Possible coupling of chemical to structural dynamics in subtilisin BPN' catalyzed hydrolysis

The viscosity dependence of enzymatic catalysis was examined in subtilisin BPN' catalyzed hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-thiobenzyl ester. The viscosity of the reaction medium was varied by added glycerol, ethylene glycol, sucrose, glucose, fructose, poly(ethylene glycol) and Ficoll-400. Responses of the Michaelis-Menten parameters associated with hydrolysis were calculated from data obtained by spectrophotometric techniques. The reactions with these two substrates have catalytic rates well below the diffusion-controlled limit and thus enable us to study the viscosity effects on catalytic steps of non-transport nature. It was found that the Km values for both amide and ester reactions remained relatively independent of cosolvents. On the other hand, while the kcat values for amide were insensitive to cosolvents, those for ester were substantially attenuated except in the case of poly(ethylene glycol). The observed rate attenuations cannot be explained by changes in proton activity, water activity, dielectric constant of the reaction medium or shifts of any kinetically important pKa. Instead, the results can be adequately described by microviscosity effects on the unimolecular deacylation step with a coupling constant of 0.65 +/- 0.11. In addition, the different viscosity dependence in the acylation vs deacylation step can be rationalized in terms of fluctuation-dependent chemical dynamics of proton transfers in the context of the Bogris-Hynes model.     

The effect of changes in the bulk dielectric constant on the DNA torsional properties

The effect of changes in the bulk dielectric constant on the DNA torsional properties was evaluated from plasmid circularization reactions. In these reactions, pUC18 previously linearized by EcoRI digestion was recircularized with T4 DNA ligase. The bulk dielectric constant of the reaction medium was decreased by the addition of different concentrations of neutral solutes: ethylene glycol, glycerol, sorbitol, and sucrose, or increased by the addition of glycine. The topoisomers generated by the ligase reaction were resolved by agarose-gel electrophoresis. The DNA twist energy parameter (kappa), which is an apparent torsional constant, was determined by linearization of the Gaussian topoisomers' distribution. It was observed that the twist energy parameter for the given solutes is almost linearly dependent on the bulk dielectric constant. In the reaction buffer, the twist energy parameter was determined to be 1100 +/- 100. By decreasing the dielectric constant to 74 with the addition of sorbitol, the value of the parameter reaches kappa = 900 +/- 100, whereas the addition of ethylene glycol leads to kappa = 400 +/- 50. Upon addition of glycine, which resulted in a dielectric constant equal to 91, the value of the twist energy parameter increased to kappa = 1750 +/- 100.     

Generation of selective microenvironment at the cell periphery through bulk-phase hydrophilic polymers

In order to understand the previously demonstrated effect of poly(ethylene glycol) on the stimulation of lymphocyte responses to syngeneic tumor cells (Ben-Sasson, S.A. and Henkart, P.A. (1977) J. Immunol. 119, 227–231), we have investigated the effects of addition of poly(ethylene glycol) to the medium in a number of cellular systems. The binding of trimeric IgG to tumor-lymphocyte Fc receptors was greatly enhanced by poly(ethylene glycol); a substantial increase in binding of trimeric IgG to non-Fc-receptor-bearing tumor cells was also observed. Similarly, the binding of labeled bovine serum albumin to lymphocyte surfaces was increased by poly(ethylene glycol), implying that nonspecific binding of proteins to cells was generally enhanced. The dose-response curve of concanavalin A mitogenesis was shifted to the right, as would be expected from a local increase in concanavalin A concentration. Antibody binding to erythrocytes as detected by complement lysis was similarly increased. It was found that in aqueous two-phase mixtures created by poly(ethylene glycol) and dextran, erythrocytes partition into the dextran phase through exclusion into dextran-rich microdroplets. It is proposed that addition of poly(ethylene glycol) to cell culture media creates a similar separate phase around the cell surface in which the local concentration of proteins is greater than that in the bulk medium. This concept explains many of the diverse effects of addition of poly(ethylene glycol) to the medium. It also can partially explain the requirement for serum to observe the poly(ethylene glycol) effect on the lymphocyte response to syngeneic tumor cells.     

Electric-field orientation of sodium poly(l-glutamate) in methanol/water and ethylene glycol/water mixtures

Transient electric birefrinqence and circular dichroism measurements have been made on sodium poly(l-glutamate) in methanol/waer and ethylene glycol/water mixtures of various compositions. The specific Kerr constant increased upon the transition from coil to helix, but decreased with further increase in methanol or ethylene glycol content on the helix side.     

Microwave absorption in oligomers of ethylene glycol

The dielectric properties of biologically and pharmaceutically important low-molecular weight ethylene glycols H(-OCH2CH2-)n -OH (n = 1,2,4,6) were investigated to clarify the effect of chain length on the dielectric properties. The measurement of dielectric constant and dielectric loss was carried out over the frequency range 200 MHz to 20 GHz at temperatures of 25 degrees C to 55 degrees C. It is found that in these molecules microwave dielectric losses are significant. The dispersion behaviour of these molecules can be represented by Cole-Cole equation. The dielectric properties of these homologous ethylene glycols are discussed in terms of the effects of chain length and intermolecular hydrogen bonds regarding the molecular conformations. These wide frequency range dielectric data have also been discussed in view of the suitable selection of the oligomer of ethylene glycol for cosmetic preparations and other pharmaceutical applications with the intention of protection of the skin from weak microwave radiations present in the surrounding environment. These systematic microwave dielectric data with frequency and temperature variation are not available and are provided in this paper.     

Effect of poly(ethylene glycol) on phospholipid hydration and polarity of the external phase

The hydration properties of phosphatidylcholine (PC)/water dispersions on the addition of poly(ethylene glycol) were studied by means of ²H-NMR. The quadrupole splittings and their temperature dependences correspond to measurements of PC/water dispersions at low water content. It is concluded that the bound water is partly extracted by poly(ethylene glycol) but the binding properties of the water in the inner hydration shell of about five water molecules are not changed. The ability of some phospholipid/water dispersions to undergo phase transitions to nonlamellar structures upon dehydration is discussed. Dipalmitoylphosphatidylcholine (DPPC) and egg phosphatidylcholine do not form nonlamellar structures on addition of purified poly(ethylene glycol), as was demonstrated by means of ³¹P-NMR. Poly(ethylene glycol) decreases the polarity of the aqueous phase and the partition of hydrophobic molecules between the membrane and the external phase is changed. This was demonstrated using the excimer fluorescence of pyrene in a ghost suspension. It is suggested that the changes in polarity and hydration on the addition of poly(ethylene glycol) can contribute to the alterations in the membrane surface observed under conditions of membrane contact and fusion.     

Mechanism of KCl enhancement in detection of nonionic polymers by nanopore sensors

The mechanisms of KCl-induced enhancement in identification of individual molecules of poly(ethylene glycol) using solitary α-hemolysin nanoscale pores are described. The interaction of single molecules with the nanopore causes changes in the ionic current flowing through the pore. We show that the on-rate constant of the process is several hundred times larger and that the off-rate is several hundred times smaller in 4 M KCl than in 1 M KCl. These shifts dramatically improve detection and make single molecule identification feasible. KCl also changes the solubility of poly(ethylene glycol) by the same order of magnitude as it changes the rate constants. In addition, the polymer-nanopore interaction is determined to be a strong non-monotonic function of voltage, indicating that the flexible, nonionic poly(ethylene glycol) acts as a charged molecule. Therefore, salting-out and Coulombic interactions are responsible for the KCl-induced enhancement. These results will advance the development of devices with sensor elements based on single nanopores.     

Effect of poly(ethylene glycol) on the polarity of aqueous solutions and on the structure of vesicle membranes

The partitioning of TEMPO into phosphatidylcholine vesicle membranes is reduced upon addition of poly(ethylene glycol). This is caused by reduced polarity of the aqueous phase as well as decreased membrane fluidity in the presence of poly(ethylene glycol). The isotropic hyperfine splitting of TEMPO in aqueous poly(ethylene glycol) solutions was used as a measure of solvent polarity. The alterations of the membrane fluidity were detected by means of two different fatty acid spin labels. The influences of physicochemical properties of an aqueous poly(ethylene glycol) phase on the membrane structure of cells and vesicles are discussed in the light of membrane fusion.     

Aggregation and fusion of unilamellar vesicles by poly(ethylene glycol)

Various aspects of the interaction between the fusogen, poly(ethylene glycol) and phospholipids were examined. The aggregation and fusion of small unilamellar vesicles of egg phosphatidylcholine (PC), bovine brain phosphatidylserine (PS) and dimyristoylphosphatidylcholine (DMPC) were studied by dynamic light scattering, electron microscopy and NMR. The fusion efficiency of Dextran, glycerol, sucrose and poly(ethylene glycol) of different molecular weights were compared. Lower molecular weight poly(ethylene glycol) are less efficient with respect to both aggregation and fusion. The purity of poly(ethylene glycol) does not affect its fusion efficiency. Dehydrating agents, such as Dextran, glycerol and sucrose, do not induce fusion. 31P-NMR results revealed a restriction in the phospholipid motion by poly(ethylene glycol) greater than that by glycerol and Dextran of similar viscosity and dehydrating capacity. This may be associated with the binding of poly(ethylene glycol) to egg PC, with a binding capacity of 1 mol of poly(ethylene glycol) to 12 mol of lipid. Fusion is greatly enhanced below the phase transition for DMPC, with extensive fusion occurring below 6% poly(ethylene glycol). Fusion of PS small unilamellar vesicles depends critically on the presence of cations. Large unilamellar vesicles were found to fuse less readily than small unilamellar vesicles. The results suggest that defects in the bilayer plays an important role in membrane fusion, and the 'rigidization' of the phospholipid molecules facilitates fusion possibly through the creation of defects along domain boundaries. Vesicle aggregation caused by dehydration and surface charge neutralization is a necessary but not a sufficient condition for fusion.     

Evaluation of viscosities of polymer-water solutions used in aqueous two-phase systems

The dynamic viscosities of dilute aqueous poly(ethylene glycol) and dextran, and poly(ethylene glycol)-dextran-water solutions have been measured. The poly(ethylene glycol) and dextran samples had average molecular masses of 8000 Da and 580 000 Da, respectively. To estimate the values of viscosity of poly(ethylene glycol)-dextran-water solutions, a Grunberg like equation has been proposed which takes into account the influence of poly(ethylene glycol) and dextran concentrations. The relative errors vary between 0.76 and 11.64 in absolute value.     

Synthesis and bioactivities of poly(ethylene glycol)–chitosan hybrids

Poly(ethylene glycol)–chitosan hybrids of various molecular weights having different degree of substitution were synthesized, by reductive N-alkylation of chitosan with poly(ethylene glycol) aldehyde, to study their bioactivities. The influence of these chitosan derivatives on the reactive oxygen species generation from canine polymorphonuclear leukocyte cells was investigated in vitro by chemiluminescence response. Reactive oxygen species generation by the influence of poly(ethylene glycol)–chitosan hybrids was decreased with the increase of degree of substitution. The reduction of interaction of poly(ethylene glycol)–chitosan hybrids with polymorphonuclear leukocyte cells might be caused by the decrease of amino group in chitosan main chain and increase of the steric hindrance by poly(ethylene glycol) chain. The influence of the poly(ethylene glycol)–chitosan hybrids on complement component C3 activation was investigated by single radial immunodiffusion method. Influence on complement component C3 activation by poly(ethylene glycol)–chitosan hybrids was almost same as chitosan.     

Synthesis of novel cationic poly(ethylene glycol) containing lipids.

In this paper, the synthesis of novel divalent cationic lipids with poly(ethylene glycol) segments is described. The lipids consist of an unsaturated double-chain hydrophobic moiety based on 3, 4-dihydroxy benzoic acid, attached to a hydrophilic poly(ethylene glycol) spacer which contains a divalent cationic end group. As poly(ethylene glycol) spacers monodisperse triethylene glycol and telechelic poly(ethylene glycol)s with an average degree of polymerization of 9, 23, and 45 were used. The divalent cationic end group was attached by coupling a protected dibasic amino acid to the PEG spacer and following cleavage of the protecting groups. These novel class of cationic lipids is of particular interest for nonviral gene delivery applications.     

Synthesis of polyamidoamine dendrimers having poly(ethylene glycol) grafts and their ability to encapsulate anticancer drugs

Polyamidoamine dendrimers having poly(ethylene glycol) grafts were designed as a novel drug carrier which possesses an interior for the encapsulation of drugs and a biocompatible surface. Poly(ethylene glycol) monomethyl ether with the average molecular weight of 550 or 2000 was combined to essentially every chain end of the dendrimer of the third or fourth generation via urethane bond. The poly(ethylene glycol)-attached dendrimers encapsulating anticancer drugs, adriamycin and methotrexate, were prepared by extraction with chloroform from mixtures of the poly(ethylene glycol)-attached dendrimers and varying amounts of the drugs. Their ability to encapsulate these drugs increased with increasing dendrimer generation and chain length of poly(ethylene glycol) grafts. Among the poly(ethylene glycol)-attached dendrimers prepared, the highest ability was achieved by the dendrimer of the fourth generation having the poly(ethylene glycol) grafts with the average molecular weight of 2000, which could retain 6.5 adriamycin molecules or 26 methotrexate molecules/dendrimer molecule. The methotrexate-loaded poly(ethylene glycol)-attached dendrimers released the drug slowly in an aqueous solution of low ionic strength. However, in isotonic solutions, methotrexate and adriamycin were readily released from the poly(ethylene glycol)-attached dendrimers.     

Mechanism of poly(ethylene glycol) interaction with proteins

Poly(ethylene glycol) (PEG) is one of the most useful protein salting-out agents. In this study, it has been shown that the salting-out effectiveness of PEG can be explained by the large unfavorable free energy of its interaction with proteins. Preferential interaction measurements of beta-lactoglobulin with poly(ethylene glycols) with molecular weights between 200 and 1000 showed preferential hydration of the protein for those with Mr greater than or equal to 400, the degree of hydration increasing with the increase in poly(ethylene glycol) molecular weight. The preferential interaction parameter had a strong cosolvent concentration dependence, with poly(ethylene glycol) 1000 having the sharpest decrease with an increase in concentration. The preferential hydration extrapolated to zero cosolvent concentration increased almost linearly with increasing size of the additive, suggesting steric exclusion as the major factor responsible for the preferential hydration. The poly(ethylene glycol) concentration dependence of the preferential interactions could be explained in terms of the nonideality of poly(ethylene glycol) solutions. All the poly(ethylene glycols) studied, when used at levels of 10-30%, decreased the thermal stability of beta-lactoglobulin, suggesting that caution must be exercised in the use of this additive at extreme conditions such as high temperature.     

Synthesis and biopharmaceutical characterisation of new poly(hydroxyethylaspartamide) copolymers as drug carriers.

Four new poly(hydroxyethylaspartamide)-based copolymers bearing (a) poly(ethylene glycol) 2000, (b) poly(ethylene glycol) 5000, (c) poly(ethylene glycol) 2000 and hexadecylalkyl, (d) poly(ethylene glycol) 5000 and hexadecylalkyle, as pendant groups were synthesised. The copolymers were obtained by partial aminolysis of polysuccinimide with poly(ethylene glycol) and hexadecylalkyl amino derivatives followed by reaction with ethanolamine. Naked polyhydroxyaspartamide was obtained by polysuccinimide reaction with ethanolamine. The nuclear magnetic resonance, infrared, light scattering and elemental analysis allowed for the extensive physico-chemical characterisation of the carriers. The molecular mass of all the polymers was in the range of 27000-34000 Da, and the polydispersivity was in the range of 1.5-1.7. By intravenous injection to mice bearing a solid tumour, all the polymeric carriers displayed a bi-compartmental pharmacokinetic behaviour. Both the poly(ethylene glycol) and the hexadecylalkyle conjugation prolonged and enhanced the distribution phase of poly(hydroxyethylaspartamide). The poly(ethylene glycol) conjugation was found to promote the carrier elimination by kidney ultrafiltration and to prevent partially the accumulation in the spleen and in the liver. The poly(ethylene glycol)/hexadecylalkyle conjugates localised preferentially in the liver were over 30% of the dose/g of tissue was determined after 144 h from administration. In the tumour all the polymers displayed a relevant accumulation that significantly increased throughout the time to reach high concentrations after 24 h. In particular, the poly(ethylene glycol)/hexadecylalkyle conjugates achieved a concentration of 15-25% of the dose/g of tissue after 24 h from administration that was maintained up to 144 h.     

Adsorption of lipoproteins with the aid of carboxymethylchitosan mircrospheres crosslinked with poly(ethylene glycol) bisglycidyl ether

Carboxymethylchitosan microspheres crosslinked with poly(ethylene glycol) bisglycidyl ether were prepared and then tested as an adsorbent for selective removal of low-density lipoprotein (LDL) in human plasma. The microspheres were formed by a method of electrostatic instillation and crosslinked with poly(ethylene glycol) bisglycidyl ether. FTIR spectral analyses and X-ray photoelectron spectroscopy revealed that carboxymethylchitosan was crosslinked through amino groups to poly(ethylene glycol) bisglycidyl ether. The plasma lipoprotein sorption tests showed that the adsorption properties of the crosslinked microspheres for LDL were dependent on the concentrations of carboxymethylchitosan and poly(ethylene glycol) bisglycidyl ether. When the concentrations of carboxymethylchitosan and poly(ethylene glycol) bisglycidyl ether were 3.5% and 6%, respectively, 40% LDL and lower than 10% high density lipoprotein in plasma could be removed and the adsorption could be reach an equilibrium in 30 min.     

Coupling of poly(ethylene glycol) to albumin under very mild conditions by activation with tresyl chloride: characterization of the conjugate by partitioning in aqueous two-phase systems

Poly(ethylene glycol) activated with tresyl chloride has been covalently linked to albumin as a result of a 2-h incubation in 0.05 M sodium phosphate buffer, pH 7.5, containing 0.125 M sodium chloride (0.344 OSM). The coupling of poly(ethylene glycol) to albumin was demonstrated by the increase in the partition coefficient of the protein in poly(ethylene glycol)-dextran aqueous two-phase systems. A linear relationship between the log of the partition coefficient of the poly(ethylene glycol)-albumin conjugate and the degree of modification (measured as the amino groups consumed during the coupling step) has been demonstrated. Countercurrent distribution in the two-phase system showed that poly(ethylene glycol)-albumin was heterogeneous with respect to its partitioning behavior, indicating that the albumin was not uniformly modified with poly(ethylene glycol).     

Enzyme action in polymer and salt solutions. I. Stability of penicillin acylase in poly(ethylene glycol) and potassium phosphate solutions in relation to water activity

The stability of penicillin acylase (penicillin aminohydrolase, EC 3.5.1.11) was studied in poly(ethylene glycol) and potassium phosphate solutions. Enzyme stability measured as the half-life of the enzymatic activity and the transition temperature determined by differential scanning calorimetry, correlated well. The enzyme stability could not be related to the water activity as a measure of solute-solvent interaction. It seems to be related more to the concentration of the solutes and much less to the molecular weight of poly(ethylene glycol). The stabilizing effect of poly(ethylene glycol) is also discussed in terms of poly(ethylene glycol)-protein interactions.     

Poly(ethylene glycol) quantitation by laser nephelometry

When poly(ethylene glycol) 3350 is estimated by the method of Skoog [(1979) Vox Sang. 37, 345-349], fine particles form. The particles are not attributable to residual protein but to a poly(ethylene glycol)/barium/iodine complex that can be quantitated by means of a laser nephelometer. The method is sensitive to at least 10 mg% poly(ethylene glycol) 3350 (4 micrograms in the cuvette) in 2500 mg% protein, and nephelometer response is approximately linear between 30 and 200 mg% of the polymer. The coefficient of variance is about 8%. Triton X-100, Pluronic F-68, Varonic 1000MS, and poly(ethylene glycol) of higher and lower molecular weight react well. Alkylated celluloses, dextrans, glycerol, glycine, and sodium dodecyl sulfate do not react significantly. Barium can be replaced with Mg, Ca, Ni, Fe, and other divalent cations in the reaction, but other than for Hg, light-scattering is most intense with Ba. The reaction goes to completion in about 5 min and is most intense when the barium is added before the iodine.     

Preparation and kinetic properties of 5-ethylphenazine-poly(ethylene glycol)-NAD+ conjugate, a unique catalyst having an intramolecular reaction step

5-Ethylphenazine-poly(ethylene glycol)-NAD+ conjugate (EP+-PEG-NAD+) was prepared by linking 1-(3-carboxypropyloxy)-5-ethylphenazine (I) to poly(ethylene glycol)-bound NAD+ (PEG-NAD+) and its kinetic properties were studied. As a reference compound, poly(ethylene glycol)-bound 5-ethylphenazine derivative (III) was also prepared and the effects of poly(ethylene glycol) on the reaction rate of the 5-ethylphenazine moiety with NADH was investigated. The second-order rate constant, k1, of the reaction of III with NADH is 2.78 mM-1 s-1 and is about 1.7 times that of 1-(3-ethoxycarbonylpropyloxy)-5-ethylphenazine (II) with NADH. A similar effect of the attached poly(ethylene glycol) was observed for the reaction of PEG-NADH with I or II. The second-order rate constants, k2 and k3, of the reactions of the reduced form of III with oxygen and with 3-(4',5'-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium ion, respectively, were k2 = 1.22 mM-1 s-1 and k3 = 32 mM-1 s-1; the k2 value is not changed but the k3 value is decreased by the attachment of the polymer. EP+-PEG-NAD+ works as a unique catalyst having an intramolecular reaction step within its turnover cycle in a coupled multi-step reaction system containing malate dehydrogenase, malate, EP+-PEG-NAD+, a tetrazolium salt and oxygen. The first-order rate constant, k4, of the intramolecular reaction was 1.1 s-1. The effects of the covalent linking of the 5-ethylphenazine and the NAD+ moieties were estimated by comparing the value of k4 with that of k1 for the reaction of III with NADH; the effective concentration of the NADH moiety for the 5-ethylphenazine moiety on the same EP+-PEG-NADH molecule (or vice versa) was calculated to be 0.40 mM from the ratio of k4/k1. The values of the rate constants in the coupled multi-step reaction system enable us to understand the dynamic features of the system and the characteristics of EP+-PEG-NAD+ as a catalyst are discussed.