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The interactions of high molecular weight hyaluronan (HA) with di-palmitoyl phosphatidylcholine (DPPC) liposomes in aqueous buffer have been investigated. The superstructures resulting from co-incubation in vitro were observed by means of negative staining and rotary shadowing electron microscopy and were also characterized by rheometric techniques. The existence of a supramolecular complex between the two partners is clearly demonstrated; this complex would exist in vivo in the synovial fluid (whose major components are, in fact, HA and DPPC) and should be responsible for the unique physiological properties of the latter.

Attention has been focused on two uronans, namely, mannuronan and galactose-depleted C-6 oxidized guar, the former of microbial origin and the latter of artificial nature, to provide original data on the extent of epimerization they can undergo in dilute aqueous solution using two C-5 mannuronic acid epimerizing enzymes, that is, AlgE-4 and AlgE-6, alone or in admixture. Original circular dichroism data coupled with 1H NMR spectra clearly point out that both uronans can be epimerized, depending on the enzyme or enzyme mixture used, to high levels yielding guluronic-rich alginate samples and guluronic-rich heteropolysaccharides, respectively. Mannuronan and its epimerization products can easily form clear, firm aqueous gels when an excess of HCl is added or when mixed with aqueous CaCl2, respectively. Depleted-guarox does not gel upon addition of excess HCl, while the heterouronan derived from it having a percent of epimerization nearly identical to that of epimerized mannuronan, that is, ca. 70%, can form gel in the presence of Ca(II) only at higher polymer and Ca(II) concentrations. With the latter, heterouronan alpha-D-galacturonic side groups exert hindrance to "junction zone" formation.

The sequence encoding the endopolygalacturonase (PG) of Fusarium moniliforme was cloned into the E. coli/yeast shuttle vector Yepsec1 for secretion in yeast. The recombinant plasmid (pCC6) was used to transform Saccharomyces cerevisiae strain S150-2B; transformed yeast cells were able to secrete PG activity into the culture medium. The enzyme (wtY-PG) was purified, characterized, and shown to possess biochemical properties similar to those of the PG purified from F. moniliforme. The wtY-PG was able to macerate potato medullary tissue disks and was inhibited by the polygalacturonase-inhibiting protein (PGIP) purified from Phaseolus vulgaris. The sequence encoding PG in pCC6 was subjected to site-directed mutagenesis. Three residues in a region highly conserved in all the sequences known to encode PGs were separately mutated: His 234 was mutated into Lys (H 234-->K), and Ser 237 and Ser 240 into Gly (S 237-->G and S 240-->G). Each of the mutated sequences was used to transform S. cerevisiae and the mutated enzymes were purified and characterized. Replacement of His 234 with Lys abolished the enzymatic activity, confirming the biochemical evidence that a His residue is critical for enzyme activity. Replacement of either Ser 237 or Ser 240 with Gly reduced the enzymatic activity to 48% and 6%, respectively, of the wtY-PG. When applied to potato medullary tissue, F. moniliforme PG and wtY-PG caused comparable maceration, while the variant PGs exhibited a limited (S 234-->G and S 240-->G) or null (H 234-->K) macerating activity. The interaction between the variant enzymes and the P. vulgaris PGIP was investigated using a biosensor based on surface plasmon resonance (BIAlite). The three variant enzymes were still able to interact and bind to PGIP with association constants comparable to that of the wild type enzyme.

The interaction between endo polygalacturonase from Fusarium moniliforme and PGIP, from Phaseolus vulgaris L. was investigated using a biosensor technique based on surface plasmon resonance (BIAlite). This new analytical system provides information on the strength and the kinetics of biomolecular interactions. PGIP (the ligand) was covalently attached to the dextran matrix of a sensor chip while endo polygalacturonase (the analyte) was introduced in a flow passing over the surface. Results from real-time BIA are presented as a sensorgram, which is a plot of changes in the resonance signal as a function of time. The experimental data were evaluated with an appropriate software to derive the values of kinetic rate constants and to compare the affinities of PGIP for several variant endo polygalacturonases obtained by site-directed mutagenesis of the Fusarium moniliforme enzyme.

Results are reported for spectroscopic and calorimetric studies of ethidium bromide (EBr) in water and in aqueous solutions of three alternating maleic acid copolymers. The dimerization constant, dimerization enthalpy and the dimer spectrum of EBr are derived. The interaction of EBr with the polycarboxylates is characterized on the basis of spectral and microcalorimetric data according to two limiting mechanisms: at low dye-to-polymer concentration ratios, EBr would be bound essentially in monomeric form with small exothermic effects: at high concentration ratios, the observed comparatively large exothermic heat is attributed to bound dye stacking.

A soil bacterium grown on propan-2-yl sulphate as sole source of carbon and sulphur yielded extracts containing an enzyme capable of liberating sulphate from racemic lactate-2-sulphate. The enzyme was purified to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, batch treatment with DEAE-cellulose, and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose. The protein was monomeric with an Mr of 55 000-60 000. The enzyme activity was specific for D-lactate-2-sulphate (Km 6.6 nM; maximal specific activity 14.3 mumol/min per mg of protein) and showed no activity towards the L-isomer. The products of the enzyme's action were inorganic sulphate and D-lactate which were released in equimolar amounts and stoicheiometrically with the amount of ester hydrolysed. No L-lactate was formed. Retention of configuration implied cleavage of the O-S bond of the C-O-S ester link and this was confirmed by 18O-incorporation experiments in which 18O from 18O-enriched water in the incubation medium was incorporated exclusively and quantitatively into inorganic sulphate. Only two other esters (serine-O-sulphate and p-nitrophenyl sulphate) of a total of 29 compounds tested were substrates for the enzyme. D-Lactate, L-lactate-2-sulphate and the substrate analogues glycollate-2-sulphate and butyrate-2-sulphate were significantly inhibitory.

Potentiometric, calorimetric, and dilatometric data on the ionization of several saturated dicarboxylic acids in water at 25°C are reported. A correlation is attempted between structure and thermodynamic evidences for the two dissociation stages of the diacids considered, in particular in terms of the propensity toward intramolecular hydrogen bond formation of some singly ionized species.

SUMMARY: A strain of Pseudomonas syringae was isolated from garden soil for its ability to utilize propan-2-yl sulphate as sole source of carbon and energy. Growth was accompanied by disappearance of the ester from culture fluids and formation of stoicheiometric amounts of inorganic sulphate. However, enzyme activity capable of desulphating propan-2-yl sulphate was not detected under a wide range of assay conditions, either intracellular or extracellular, or at different phases of batch culture. No organic radioactive metabolites were detected during growth on propan-2-yl [35S]sulphate or [1,3-14C]propan-2-yl sulphate alone, but when the latter compound was supplemented with unlabelled lactate, small amounts of 14C-labelled lactate were detected. This and the induction by propan-2-yl sulphate of a specific d-lactate-2-sulphatase led to a proposed pathway in which propan-2-yl sulphate undergoes a stereospecific oxidation to d-lactate-2-sulphate before rapid desulphation to d-lactate and inorganic sulphate.

The changes in enthalpy, entropy and volume upon melting have been determined by dilatometric and differential scanning calorimetry measurements for four polyesteramides of the type: -[- COφCONH(CH 2 ) n  NHCOφCOO(CH 2 ) m  O -]- x with the following values for n and m; 6-6, 12-2, 12-6 and 12-12. The changes in each state function vary quite regularly with the number of CH2 groups/repeating unit. A comparison is made between experimental data on the entropy of fusion and theoretical predictions. There is emphasis on the influence on the thermodynamics of melting of the rigidity of the -OCO-φ-CONH- residues and, in particular, of the persistence in the molten state of many interchain hydrogen bonds.

The results obtained in a study of the heat and entropy of fusion, and a few data on the crystallization kinetics, of poly-β-propiolactone and poly-ϵ-caprolactone are reported. Attention is focused on the entropy data for the two polymers. A comparative, theoretical analysis of the constantvolume entropy of melting of the polylactones and of three aliphatic polyesters is made. The remainder of the entropy of melting of the polymers considered is qualitatively correlated with chemical constitution and crystal structures.

A detailed uv spectral investigation of the interaction of methyl orange (and other similar anionic azo-dyes) with four different synthetic cationic polyelectrolytes, with colloidal electrolytes, as well as with two proteins in dilute aqueous solutions, has been carried out. Under appropriate conditions, binding of methyl orange, ethyl orange, and butyl orange to poly( l -ornithine, poly- l -lysine), poly ( N -methylvinyl pyridinium chloride), and trimethyldodecylammonium chloride, gives rise to marked changes in the spectrum of each of the three dyes (but not of other dyes examined). These spectral changes are quite different from those which result from the binding of the azo-dyes on to the strongly ionized poly(vinylbenzyltrimethyl ammonium chloride) chains. The effects observed with the four synthetic polyelectrolytes mentioned above and with the colloidal electrolytes, are postulated to be connected with a conformational change of bound dye molecules. Interaction of methyl orange with bovine serum albumin and with lysozyme would mainly result in a partial solubilization of the dye in the hydrophobic regions of the proteins.

Calculations of the unperturbed dimensions of Na-polyacrylate have been carried out, for various concentrations of meso and racemic diads, allowing for coulombic interactions both between side charges within the chain and between side charges and the counter-ions. Contrary to previous calculations where the counter-ion was neglected, the present approach can explain the rather high experimental value of the characteristic ratio. Both the Monte Carlo method and the method suggested by one of the authors, based on the hypothesis of a conditional stereo chemical equilibrium within the chain, are employed; the results are in substantial agreement.

The unperturbed dimensions of poly-ϵ-caprolactone (PϵL), poly-β-propiolactone (PβL) and polypivalolactone (PPL) have been evaluated theoretically. We find that the values of the characteristic ratio, C∞, are 6 · 66 (PϵL), 3 · 98 (PβL) and 8 · 10 (PPL). The calculations are based, as usual, on the isomeric rotational state model. Statistical weights are assigned to allowed rotational states around the different bonds along polylactone chains on the basis of indications derived from conformational van der Waals energy maps and by analogy with the cases of other similar chains studied by other authors. Dipolar interactions were not explicitly taken into account. Theoretical evaluations of the mean square dipole moments of PϵL, PβL and PPL chains are also made. Correlation of theory and experiment must await more detailed investigations, as experimental data are limited to the PϵL case for which there is apparently a disagreement between our C∞ value and that extrapolated from intrinsic viscosity data (in good solvents). Theoretical C∞ values, however, depend critically on the choice of the values of one or two statistical weights: theory is thus flexible enough to lead to ready agreement with information from future and reliable experimental data.

The results are reported from a study of the heat and entropy of melting of polypivalolactone (PPL) as well as of the kinetics of PPL crystallization. The study was performed by differential scanning calorimetry. Thermodynamic and kinetic parameters of PPL fusion and crystallization are discussed; a brief account of the constant-volume entropy of melting of PPL is presented in terms of an approximate theoretical analysis.

THE gas-density balance apparatus developed by Martin and James1 for detection and estimation of components in gas-phase chromatography might be used for a rapid process for evaluating the molecular weights of single components.

Summary The interaction of two natural tetra-azacyclopentazulene dyes with native calf thymus DNA was studied by means of microcalorimetric, viscosimetric, and spectroscopic measurements. The results are consistent with the hypothesis of an intercalative-type binding. However, comparison of calorimetric studies shows that the changes in enthalpy associated with the interaction of these compounds with DNA are, in absolute value, significantly lower than those found with known intercalating agents (daunomycin, ethidium bromide). The influence of these dyes on the template capacity of DNA in the in vitro synthesis of nucleic acids was also determined. Under the conditions used, these compounds selectively inhibited DNA synthesis. No appreciable inhibitory effect upon E. coli RNA polymerase was observed. Both compounds had greater inhibitory effect on rat liver high molecular weight DNA polymerase than E. coli DNA polymerase I. Zoanthoxanthin was a more effective inhibitor than 3-norzoanthoxanthin.

Four carboxylated derivatives of scleroglucan have been obtained by oxidation, to different extents, of the glucopyranose side chains of the natural polysaccharide. The diffusion of model molecules through aqueous solutions of these new products was measured at various pH values. The reversible pH induced sol-gel transition of some of the polyelectrolyte solutions tested effects a remarkable variation in the diffusion rate of the permeating species; in this sense the most interesting polysaccharide appears to be the product with 70% oxidized glucopyranose moieties. The behaviour of scleroglucan and its derivatives has been compared with that of carboxymethylcellulose and the mechanism involved in drug permeation and release discussed. The possible application of one of the new products in controlled release formulations for oral use is also reported.

Analytical expressions are obtained to evaluate the electrostatic enthalpy of mixing the univalent-salt form of a polyelectrolyte with a neutral salt of the same valency in aqueous solution, following the theory developed by G. S. Manning, avoiding restrictive assumptions regarding the ionic parameters. An endothermic effect is always predicted for the enthalpy of mixing (corrected for the heat of dilution of the polyelectrolyte), which is function of the linear charge density parameter, ξ, of the ratio of neutral salt to the monomoles of polymer, and of the amount of salt initially present in the system. In this way a quantitative method is suggested to test the conformational parameters of polyelectrolytes and their dependence on ion-induced changes, provided that the main non-conformational effect of added salt on the polymer is mostly electrostatic. Specific effects (as site-binding), if present, should be additionally accounted for. However, in the case of the pH-induced conformational transition of polygalacturonate the electrostatic approach is able to give a self-consistent reference for the excess heat effects therein involved. The ionic polysaccharides which have been chosen for confronting the theoretical predictions with the experimental microcalorimetric data are polygalacturonate, polyguluronate, polymannuronate and alginate.

The acid fading of Auramine O (AuO) at 25° in water and in the presence of five different maleic acid copolymers has been studied. The pseudo-first order kinetic constants show that the polyelectrolytes exert quite distinct effects on the fading of AuO. The data are tentatively interpreted in terms of chemical structure and hydrophobicity of the macroions.

The results of spectroscopic and microcalorimetric measurements on dilute aqueous solutions of phosvitin are described. Our infrared and circular dichroism spectra show that the phosphoprotein chains may assume either random-coiled or partially helical or β-structures depending upon the pH. The transition to the low pH β-form occurs with a distinct cooperative character. The strongly interdependent ionization of fixed charges densely spaced along phosvitin chains, typical of highly charged polyelectrolytes, is well illustrated by the calorimetric enthalpy of protonation data. The interaction of phosvitin with acridine orange in dilute aqueous solution (pH ∼5) has been studied by means of spectral and equilibrium dialysis experiments. For relatively low polymer to dye concentration ratios, bound acridine orange exhibits γ-type spectrum thus suggesting aggregation of dye molecules onto the protein. Gradual decrease in the extent of bound acridine orange aggregation is produced by increasing the concentration of the protein; the monomerically bound form is hardly obtained, however, even at relatively high phosvitin concentrations. Binding curves reveal a cooperativity character for the dye-protein interaction. It is suggested that about 60% only of phosphomonoester side groups of phosvitin are available for acridine orange binding, these being characterized by a high “stacking” coefficient. The results are tentatively discussed on the basis of present information on phosvitin chemical composition and conformation.

The pH-induced conformational transition of the exocellular polysaccharide from Rhizobium trifolii strain TA-1 (TA1EPS) has been studied by chiroptical, calorimetric and potentiometric methods. By lowering the pH of neutral aqueous TA1EPS solutions, the specific rotation shows an abrupt change from positive to negative values of [α]. The same trend is also observed in the molar ellipticity. No evidence of a pH-induced change in the chain conformation of TA1EPS has been noticed using aqueous 40 mM NaCl as solvent. From isothermal calorimetric and differential scanning calorimetric experiments, the value of the enthalpy change for the pH-induced ordering process is −6 kJ equiv.−1. Potentiometric titration data suggest that the pH-induced transition involves only a small change in the overall charge density. The possible differences between the ordered form at low pH and that previously found in salt solution are discussed.

Several samples of polyglucuronan have been prepared by oxidation of cellulose with nitrogen oxides. Optimisation of the reaction conditions in terms of degree of oxidation and molecular weight of the products was carefully performed.

Solution rheology of an extracellular polysaccharide from the cyanobacterium Cyanospira capsulata has been investigated in water and in aqueous 0·1 m NaCl at 25°C. The study has been carried out within a range of defined experimental conditions aiming at providing both steady-shear and transient rheological behaviour. The behaviour of the polysaccharide solutions is reminiscent of that of concentrated guar for the time-dependent properties alone, while for the rheological effectiveness and for the concentration-dependent properties, similarities with xanthan are found. The whole set of preliminary data are discussed within the framework of molecular viscoelastic theories and some inconsistencies are disclosed. These may be solved eventually when a larger number of data are also obtained on different systems.

Viscosity, optical activity, and differential scanning calorimetry data clearly point out that partial and/or total removal of charged substituent groups, i.e. succinate and pyruvyl residues, from succinoglycan lead to water soluble derivatives exhibiting a higher stability order → disorder conformational changes with respect to the native polysaccharide. The new succinoglycan derivatives also exhibit very little, if any, hysteresis upon ‘renaturation’ (cooling) as opposed to the case of the parent polymer. The absence of ionized groups is thus beneficial, thermodynamically and kinetically, to the attainment in dilute aqueous solution of an ordered conformation by the uncharged succinoglycan backbone, as allowed by the regular enchainment of its constituent sugar residues.

Selective, quantitative oxidation of pendant 1,6-β-linked glucopyranose residues along scleroglucan chains has yielded a new carboxylated polyelectrolyte. Study of the aqueous solution properties of the latter (named sclerox-I) has disclosed rather anomalous behaviour. Viscosity, calorimetric and chiro-optical measurements show that the interaction of sclerox-I with Ca2+ ions leads to a change in conformation of the polyanions, probably of the type random coils → ordered, elongated forms.

An extension of the counterion-condensation (CC) theory of linear polyelectrolytes has been developed for the case of a system containing a mixture of counterions of different valency, i and j. The main assumption in the derivation of the model is that the relative amount of the condensed counterions of the type i and j is strongly correlated and it is determined by the overall physical bounds of the system. The results predicted by the model are consistent, in the limiting cases of single species component, with those of the original CC theory. The most striking results are obtained for the cases of low charge density and excess of counterion species: in particular, an apparent positive "binding" cooperativity of divalent ions is revealed for small, increasing additions of M2+ ions to a solution containing a swamping amount of monovalent salt and a polyelectrolyte of low charge density. Apparent "competitive binding" of mono- and divalent ions derives as a bare consequence of the electrostatic interactions. Theoretical calculations of experimentally accessible quantities, namely single-(counter) ion activity coefficients, confirm the surprising predictions at low charge density, which qualitatively agree with the measured quantities.