Resonance Raman spectroscopy has been used as a probe of the structure of ligands at the active site of hemerythrin. Molecularly revealing insights have been obtained with oxyhemerythrin and with metazidohemerythrin. This spectroscopic technique has also facilitated a comparison of oxygen carrier within erythrocytes with that in solution. The electronic state of the bound O2 is the same in the natural environment as in the artificial one.     

The partition of glycosaminoglycan-quaternary ammonium complexes: I. The effect of phase composition

The complexes formed between quaterny ammonium cations and polyanionic glycosaminoglycans can be partitioned between partially miscible aqueous inorganic salt and alcohol phases. Small changes in salt concentration can completely shift the complex from one phase to the other. The effect of the phase composition variables: the type of inorganic salt, the type of quaternary ammonium salt, and the alcohol used, were systematically investigated. The sharp transition from solubility in the upper non-aqueous phase to solubility in the lower, aqueous phase was found to be strongly affected by the type of inorganic salt. This transition occurred at higher salt concentrations when NaCl, KCl, or LiCl were used than when CaCl₂ or MgCl₂ were used. Differences in behavior among glycosaminoglycans were larger for NaCl than for CaCl₂. The complex is stabilized to dissociation by salt by increasing hydrophobicity of the non-aqueous phase. However, aggregation of the complex into an insoluble form is also favored by an increasingly hydrophobic environment. The most consistent partition was observed with 1- and 2-butanol. The partition isotherm of chondroitin 4-sulfate was investigated at constant salt concentration. It was found that the partition coefficient varies with the concentration of chondroitin 4-sulfate, although the magnitude of this effect could be diminished by increasing the quaternary ammonium salt concentration.     

The partition of glycosaminoglycan-quarternary ammonium complexes II. The effects of polymer molecular weight and sulfation

Previous results have shown the possibility for obtaining high-resolution separations of glycosaminoglycans by partition in butanol/aqueous two-phase systems containing quarternary ammonium salts. In this paper, the effects on partition behavior of both polymer molecular weight and sulfation were examined. Two series of fractioned chondroitin sulfate polymers were isolated in which the molecular weight and sulfation varied systematically. In the molecular weight series the six samples, spanned the range from 3200 ± 300 to 19 700 ± 500 and each sample carried 0.8 sulfate groups per uronic acid residue. In the sulfation series, each sample had an essentially constant molecular weight of 13 000, but the sulfation varied from 0.58 to 0.88 sulfate groups per uronic acid. The $\text{C}{}_{50}{}^1$ of each of these samples was determined in the 1-butanol/aqueous NaCl phae system containing 1% hexadecylpyridinium chloride. In the series wherein the molecular weight varied, the C₅₀ increased with molecular weight up to 12 000 where a limiting value was reached. In the series wherein the sulfation varied, a linear relationship was found between the C₅₀ and the square of the number of anionic substituents per disaccharide. These results show that fractionation by partition techniques will be sensitive to the anionic nature of the polymer, but for the common connective tissue glycosaminoglycan, there will be no fractionation according to molecular weight.     

The partition behavior of complexes of glycosaminoglycans and quaternary ammonium salts

Complexes of purified glycosaminoglycans and hexadecylpyridinium chloride are shown to be capable of partition between aqueous and butanol phases. The partition coefficient of these complexes is dependent upon the concentration of the supporting electrolyte as well as the concentration of the quaternary ammonium salt. A sharp transition, during which the complex changes from complete solubility in the butanol phase to complete solubility in the aqueous phase, occurs over a very narrow range of salt concentrations. The various glycosaminoglycans show differences sufficient to permit fractionation at least into nonsulfated, monosulfate, and polysulfated classes by simple partition.     

Separation of individual sulfated bile acid conjugates as calcium complexes using reversed-phase partition thin-layer chromatography.

A method for separating individual monosulfated primary bile acid conjugates by reversed-phase partition thin-layer chromatography on octadecyl-bonded silica gel is described. The solvent system is acetonitrile containing calcium, probably as calcium carbamate. Excellent resolution of the 3- and 7-monosulfated glycine conjugates, as well as 3- and 7-monosulfated taurine conjugates of cholic and chenodeoxycholic acids is reported. A convenient class separation of sulfated from nonsulfated primary bile acid conjugates by adsorption thin-layer chromatography on low-polarity silica gel is also described.     

Two-phase partition studies of alkali cation complexation by ionophores

Summary The partition of alkali cations and anions between an aqueous and an immiscible organic phase has been studied in the absence and presence of neutral and carboxylic ionophores of the valinomycin and nigericin types, respectively. Cation extraction into the organic phase was augmented considerably by the ionophores, and a cation specificity of K⁺Rb⁺>Cs⁺Na⁺ was found for all the neutral ionophores tested. Evidence is given that the actual values of ion specificity are a function of the solvent polarity, especially for valinomycin where an inversion of the K⁺/Rb⁺ specificity was observed. The ionophores examined have the following rank order of effectiveness for K⁺ extraction into a standard organic phase consisting of 70% toluene-30%n-butanol: valinomycin>18-crown-6trinactin>enniatin Bdinactin>monactin>nonactin. The ion affinity and selectivity data thus obtained have been compared with data previously reported.In a toluene-butanol solvent, extraction of cations in the absence of ionophores occurs as ion pairs. On the other hand, the neutral ionophores extract the cations by the mechanism of complexation, with the lipophilic anions coextracted as free gegenionic species at lower ionophore complex concentrations. When the concentration of extracted cations exceeds 1×10^–4 m, ion pairing between the ionophore complex and the anion occurs, and this tendency increases with increasing concentration and decreasing polarity of the organic phase. Anion pairing with the complexed cations is much less than for the free cations and this effect appears to be due to the larger distance of closest approach of the anion for the complexed cation.     

Optical studies on complexes between DNA and pseudoisocyanine.

Linear dichroism (LD) results when pseudoisocyanine=PIC (1,1-diethy1-2,2-cyannine iodide) binds to flow-oriented DNA. LD may be used to follow the complexation both stoichmetrically and structurally, since when specified to unti complex concentration LD provides a measure of the average orientation of the absorbing transition dipole. Two different types of complexes can be distinguished: I. One strong, ionic-strength insensitive complex with monomeric PIC with an orientation indication intercalation. II. Several weaker complexes of electrostatic nature (only observed at I less than 0.2M Na Cl) among which those with dimeric (IIa) and with polymeric (IIb) PIC are concluded both from LD and from circular dichroism (CD). The dimer is probably formed by employing one intercalated PIC as a second site. The polymer complex is characterized by a very sharp absorption band at 553 nm polarized parallel to the DNA-axis (with positive LD and positive CL). The structure, a right-handed helical array of PIC molecules, is discussed in terms of exciton theory in relation to that of polymeric free PIC ("Scheibe polymer") which is also shown to interact with DNA (IIc) yielding a large aggregate which is degraded at a distinct flow force field...     

Structural studies of iron and cobalt tetrasulfonated phthalocyanine-globin complexes.

The structure of the complexes of iron and cobalt tetrasulfonated phthalocyanines with globin has been investigated by circular dichroism (CD), electron paramagnetic resonance (EPR) and polyacrylamide gel electrophoresis. Electrophoretic investigations and the molecular weight estimation indicates that the model complexes in the solutions are dimers. It is evident from the results of CD measurements that the incorporation of the iron or cobalt tetrasulfonated phthalocyanine into apohemoglobin significantly increases the helical structure of the protein and causes an appearance of the induced Soret and visible Cotton effects. Unlike methemoglobin, several discrete transition energies in the CD Soret band of Fe(III)L-globin are observed which suggest an inequivalence of the subunits within this complex. This suggestion is supported by EPR studies, which show that the iron atoms in Fe(III)L-globin are in two low electronic states. Electronic structures of the cobalt ions in Co(II)L-globin and oxyCo(II)L-globin are similar to those of coboglobin and oxycoboglobin, respectively, as is proved by EPR results. On this basis we conclude that the oxygen adduct of Co(II)L-globin can be described as a superoxide ion corrdinated to a formally cobaltic phthalocyanine compound.     

Spectroscopic studies of enantiomeric discrimination in jet-cooled chiral complexes.

Van der Waals complexes formed between chiral molecules in the isolated gas phase were studied by combining supersonic expansion techniques with laser spectroscopy. The weakly bound diastereoisomers formed between a chiral secondary alcohol, butan-2-ol, and a chiral aromatic derivative such as 2-naphthyl-1-ethanol or 1-phenylethanol used as a resolving agent were discriminated on the basis of the spectral shifts of the UV S(0)-S(1) transition of the chromophore. Ground-state depletion spectroscopy (hole burning) has shown that, while only one structure was detected for the 1-phenylethanol/butan-2-ol homochiral complex, the heterochiral complex is trapped in the jet under two different conformations. Two isomers have also been shown for each diastereoisomeric pair of the 2-naphthyl-1-ethanol/butan-2-ol complexes. Using a semiempirical potential model, these isomeric forms were related to calculated structures which exhibit a folded or extended geometry depending on the solvent conformation (anti or gauche). The relative binding energy of the complexes involving R-1-phenylethanol and R- or S-butan-2-ol were obtained from fragmentation threshold measurements following two-color photoionization. Comparison of the diastereoisomers exhibiting a similar spectral signature shows that the homochiral pair is more stable than the heterochiral one by about 0.7 kcal/mol. The fragmentation threshold has been shown to depend on the jet-cooled isomer and this result addresses the role of conformational control in enantioselective interactions.     

Drug-metal interactions: spectroscopic studies of copper hydantoin complexes.

The preparation and spectral properties of copper(II) complexes of two hydantoins are reported. Complexes of the general formula Cu(hyd)2(py)2, where hyd = phenytoin or nirvanol; and py = pyridine were prepared and characterized by infrared and ESR. Spectral data show that the copper atom is bound to the nitrogen atom of the hydantoin anion and to the nitrogen atom of the pyridine molecule to form 2:2:1 hydantoin:pyridine:copper complexes. The ESR data indicate that both complexes have tetragonal symmetry (g11 greater than g perpendicular greater than g e) with the unpaired electron in the d x2-y2 orbital.