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.     

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.     

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.     

Enzymes in polyelectrolyte complexes. The effect of phase transition on thermal stability

Penicillin amidase, alpha-chymotrypsin and urease have been immobilized in water-soluble nonstoichiometric polyelectrolyte complexes (N-PEC). N-PEC are formed by modified poly(N-ethyl-4-vinyl-pyridinium bromide) (polycation) and excess poly(methylacrylic acid) (polyanion). N-PEC are a new class of polymers capable, characteristically, of phase transitions solution in equilibrium precipitate induced by slight change in pH or ionic strength. Neither the chemical structure of the carrier nor the number of cross-linkages between an enzyme and a carrier change on phase transition. That gives an unique opportunity to elucidate the difference between enzymes immobilized on water-soluble and water-insoluble supports. A detailed study of the phase transition effect on thermal stability of the enzymes and protein-protein interactions has been carried out. The following effects were found. Pronounced thermal stabilization of penicillin amidase and urease may be achieved on two conditions: the enzyme is in the precipitate; (b) the enzyme is linked to the N-PEC nucleus. Then the thermal stability of N-PEC-bound penicillin amidase increases 7-fold at pH 5.7, 60 degrees C, and 300-fold at pH 3.1, 25 degrees C, compared to the native enzyme. For urease, the thermal stabilization increases 20-fold at pH 5.0, 70 degrees C. The localization of enzyme on N-PEC has been established by titration of alpha-chymotrypsin bound to a polycation or polyanion with basic pancreatic trypsin inhibitor. Both in solution (pH 6.1) and in N-PEC precipitate (pH 5.7), an alpha-chymotrypsin molecule bound to a polyanion is fully exposed to the solution. If the enzyme is bound to a polycation, only 20% of alpha-chymotrypsin molecules in the precipitate and 40% in solution retain their ability for protein-protein interactions. This means that a polycation-bound enzyme is localized in the hydrophobic nucleus of the complex, whereas the polyanion-bound enzyme sits on the hydrophilic shell of the complex. On pH-induced phase transition (pH decreases from 6.1 to 5.7), there occurs a stepwise decrease in penicillin amidase activity which is due to a 9.8-fold increase in the Km for 2-nitro-4-phenylacetamidobenzoic acid. Change of the catalytic activity and thermal stability of N-PEC-bound penicillin amidase is fully reversible and reproducible. Such soluble-insoluble immobilized enzymes with controllable thermal stability and activity may be used for simulating events in vivo and in biotechnology.     

Silver(I) complexes with heterocyclic thiones and tertiary phosphines as ligands. Part 4. Dinuclear complexes of silver(I) bromide: the crystal structure of bis[bromo-(pyrimidine-2-thione)(triphenylphosphine)silver(I)]

Mixed-ligand complexes of the formula [Ag(PPh₃)(L)Br]₂ were obtained by treatment of various heterocyclic thiones L {L=pyridine-2-thione (py2SH), pyrimidine-2-thione (pymtH), benz-1,3-imidazoline-2-thione (bzimtH₂), benz-1,3-thiazoline-2-thione (bztztH), 1-methyl-1,3-imidazoline-2-thione (meimtH) and 5-methoxy-benz-1,3-imidazoline-2-thione (5MeObzimtH₂)} with equivalent quantities of silver(I) bromide and triphenylphosphine in dry acetone. The compounds were characterized by their IR, far-IR, UV–Vis and ¹H NMR spectroscopic data. The crystal structure of [Ag(PPh₃)(pymtH)Br]₂ was determined by single-crystal X-ray diffraction methods. The complex exhibits a planar Ag₂Br₂ moiety in which each of the doubly bromine-bridged Ag(I) centres is further bonded to one phosphine P and one thione S atom.     

The effect of the lipid composition on the partition of liposomes in aqueous two-phase systems

Liposomes have been partitioned in aqueous two-phase systems consisting of water, dextran, poly(ethylene glycol), salt and buffer. Liposomes were used as a model system in order to determine the contribution of the lipids on the partition of membrane particles. The liposomes were composed of phospholipids with different polar head groups and different degrees of unsaturation. The role of cholesterol was also investigated.The polar head group of the phospholipid plays a dominant role in determining the partition of liposomes, while the degree of unsaturation is of less importance, thereby indicating that partition in two-phase systems is a surface dependent method. Incorporation of cholesterol in liposomes reduces differences in partition between liposomes of various composition.     

The genome of Plasmodium falciparum. I: DNA base composition.

Some structural properties of the DNA of Plasmodium falciparum were studied thoroughly using several techniques. Its G+C content was found to be extremely low (17-19%), the lowest reported for a living organism. The DNA seems to be composed only of the four major bases as no methylated bases were detected. This DNA had a Tm value of 62.5 degrees C and its denaturation profile showed no marked intramolecular heterogeneity.     

The dynamics of phase partition. A study of parameters affecting rat liver organelle partitioning in aqueous two-polymer phase systems.

Separation of subcellular organelles by two-phase partition is thought to reflect differential partition of the organelles between the two phases or between one of the phases and the interface. Studies by Fisher and colleagues [Fisher & Walter (1984) Biochim. Biophys. Acta 801, 106-110] suggest that cell separation by phase partition is a dynamic process in which the partition changes with time. This is mainly due to association of the cells with sedimenting droplets of one phase in the bulk of the other. Rat liver organelle partition was studied to determine whether the same dynamic behaviour is observed. Partition was clearly time-dependent during 24 h at unit gravity, and was also affected by altering the volume ratio of the two phases and the duration of phase mixing. These results indicate that, as with cells, the partition of organelles between phases is a dynamic process, and is consistent with the demonstration that organelles adhere to the phase droplet surfaces. Optimization of the volume ratio between phases may lead to significant processing economies. Organelle sedimentation in the upper phase was significantly faster than in the isoosmotic sucrose. Theoretical modelling of apparent organelle sizes indicates that aggregation occurs in the poly(ethylene glycol)-rich upper phase. This phenomenon is likely to limit the use of this technique in organelle separations unless means can be found to decrease aggregation.     

Bimolecular systems: I. Linear systems of complexes

A vast number of biologically important processes are based upon bimolecular systems. In these systems intermediate complexes are formed. Bimolecular systems in which no complex-complex interactions occur are called linear systems of complexes. A definition and some characteristic properties of these systems are given here. There may exist a contradiction of Onsager's principle of detailed balancing in these systems; however, no principal differences are found between the steady state behavior of an open system and that of a closed system. It is shown that the steady state behavior of a linear system of complexes of arbitrary complexity has some similarities with the steady state behavior of a simple bimolecular system, e.g., Michaelis-Menten enzymatic reaction. Multiplicity of action of the substances participating in biomolecular processes may produce some qualitative differences in the steady state behavior of the system.     

Two-dimensional spreads of synaptonemal complexes from solanaceous plants. I. The technique

Using beta-glucuronidase the cell walls of tomato and potato primary microsporocytes can be digested. When the resulting protoplasts are exposed to distilled water, they burst, and complete sets of synaptonemal complexes are released to settle on plastic coated slides. After drying and formalin fixation, the synaptonemal complexes can be stained with silver or phosphotungstic acid and observed in the light and/or electron microscope. Silver staining gives better contrast for both light and electron microscopy but stains only lateral elements and kinetochores. Phosphotungstic acid staining gives little or no contrast for light microscopy, but stains both the lateral and central elements of the synaptonemal complex, kinetochores, and structures that are probably recombination nodules for electron microscopy. This technique offers a powerful tool for genome analysis by allowing (1) the determination of relative and absolute lengths of synaptonemal complexes and chromosome arm ratios at pachytene, (2) the analysis of complex patterns of synapsis, and (3) the location of what are probably recombination nodules along the length of synaptonemal complexes.