Ionic Strength Mu Research Articles

Solubility, stability and ionization behaviour of famotidine.

The pKa of famotidine was determined at 23 +/- 0.2 degrees C to be 6.76, 6.98 and 6.89 by a spectrophotometric, solubility, and partitioning method, respectively. The pH-solubility profile of famotidine indicated an intrinsic solubility of 2.7 mM at 23 degrees C. Degradation of famotidine followed pseudo-first-order kinetics over a pH range of 1-11 at 37 +/- 0.2 degrees C and at an ionic strength (mu) of 0.5. The pH-rate profile was accounted for by the specific acid and base catalysed reactions as well as water-catalysed decomposition of both protonated and free famotidine. A pKa of 6.60, determined by potentiometry at 37 +/- 0.5 degrees C (mu = 0.5), was used in the kinetic calculations. Maximum stability occurred at pH 6.3. Undissociated acetic acid, dihydrogen phosphate ion and glycine anion were found to contribute to the general acid and base catalyses. pH-dependency of the apparent octanol-water partition coefficient of famotidine indicated a partition coefficient of 0.23 for free famotidine at 23 +/- 0.2 degrees C.

Uranyl-Se(IV) interaction in aqueous acid solutions studied by time-resolved laser-induced fluorescence spectroscopy (TRLFS) and UV-Vis spectrophotometry

The uranyl-selenium(IV) interaction was studied by time-resolved laser-induced fluorescence spectroscopy in aqueous perchloric acid solutions containing 1 x 10(-4) M U(VI) and Se(IV) at different concentrations (from 0 up to 0.3 M) at pH 1 and 2. The quenching of uranyl fluorescence is observed. HSeO(3)(-) is demonstrated to be responsible for the fluorescence quenching. Stern-Volmer analysis gives the dynamic quenching rate constant value (k(q)) (5.0 +/- 0.4) x 10(9) L M(-1) s(-1) at ionic strength (mu) 0.05 M. The bimolecular excited-state process is shown to be diffusion-controlled as k(q) is practically identical to the diffusion rate constant as calculated for uranyl and hydroselenite charged species. With an increase of HSeO(3)(-) concentration, static quenching occurs in addition to the dynamic quenching. The hypothesis of the formation of a weak fluorescent ground state complex between uranyl and HSeO(3)(-) is supported. The logarithm of the stability constant value (beta) is found to be 3.35 +/- 0.12 (mu= 0.05 M) and 3.32 +/- 0.15 (mu = 0.2 M). These constants are confirmed by UV-Vis spectrophotometry. The modelling factor analysis of absorption spectra gives logbeta = 3.35 +/- 0.17 (mu = 0.035 M). Extrapolation to mu = 0 by the specific ion interaction theory (SIT) gives logbeta degrees = 3.62 +/- 0.15 at 20 degrees C.

Determination of the Parameters Controlling Swelling of Chemically Cross-Linked pH-Sensitive Poly(N-vinylimidazole) Hydrogels

The number of variables controlling the behavior of ionic gels is large and very often some of them are unknown. The aim of this work is to interpret quantitatively the swelling behavior of pH sensitive gels, with the minimum number of simplifying assumptions. With this purpose, the equilibrium degree of swelling (S) and protonation (alpha) of chemically cross-linked poly(N-vinylimidazole) (PVI) immersed in aqueous salt solutions were measured as a function of the ionic strength (mu), in the whole range of pH. In acid solutions with pH in the range 0 to 4, imidazole moieties become protonated, and PVI behaves as a polyelectrolyte gel: S decreases upon increasing mu both for NaCl and for CaCl(2), with HCl as protonating acid. In aqueous solutions with larger pH, between 4 and 12, the hydrogel is practically neutral, and S increases as mu rises, showing a salting-in effect. From the quantitative analysis of these results, the following facts emerged. Protonation induces chain stiffness (as measured by the non-Gaussian factor) and worsening of the solvent quality of the aqueous media (as measured by the polymer-solvent interaction parameter). For alpha below 33%, swelling seems to be governed by the excess of mobile counterions inside the gel with respect to the bath, with a minor but still significantly negative contribution of the osmotic swelling pressure due to polymer-solvent mixing. Above 33% protonation, it is necessary to consider Manning counterion condensation to get parameters with physical meaning. The crossover between polyelectrolyte and salting-in effects corresponds to alpha and mu values with the same ionic and mixing contributions to the osmotic swelling pressure. The formation of ionic nonpermanent cross-links, with H(2)SO(4) as the protonating acid, was discarded.

Dependence of μ-Conotoxin Block of Sodium Channels on Ionic Strength but Not on the Permeating [Na+]: IMPLICATIONS FOR THE DISTINCTIVE MECHANISTIC INTERACTIONS BETWEEN Na+ AND K+ CHANNEL PORE-BLOCKING TOXINS AND THEIR MOLECULAR TARGETS

Mu-conotoxins (mu-CTXs) are Na+ channel-blocking, 22-amino acid peptides produced by the sea snail Conus geographus. Although K+ channel pore-blocking toxins show specific interactions with permeant ions and strong dependence on the ionic strength (mu), no such dependence has been reported for mu-CTX and Na+ channels. Such properties would offer insight into the binding and blocking mechanism of mu-CTX as well as functional and structural properties of the Na+ channel pore. Here we studied the effects of mu and permeant ion concentration ([Na+]) on mu-CTX block of rat skeletal muscle (mu1, Nav1.4) Na+ channels. Mu-CTX sensitivity of wild-type and E758Q channels increased significantly (by approximately 20-fold) when mu was lowered by substituting external Na+ with equimolar sucrose (from 140 to 35 mm Na+); however, toxin block was unaltered (p > 0.05) when mu was maintained by replacement of [Na+] with N-methyl-d-glucamine (NMG+), suggesting that the enhanced sensitivity at low mu was not due to reduction in [Na+]. Single-channel recordings identified the association rate constant, k(on), as the primary determinant of the changes in affinity (k(on) increased 40- and 333-fold for mu-CTX D2N/R13Q and D12N/R13Q, respectively, when symmetric 200 mm Na+ was reduced to 50 mm). In contrast, dissociation rates changed <2-fold for the same derivatives under the same conditions. Experiments with additional mu-CTX derivatives identified toxin residues Arg-1, Arg-13, and Lys-16 as important contributors to the sensitivity to external mu. Taken together, our findings indicate that mu-CTX block of Na+ channels depends critically on mu but not specifically on [Na+], contrasting with the known behavior of pore-blocking K+ channel toxins. These findings suggest that different degrees of ion interaction, underlying the fundamental conduction mechanisms of Na+ and K+ channels, are mirrored in ion interactions with pore-blocking toxins.

Structure-physicochemical function relationships of soybean beta-conglycinin heterotrimers.

We purified four single molecular species of beta-conglycinin heterotrimers consisting of the alpha and beta subunits or the alpha' and beta subunits from mutant soybean cultivars lacking the alpha or alpha' subunit, respectively, and examined their structural features and physicochemical functions. The extent of the hydrophobicities of the heterotrimers was related to the number of the alpha or alpha' subunit. The thermal stabilities of the heterotrimers were mainly conferred by the subunit which had lower thermal stability. Solubilities at low ionic strength (mu = 0.08) of the heterotrimers containing the alpha or alpha' subunit were very similar to those of the alpha and alpha' homotrimers, respectively. Emulsifying abilities and heat-induced associations of the heterotrimers containing one beta subunit were similar to those of the alpha or alpha' homotrimer, whereas those of the heterotrimers containing two beta subunits were similar to those of the beta homotrimer.

Thermodynamics in [Mn(II)–antibiotics–bacitracin] mixed system: a polarographic approach

Polarographic technique was used to determine the kinetic parameters, thermodynamic parameters and stability constants (log(beta)) of Mn(II) complexes with neomycin, chlortetracycline, oxytetracycline, tetracycline, penicillin V and penicillin G as primary ligands and bacitracin as the secondary ligand, at pH 7.3+/-0.01 and an ionic strength mu=1.0 M (NaClO(4)) at 25 degrees C. The study was also carried out at 35 degrees C to determine the stability constants and thermodynamic parameters viz. enthalpy change (DeltaH), entropy change (DeltaS) and free energy change (DeltaG) of complexes.

Antitumor activities of conjugates of mitomycin C with estradiol benzoate and estradiol via glutaric acid in suspension dosage form.

Conjugates of mitomycin C (MMC) with estradiol bezoate and estradiol via glutaric acid (EB-glu-MMC and E-glu-MMC, respectively) were examined for their antitumor activities against P388 leukemia and sarcoma 180. EB-glu-MMC and E-glu-MMC were suspended in 10% (v/v) propylene glycol in saline and administered intraperitoneally to mice bearing P388 leukemia intraperitoneally or to mice bearing sarcoma 180 subcutaneously. The antitumor effect against P388 leukemia was greater in the order MMC>E-glu-MMC>EB-glu-MMC, and only the former two compounds significantly increased life span. On the other hand, EB-glu-MMC and E-glu-MMC showed suppression of sarcoma 180 growth at higher doses close to or better than MMC. In the mixture of 1/15 M phosphate buffer (pH 7.4, ionic strength (mu) adjusted to 0.3 with NaCl)-propylene glycol (9:1, v/v) at 37 degrees C, MMC was released much more slowly from EB-glu-MMC suspension than from E-glu-MMC suspension. With regard to chemotherapy against sarcoma 180, both conjugates were considered to supply MMC slowly but effectively at higher doses.

Highly nonproductive complexes with Anabaena ferredoxin at low ionic strength are induced by nonconservative amino acid substitutions at Glu139 in Anabaena ferredoxin:NADP+ reductase.

Ferredoxin (Fd) and ferredoxin:NADP(+) reductase (FNR) from Anabaena function in photosynthetic electron transfer (et). The et interaction between the FNR charge-reversal mutant E139K and Fd at 12 mM ionic strength (mu) is extremely impaired relative to the reaction with wt FNR, and the dependency of k(obs) on E139K concentration shows strong upward curvature at protein concentrations > or = 10 microM. However, at values of mu > or = 200 mM, reaction rates approach those of wild-type FNR, and normal saturation kinetics are observed. For the E139Q mutant, which is also significantly impaired in its et interaction with Fd at low FNR concentrations and low mu values, the dependency of k(obs) on E139Q concentration shows a smaller degree of upward curvature at mu = 12 and 100 mM and shows saturation kinetics at higher values of mu. wt FNR and the E139D mutant both show a slight amount of upward curvature at FNR concentrations >30 microM at mu = 12 mM but show the expected saturation kinetics at higher values of mu. These results are explained by a mechanism in which the mutual orientation of the proteins in the complex formed at low ionic strength with the E139K mutant is so far from optimal that it is almost unreactive. At increased E139K concentrations, the added mutant FNR reacts via a collisional interaction with the reduced Fd present in the unreactive complex. The et reactivity of the low ionic strength complexes depends on the particular amino acid substitution, which via electrostatic interactions alters the specific geometry of the interface between the two proteins. The presence of a negative charge at position 139 of FNR allows the most optimal orientations for et at ionic strengths below 200 mM.

The roles of the N-linked glycans and extension regions of soybean beta-conglycinin in folding, assembly and structural features.

Beta-conglycinin, one of the dominant storage proteins of soybean, is a trimer composed of three subunits, alpha, alpha' and beta. All subunits are N-glycosylated and alpha and alpha' contain extension regions in addition to the core regions common to all subunits. Non-glycosylated individual subunits and deletion mutants (alpha(c) and alpha'(c)) lacking the extension regions of alpha and alpha' were expressed in Escherichia coli. All recombinant proteins were purified to near homogeneity and appeared to have the correct conformation, as judged by CD, density-gradient centrifugation and gel-filtration profiles, indicating that the N-linked glycans and extension regions are not essential for the folding and the assembly into trimers of beta-conglycinin. Density-gradient centrifugation, gel-filtration and differential scanning calorimetry profiles of the recombinant proteins and the native beta-conglycinin indicated that the N-linked glycans and extension regions contribute to the dimension of beta-conglycinin but not to the density and the thermal stability. Comparing the solubilities of the individual subunits with those of deletion mutants, only the alpha and alpha' subunits were soluble at lower ionic strength (mu < 0.25) at around the pH value of the endoplasmic reticulum. This suggests that the extension regions play an important role in the prevention of aggregation in the endoplasmic reticulum in analogy with the N-linked glycans.

Very rapid, ionic strength-dependent association and folding of a heterodimeric leucine zipper.

Leucine zippers (coiled coils) are dimerization motifs found in several DNA-binding transcription factors. A parallel leucine zipper composed of the acidic chain X1-EYQALEKEVAQLEAENX2-ALEKEVAQLEHEG-amide and the basic chain X1-EYQALKKKVAQLKAKNX2ALKKKVAQLKHKG-amide was designed to study the kinetics of folding of a heterodimeric leucine zipper and to investigate the role of electrostatic attraction between oppositely charged peptide chains to the folding reaction. Each peptide alone did not form a leucine zipper at ionic strength (mu) < 1 M because of electrostatic repulsion between like charges in a homodimer. Therefore, the formation of the heterodimeric leucine zipper could be investigated by simple mixing of acidic and basic chains. To monitor folding, a fluorescent label was located either at the N-terminus (X1 = fluorescein-GGG, X2 = Q) or in the center of the coiled coil (X1 = acetyl, X2 = W). Folding could be described by a simple two-state reaction involving the disordered monomers and the folded heterodimer. The same bimolecular rate constant (k(on)) was observed independent of the location of the fluorescent label, indicating that both fluorescence probes monitored the same reaction. Lowering of the ionic strength increased k(on) from 4 x 10(6) M-1 s-1 (mu = 525 mM) to about 5 x 10(7) M-1 s-1 (mu = 74 mM). When extrapolated to mu = O, k(on) was approximately 10(9) M-1 s-1, which is near the diffusion limit. In contrast, the rate of dissociation depended very weakly on ionic strength; k(off) decreased only by about 2-fold when mu was lowered from 525 to 74 mM. Equilibrium association constants (Ka) of the heterodimeric zippers measured directly and calculated from kinetic constants (Ka = k(on)/k(off) were in good agreement. The observed two-state mechanism, the strong dependence on ionic strength of k(on) but not of k(off), and the nearly diffusion-limited association rate at very low ionic strength point to a folding pathway in which the formation of an electrostatically stabilized dimeric intermediate may be rate-limiting and the subsequent folding to the final dimer is very rapid and follows a "down-hill" free energy landscape. The small increase of k(off) at increasing ionic strength indicates a minor contribution of electrostatics to the stability of the folded leucine zipper.

Changes in the surface charge of bacteria caused by heavy metals do not affect survival.

Bacillus subtilis and Agrobacterium radiobacter remained viable when exposed to Ni (1 x 10(-4)M; ionic strength (mu) = 3 x 10(-4)) at pH values known to cause a change of the net negative charge of the cells to a net positive charge (charge reversal). The gross morphology, as determined by scanning electron microscopy, of these and other bacteria and of Saccharomyces cerevisiae was not altered in the presence of Ni, Cu, and Zn (1 x 10(-4) M; mu = 3 x 10(-4)), which caused a charge reversal at pH values between 6.0 and 9.0. Similar results were obtained in the presence of Na and Mg, which did not cause charge reversal at the same mu and pH values. These results confirmed that cells remain viable when their surface charge is changed in the presence of some heavy metals at high pH values.

Partially-hydrophobized polymer particles derived from N,N-dimethylaminopropylacrylamide for endotoxin removal from acidic protein solution.

Novel copolymeric adsorbents for the selective removal of endotoxin from an acidic protein solution were prepared. The adsorbents comprise spherical copolymers derived from N,N-dimethylaminopropylacrylamide (DMAPAA) and divinylbenzene (DVB). When the molar ratio of DMAPAA to DVB was 80/20 (amino-group content: 5.1 meq/g) and the pore size (molecular mass exclusion of polysaccharide, Mlim) was 4000 to 10000, DMAPAA/DVB showed high endotoxin-adsorbing activity at pH 5.0 to 9.0 and ionic strengths of mu = 0.05 to 0.4. The capacity of the adsorbent (Mlim: 4000) was 390 micrograms of endotoxin (lipopolysaccharide purified from E. coli O111:B4) per ml of the adsorbent using the batchwise method. The apparent dissociation constant between endotoxin and the adsorbent was 2.2 x 10(-12) M. On the other hand, the adsorption of bovine serum albumin, an acidic protein, by the adsorbent increased with an increase in Mlim from 4000 to 10000, but decreased with an increase in ionic strength (mu) from 0.05 to 0.2. As a result, DMAPAA/DVB (80/20) (Mlim: 4000) selectively removed endotoxin from various acidic protein solutions at pH 7.0 and mu = 0.05. The residual concentration of endotoxin in the protein solution always decreased to a concentration lower than 0.1 ng/ml, and recovery of the protein was more than 97%.

A complete mechanism for steady-state oxidation of yeast cytochrome c by yeast cytochrome c peroxidase.

Steady-state oxidation of yeast cytochrome c (yCc) was monitored as a function of ionic strength (mu) for mutants of a cloned cytochrome c peroxidase [CcP(MI)]. The data are best interpreted in the context of a two binding site model, where the affinity of the two sites for yCc differs by approximately 1000-fold and rapid intracomplex electron transfer (ET) occurs only at the high-affinity site identified in the crystal structure. At low mu, catalysis is apparently limited by the rate of yCc dissociation from the reactive high-affinity site (koff). Binding of yCc at the low-affinity site increases koff and therefore increases the rate of catalysis. Mutations at the high-affinity site also increase the rate of catalysis by the 1:1 CcP(MI):yCc complex by increasing koff. Mutations at residues that interact strongly with yCc at the high-affinity site (Asp 34, Glu 290, and Ala 193) cause the greatest increase in koff (25-38-fold at mu = 20 mM). Mutations at residues that interact less strongly with yCc (Glu 32 and Glu 291) cause smaller increases in koff (10- and 3-fold, respectively, at mu = 20 mM). The results provide additional evidence that the high-affinity site formed in solution is similar to the one identified in the crystal structure and that yCc dissociation from this site limits enzyme turnover at low ionic strength. Numerical integration simulations show that the model accurately predicts enzyme turnover rates at the high-affinity site, using published rate constants for the elementary reaction steps.

C1q binding properties of monomer and polymer forms of mouse IgM mu-chain variants. Pro544Gly and Pro434Ala.

The effect of replacing proline with alanine at position 434 in the C mu 3 domain (P434A) and with glycine at position 544 in the C mu 4 domain (P544G) of the mu-chain of mouse IgM has been studied. The P434A substitution results in the loss of measurable complement-mediated cytolytic activity (CML) and a decrease in the association rate constant at low ionic strength (mu = 0.06), that results in a diminished Ka for C1q binding to P434A IgM bound to haptenated cells (0.4 x 10(9) M-1). Binding of C1(qr2s2) could not be detected. In contrast, replacement of proline at 544 had no measurable effect on the cytolytic or C1q/C1 binding properties of the polymeric molecule, supporting the view that the C mu 3 domain is important in C1q binding and CML. The secreted monomeric subunit of P544G was not able to mediate CML. Also, whereas hapten-bound P544G polymer bound C1q with a functional affinity of 1.5 x 10(9) M-1 at low ionic strength (mu = 0.06), similar to that observed with wild-type polymer (1.7 x 10(9) M-1) and wild-type IgG monomer (4.7 x 10(9) M-1), no C1q binding was detected with the P544G IgM monomer. This could not be attributed to differences in glycosylation. Inasmuch as the P544G mutation per se had no effect on the C1q binding properties of the polymer, we conclude that unlike IgG, aggregation does not sufficiently enhance the avidity of IgM monomer to enable it to activate complement. Augmentation of the site must occur during polymerization or when the IgM binds to Ag.

Recombinant human insulin. V. Optimization of the reversed-phase high-performance liquid chromatographic separation.

The applicability of reversed-phase high-performance liquid chromatography (HPLC) to the analysis of the products of recombinant insulin was studied. The influence of several mobile phases in reversed-phase and ion-pair HPLC on selectivity, resolution and sensitivity was investigated. Optimum conditions for the separation of insulin-related proteins on commercial and laboratory-made supports were established by means of three-dimensional optimizations of selectivity and resolution as a function of pH and ionic strength (mu). A mechanism for the separation of proteins with a mobile phase containing a high salt concentration and a pH near the isoelectric point of proteins is proposed. The questions of scaling up are considered. The proposed techniques allow the analysis of the main impurities and ensures a high quality of active insulin production.

Rotational dynamics of actin-bound intermediates of the myosin adenosine triphosphatase cycle in myofibrils

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to measure the microsecond rotational motion of actin-bound myosin heads in spin-labeled myofibrils in the presence of the ATP analogs AMPPNP (5'-adenylylimido-diphosphate) and ATP gamma S (adenosine-5'-O-(3-thiotriphosphate)). AMPPNP and ATP gamma S are believed to trap myosin in two major conformational intermediates of the actomyosin ATPase cycle, respectively known as the weakly bound and strongly bound states. Previous ST-EPR experiments with solutions of acto-S1 have demonstrated that actin-bound myosin heads are rotationally mobile on the microsecond time scale in the presence of ATP gamma S, but not in the presence of AMPPNP. However, it is not clear that results obtained with acto-S1 in solution can be extended to actomyosin constrained within the myofibrillar lattice. Therefore, ST-EPR spectra of spin-labeled myofibrils were analyzed explicitly in terms of the actin-bound component of myosin heads in the presence of AMPPNP and ATP gamma S. The fraction of actin-attached myosin heads was determined biochemically in the spin-labeled myofibrils, using the proteolytic rates actomyosin binding assay. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to be rotationally mobile on the microsecond time scale (tau r = 24 +/- 8 microseconds) in the presence of ATP gamma S, but not AMPPNP. Similar results were obtained at low ionic strength, confirming the acto-S1 solution studies. The microsecond rotational motions of actin-attached myosin heads in the presence of ATP gamma S are similar to those observed for spin-labeled myosin heads during the steady-state cycling of the actomyosin ATPase, both in solution and in an active isometric muscle fiber. These results indicate that weakly bound myosin heads, in the pre-force phase of the ATPase cycle, are rotationally mobile, while strongly bound heads, in the force-generating phase, are rotationally immobile. We propose that force generation involves a transition from a dynamically disordered crossbridge to a rigid and stereospecific one.

Rotational dynamics of actin-bound myosin heads in active myofibrils

We have used saturation-transfer electron paramagnetic resonance (ST-EPR) to measure the submillisecond rotational motions of actin-bound myosin heads in active myofibrils. The cross-bridges were spin-labeled with a maleimide nitroxide derivative (MSL) that has previously been shown to undergo microsecond rotational motions on actin-bound myosin heads in solution during steady-state ATPase activity at low ionic strength [Berger, C. L., Svensson, E. C., & Thomas, D. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8573]. To determine whether this is also true for cross-bridges in the myofibrillar lattice under physiological buffer conditions, we have performed ST-EPR experiments during the brief steady state following photolysis of caged ATP in a suspension of spin-labeled myofibrils. The myofibrils were partially cross-linked with EDC [1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide] to prevent their shortening upon activation. The fraction of actin-attached myosin heads was determined biochemically at physiological ionic strength in the active myofibrils, using the proteolytic rates acto-myosin binding assay [Duong, A. M., & Reisler, E. (1989) Biochemistry 28, 3502]. These data were then used to correct the ST-EPR spectra of active myofibrils for the presence of unattached myosin heads, which were assumed to undergo the same motions as in relaxation. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to have considerable microsecond rotational motion (tau r = 3.5 +/- 1.1 microseconds) in the active myofibrils. Similar results (tau r = 3.2 +/- 0.8 microseconds) were obtained with active myofibrils at low ionic strength (mu = 45 mM), confirming the work done in solution. Thus, under physiological conditions and even within the constraints of the myofibrillar lattice, actively cycling actin-attached myosin heads are rotationally mobile on the microsecond time scale. Since partially EDC-fixed myofibrils are an excellent analog of isometrically contracting muscle fibers in solution, it is likely that these microsecond rotational motions are directly related to the molecular mechanism of muscle contraction in vivo.

Reduction of endotoxin contamination of various crude vaccine materials by gram-negative bacteria using aminated poly(gamma-methyl L-glutamate) spherical particles.

We describe a method for the removal of endotoxins from various crude antigen solutions originating from gram-negative bacteria using aminated poly(gamma-methyl L-glutamate) (PMLG) spherical particles. The aminated PMLG adsorbents showed high affinity for various purified endotoxins at an ionic strength of mu = 0.1. The endotoxin-adsorbing capacity of the adsorbent increased with increase in the amino-group content of the adsorbent. The adsorbent (3.2 meq/g amino-group content) showed the highest affinity for endotoxin at ionic strengths ranging from mu = 0.025-0.8. The adsorption of Bordetella pertussis antigen to the adsorbent decreased with increasing amino-group content of the adsorbent at an ionic strength of mu = 0.2. The adsorption of B. bronchiseptica protein to the adsorbent increased with increasing amino-group content of the adsorbent, but decreased with increasing ionic strength. The adsorbent (3.2 meq/g of amino-group content) selectively reduced endotoxin in crude antigen solutions originating from gram-negative bacteria, B. pertussis, B. bronchiseptica and Pasteurella multocida, even at a high ionic strength (mu = 0.2-0.4) without affecting the recovery of the protective antigens.

Heavy metals alter the electrokinetic properties of bacteria, yeasts, and clay minerals

The electrokinetic patterns of four bacterial species (Bacillus subtilis, Bacillus megaterium, Pseudomonas aeruginosa, and Agrobacterium radiobacter), two yeasts (Saccharomyces cerevisiae and Candida albicans), and two clay minerals (montmorillonite and kaolinite) in the presence of the chloride salts of the heavy metals, Cd, Cr, Cu, Hg, Ni, Pb, and Zn, and of Na and Mg were determined by microelectrophoresis. The cells and kaolinite were net negatively charged at pH values above their isoelectric points (pI) in the presence of Na, Mg, Hg, and Pb at an ionic strength (mu) of 3 x 10(-4); montmorillonite has no pI and was net negatively charged at all pH values in the presence of these metals. However, the charge of some bacteria, S. cerevisiae, and kaolinite changed to a net positive charge (charge reversal) in the presence of Cd, Cr, Cu, Ni, and Zn at pH values above 5.0 (the pH at which charge reversal occurred differed with the metal) and then, at higher pH values, again became negative. The charge of the bacteria and S. cerevisiae also reversed in solutions of Cu and Ni with a mu of greater than 3 x 10(-4), whereas there was no reversal in solutions with a mu of less than 3 x 10(-4). The clays became net positively charged when the mu of Cu was greater than 3 x 10(-4) and that of Ni was greater than 1.5 x 10(-4). The charge of the cells and clays also reversed in solutions containing both Mg and Ni or both Cu and Ni (except montmorillonite) but not in solutions containing both Mg and Cu (except kaolinite) (mu = 3 x 10(-4)). The pIs of the cells in the presence of the heavy metals were at either higher or lower pH values than in the presence of Na and Mg. Exposure of the cells to the various metals at pH values from 2 to 9 for the short times (ca. 10 min) required to measure the electrophoretic mobility did not affect their viability. The specific adsorption on the cells and clays of the hydrolyzed species of some of the heavy metals that formed at higher pH values was probably responsible for the charge reversal. These results suggest that the toxicity of some heavy metals to microorganisms varies with pH because the hydrolyzed speciation forms of these metals, which occur at higher pH values, bind on the cell surface and alter the net charge of the cell.(ABSTRACT TRUNCATED AT 400 WORDS)

Removal of endotoxin from culture supernatant of Bordetella pertussis with aminated poly(gamma-methyl L-glutamate) spherical beads.

Attempts were made to prepare adsorbents having a high affinity for endotoxin in the culture supernatant of Bordetella pertussis. When poly(gamma-methyl L-glutamate) (PMLG) was used as a matrix and amino groups as the ligand, the highest affinity for endotoxin was attained even at a high ionic strength (mu = 0.2-0.4). PMLG beads containing amino groups of about 3.2 meq/g selectively removed endotoxin from the culture supernatant of B. pertussis without affecting the protective antigens. It was demonstrated that 1 ml of the wet adsorbent adsorbed 4.5 mg of endotoxin. The beads of PMLG derivatives, therefore, are considered to be a useful adsorbent for the removal of endotoxin from the pertussis vaccine, affecting neither filamentous hemagglutinin nor pertussis toxin.