Determination of surface charge of some bacteria by colloid titration

The surface charge of bacteria is always negative except below pH 2. The negative charge depends mainly upon phosphate and carboxyl groups. The charge of six kinds of bacteria was determined by colloid titration between pH 2 and 11. When there is amino group on the cell surface, it is positive as ammonium cation between pH 2 and 10, and it combines with the negative groups to neutralize the negative charge. In the presence of formalin, the colloid titration results show that the free amine is blocked by formalin, and the amino group can be estimated by the difference between the two titration results. Above pH 10, the titration results of Escherichia coli and Salmonella typhi in the presence of formalin implied the existence of guanidyl group. The titration results of living bacterial suspensions were quite the same as those of heat-killed ones.     

A new solid-phase support for oligonucleotide synthesis.

An alternative solid support for oligonucleotide synthesis was developed by coupling a polymer colloid to a modified polyethylene filter disc. The functions on the polymer colloid not used for attachment to the surface were derivatized with a Jeffamine diamine and loaded with appropriate deoxynucleoside succinates. The performance of this support system was evaluated and compared to existing resins.     

Interaction of heparin with two synthetic peptides that neutralize the anticoagulant activity of heparin

Two synthetic analogues of the heparin-binding domain of heparin/heparan sulfate-interacting protein (Ac-SRGKAKVKAKVKDQTK-NH2) and the all-d-amino acid version of the same peptide (l-HIPAP and d-HIPAP, respectively) were synthesized, and their efficacy as agents for neutralization of the anticoagulant activity of heparin was assayed. The two analogue peptides were found to be equally effective for neutralization of the anticoagulant activity of heparin, as measured by restoration of the activity of serine protease factor Xa by the Coatest heparin method. The finding that l-HIPAP and d-HIPAP are equally effective suggests that d-amino acid peptides show promise as proteolytically stable therapeutic agents for neutralization of the anticoagulant activity of heparin. The interaction of l-HIPAP and d-HIPAP with heparin was characterized by 1H NMR, isothermal titration calorimetry (ITC), and heparin affinity chromatography. The two peptides were found to interact identically with heparin. Analysis of the dependence of heparin-peptide binding constants on Na+ concentration by counterion condensation theory indicates that, on average, 2.35 Na+ ions are displaced from heparin per peptide molecule bound and one peptide molecule binds per hexasaccharide segment of heparin. The analysis also indicates that both ionic and nonionic interactions contribute to the binding constant, with the ionic contribution decreasing as the Na+ concentration increases.     

Biophysical insight into the heparin‐peptide interaction and its modulation by a small molecule

The heparin‐protein interaction plays a vital role in numerous physiological and pathological processes. Not only is the binding mechanism of these interactions poorly understood, studies concerning their therapeutic targeting are also limited. Here, we have studied the interaction of the heparin interacting peptide (HIP) from Tat (which plays important role in HIV infections) with heparin. Isothermal titration calorimetry binding exhibits distinct biphasic isotherm with two different affinities in the HIP‐heparin complex formation. Overall, the binding was mainly driven by the nonionic interactions with a small contribution from ionic interactions. The stoichiometric analysis suggested that the minimal site for a single HIP molecule is a chain of 4 to 5 saccharide molecules, also supported by docking studies. The investigation was also focused on exploiting the possibility of using a small molecule as an inhibitor of the HIP‐heparin complex. Quinacrine, because of its ability to mimic the HIP interactions with heparin, was shown to successfully modulate the HIP‐heparin interactions. This result demonstrates the feasibility of inhibiting the disease relevant heparin‐protein interactions by a small molecule, which could be an effective strategy for the development of future therapeutic agents.     

Histidine-rich glycoprotein modulation of the anticoagulant activity of heparin. Evidence for a mechanism involving competition with both antithrombin and thrombin for heparin binding

Heparin binding to rabbit histidine-rich glycoprotein (HRG) was studied in a purified system, allowing for determination of a heparin dissociation constant of approximately 5.5 X 10(-8) M for the interaction with HRG at pH 7.0. The strong interaction between heparin and HRG was demonstrated to be competitive with the binding of both antithrombin and thrombin to the heparin chain. HRG was further tested as a modulator of the anticoagulant activity of heparin by comparing rates of the heparin-catalyzed reaction between antithrombin and thrombin in the presence and absence of added HRG. The heparin-antithrombin-thrombin reaction was modeled using the formalism of a two-substrate enzyme-catalyzed reaction with heparin as the enzyme and HRG analyzed as an enzyme inhibitor. HRG was shown to compete with both antithrombin and thrombin for binding to heparin by this kinetic analysis. Thus, both the kinetic and heparin-binding data indicate that the mechanism by which HRG modulates heparin anticoagulant activity involves competition for heparin with both the inhibitor and the protease. Inhibition by HRG of the heparin-catalyzed reaction was found to be highly dependent on pH, with a sharp increase in inhibition from about 15% to greater than 90% observed as pH was lowered from 7.4 to 7.0. Since little change in the rate of the heparin-catalyzed inhibition of thrombin by antithrombin occurs in this pH region, the dramatic change in HRG inhibition seen upon pH titration may reflect increasing ionic interaction between heparin and HRG due to the protonation of histidine residues which occurs in this pH region.     

Interaction of soluble and surface-bound heparin binding growth-associated molecule with heparin.

The interaction of heparin with heparin binding growth-associated molecule (HB-GAM) was studied using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). ITC studies showed that, in solution, heparin bound HB-GAM with a deltaH of -30 kcal/mole corresponding to a dissociation constant (Kd) of 460 nM. The stoichiometry of interaction was 3 moles of HB-GAM per mole of heparin, corresponding to a minimum heparin binding site for HB-GAM of 12-16 saccharide residues. Kinetic measurements of heparin interaction with HB-GAM made by SPR afforded a Kd of 4 nM, suggesting considerably tighter binding when HB-GAM was immobilized on a surface. Affinity chromatography of a sized mixture of heparin oligosaccharides, having a degree of polymerization (dp) of > 14 saccharide units, on HB-GAM-Sepharose demonstrated that oligosaccharides having more than 18 saccharide residues showed the tightest interaction.     

Multiple complexes of thrombin and heparin

Fluorescence polarization has been used to study the interaction of thrombin and heparin, and the catalysis by heparin of the combination of thrombin and antithrombin. At low ionic strength (20 mM Tris, pH 7.4), the addition of heparins of known molecular weights to thrombin led to the formation of large complexes (defined as 'complex 1'). Further addition of heparin led to a rearrangement of these large complexes to form smaller complexes (defined as 'complex 2'). The molar ratio of thrombin to heparin in complex 1 increased with increasing heparin molecular weight, and corresponded to one thrombin molecule for every heparin segment of Mr 3000. The stoichiometry of complex 2 was 1 heparin to 1 thrombin, irrespective of the heparin molecular weight. At higher ionic strength (150 mM NaCl) some complex 1 was still formed. However, by reversing the titration and adding thrombin to fluorescein-heparin the dissociation constant for complex 2 was estimated to be 1-3 microM and independent of the heparin molecular weight. The complex formed between thrombin and heparin, to which antithrombin was attached, has a dissociation constant of 1-2 microM, again irrespective of the heparin molecular weight. In the heparin-catalysed thrombin-antithrombin reaction, an increase in the size of heparin leads to a lowering of the observed Km for thrombin. A possible explanation is that thrombin, after initial binding to the heparin, moves rapidly to the site where it combines with antithrombin.     

The interaction of platelet factor 4 with heparins of different chain length

The interaction of platelet factor 4 with heparin of varying chain lengths has been investigated by labelling the heparin with fluorescein isothiocyanate and monitoring the change in anisotropy of fluorescence when the protein is added to a solution of the polysaccharide. The shape of the titration curve depends on the Mr of the heparin and chains of Mr greater than 10 000 showed a definite break when the concentration of polysaccharide and protein became equimolar. Evidence is presented to show that most of the fluorescein label is linked to residual serines on the heparin. Similar break-points were observed if total fluorescence or light-scattering was used to monitor the interaction. Unlabelled heparin was used for the latter method. These results together with those obtained in buffer of high ionic strength lead us to propose a model where the heparin is wrapped around the tetrameric protein.     

Kinetic analysis of various heparin fractions and heparin substitutes in the thrombin inhibition reaction

Kinetic characteristics of several heparin preparations and substitute heparins were determined to help understand the bases for activity differences. Several materials were highly active in factor Xa inhibition and the reaction rate at constant factor Xa concentration appeared to be predicted by the extent of intrinsic antithrombin III fluorescence change induced by the polysaccharide. Heparin fractions of different molecular weight and affinity for antithrombin III showed similar kinetic parameters in catalysis of the thrombin-antithrombin III reaction when these parameters were expressed on the basis of antithrombin III-binding heparin. The latter was determined by stoichiometric titration of the antithrombin III fluorescence change by the heparin preparation. However, the various heparin fractions showed very different specific activities per mg of total polysaccharide. This indicated that functional heparin molecules had similar kinetic properties regardless of size or antithrombin III-binding affinity and is possible because the Km for antithrombin III is determined by diffusion rather than by binding affinity. Substitute heparins and depolymerized heparin were poor catalysts for thrombin inhibition, due at least partially to their affinity for thrombin. This latter binary interaction inhibits thrombin reaction in the heparin-catalyzed reaction.     

Interaction of heparin with annexin V

The energetics and kinetics of the interaction of heparin with the Ca2+ and phospholipid binding protein annexin V, was examined and the minimum oligosaccharide sequence within heparin that binds annexin V was identified. Affinity chromatography studies confirmed the Ca2+ dependence of this binding interaction. Analysis of the data obtained from surface plasmon resonance afforded a Kd of approximately 21 nM for the interaction of annexin V with end-chain immobilized heparin and a Kd of approximately 49 nM for the interaction with end-chain immobilized heparan sulfate. Isothermal titration calorimetry showed the minimum annexin V binding oligosaccharide sequence within heparin corresponds to an octasaccharide sequence. The Kd of a heparin octasaccharide binding to annexin V was approximately 1 microM with a binding stoichiometry of 1:1.     

Randomness in the heparin polymer: computer simulations of alternative action patterns of heparin lyase

Heparin is a mixture of linear polysaccharides of undetermined sequence. Both biosynthetic data and computer simulation studies have established that each heparin polymer chain is comprised of oligosaccharides of defined sequence, representing ordered domains. One such ordered domian is a pentasaccharide corresponding to heparin's antithrombin III binding site. Previous computer simulation studies, performed under the assumption that heparin lyase (heparinase, EC 4.2.2.7), has a random endolytic action pattern, suggested that certain of these ordered oligosaccharide domains may themselves be nonrandomly arranged in the heparin polymer. The present work presents computer simulations of alternative action patterns for heparin lyase while assuming a random distribution of these oligosaccharide units within the heparin polymer. We consider action patterns that are determined solely by the primary structure of the substrate molecules. Results of the simulations are compared to (1) the experimental measurements of product chains formed throughout the reaction and (2) the change in weight average molecular weight Mw as a function of reaction completion as determined by absorbance at 232 nm. From the simulation of 60 action patterns for heparin lyase, we infer that one of the following statements concerning heparin and heparin lyase is true: (1) Heparin is a random arrangement of a small number of structurally defined oligosaccharide units. Heparin lyase changes its action pattern during the depolymerization of heparin (perhaps influenced by the secondary structure of substrate). (2) Heparin contain clusters of oligosaccharide sequences that are present in low concentrations (overall) in the polymer. Heparin lyase has a specificity for cleaving glycosidic linkages either exolytically at the nonreducing terminus of a chain or (endolytically) at the reducing side of these rare oligosaccharide sequence.     

Physical polymer matrices based on affinity interactions between peptides and polysaccharides

A rapidly forming polymer matrix with affinity-based controlled release properties was developed based upon interactions between heparin-binding peptides and heparin. Dynamic mechanical testing of 10% (w/v) compositions consisting of a 3:1 molar ratio of poly(ethylene glycol)-co-peptide (approximately 18,000 g/mol) to heparin (approximately 18,000 g/mol) revealed a viscoelastic profile similar to that of concentrated, large molecular weight polymer solutions and melts. In addition, the biopolymer mixtures recovered quickly following thermal denaturation and mechanical insult. These gel-like materials were able to sequester exogenous heparin-binding peptides and could release these peptides over several days at rates dependent on relative heparin affinity. The initial release rates ranged from 3.3% per hour for a peptide with low heparin affinity to 0.025% per hour for a peptide with strong heparin affinity. By altering the affinity of peptides to heparin, a series of peptides can be developed to yield a range of release profiles useful for controlled in vivo delivery of therapeutics.     

Polymer concentration dependence of the helix to random coil transition of a charged polypeptide in aqueous salt solution.

The helix to coil transition of poly(L-glutamic acid) was investigated in 0.05 and 0.005 M aqueous potassium chloride solutions by use of potentiometric titration and circular dichroism measurement. Polymer concentration dependence of the transition was observed in the range from 0.006 to 0.04 monomol/e in 0.005 M KG1 solution. The polymer concentration dependence can be interpreted by current theories of the transition of charged polypeptides and of titration curves of linear weak polyelectrolytes taking the effect of polymer concentration into consideration.     

Protamine — antagonist to heparin

Protamine is used for titration of heparin in vitro for diagnosis of hemorrhagic states and for neutralization of heparin in vivo to terminate heparinization. The protamine equivalent varies with the heparin preparation, conditions of testing and, in vivo, with the amount of heparin present in the circulation. The latter depends on time after administration and the hemodynamic and metabolic state of the patient. Protamine, when injected rapidly, will release histamine and agglutinate platelets. Bleeding (spontaneous hemorrhage) demonstrates a multiple breakdown of hemostatic mechanisms due to surgical stress, drugs, exposure of the blood to foreign surfaces, etc. Simple rules for the amount of protamine required for an individual patient based on clinical judgement will be satisfactory in most cases. When hemostasis is not achieved, it must be appreciated that heparin and protamine are only part of a complex deteriorating situation.     

Heparin conjugated polylactide as a blood compatible material

A heparin-conjugated biodegradable polymer (PLA-heparin) by the direct coupling of heparin to polylactide (PLA) was synthesized and characterized. The surface exposed heparin content associated PLA-heparin was measured to be 0.067 microg/cm2. PLA-heparin coated surface has shown higher hydrophilicity rather than control PLA surface. The clotting time of PLA-heparin conjugate measured by activated partial thromboplastin time (APTT) was significantly prolonged as compared to PLA. The bioactivity of bound heparin measured by APTT corresponds to 17.4% of free heparin. It has been also demonstrated that the conjugation of heparin suppresses the protein adsorption as well as the platelet adhesion. These results indicate that the unique property of bound heparin has an inhibiting influence on the coagulation, plasma protein adsorption, and subsequent platelet adhesion systems. This novel PLA-heparin conjugate could be applied as blood/tissue compatible biodegradable materials for implantable medical devices and tissue engineering.     

Morphological Changes in Chilling Injured Sweet Potato Root

Hydrophobicity and amounts of polar groups on the cell surface were determined by the use of fluorescent probe, colloid titration, and acid titration. Surface hydrophobicity of hydrocarbon-grown cells was about 7 times greater than that of glucose-grown cells of the same strain, while amounts of polar groups did not differ so much. Adsorption of cells to air bubbles was maximum at pH 3. Langmuir’s adsorption isotherm held for the adsorption. Affinity to air bubbles of hydrocarbon-grown cells was 1.8 times greater than that of glucose-grown cells, while the theoretical maximum amounts of cells to adsorb per unit area of bubbles were equal for the both cells.     

Heparin/polypyrrole (PPy) composite on gold-coated matrix for the neurite outgrowth of PC12 cells by electrical stimulation

A heparin/polypyrrole (PPy) composite, an electrical conducting polymer, was designed to enhance the interactions between a gold-coated matrix and nerve cells, with the cell (PC12 cells) interactions investigated under different conditions, both with and without electrical stimulation. The heparin concentration in the composites increased with increasing current density under the preparation condition, indicating that the heparin concentration in the composite could be controlled by managing the current density. Optical imaging showed that PC12 cells well attached to the PPy surfaces covered with heparin, but were poorly interacted to PPy surfaces without the heparin and gold coated matrix. The neurite length of the PC12 cells on the surfaces with an electrical stimulation (100 mV for 1h) significantly increased, with a median length of 77.5 μm; whereas, that without electrical stimulation was 10∼20 μm. Therefore, the heparin/polypyrrole (PPy) composite may provide insight for the development of an ideal nerve guidance channel.     

Potentiometric titration of poly-S-carboxymethyl-L-cysteine in aqueous NaCl solution

pH titration measurements of poly- S-carboxymethyl-L -cysteine were undertaken in the aqueous Nacl solution in relation to the β form–random coil transition. The titration curves show a marked molecular weight dependence because of the shortened chain length of materials. Comparison of the optical rotatory dispersion parameter a₀ with the titration curve reveals that the titration curve apparently reflects a β structure–random coil transition. The β form of this polymer is assumed to be an intramolecular β form, rather than a β structure stabilized by an intermolecular hydrogen bond, at least in the polymer concentration range considered here. The standard free energy change per amino acid residue for the transition from un-ionized random coil to un-ionized β form is estimated to be about ?750 cal/mole residue in the range of 0.005–0.2M NaCl concentration.     

Isothermal titration calorimetry study of the polyelectrolyte complexation of xanthan and chitosan samples of different degree of polymerization

Mixing oppositely charged polyelectrolytes in aqueous solutions leads to the spontaneous formation of polyelectrolyte complexes. Here, we characterize the interaction between xanthan of two different chain lengths, a tri-glucosamine and a chitosan polymer by isothermal titration calorimetry (ITC). Analysis of the experimental thermodynamic data assuming a single set of identical sites indicated both enthalpic and entropic contributions to the overall interaction in the interaction between xanthan and tri-glucosamine. The relative contribution of entropy compared to enthalpy was found to be largest for the shortest chain length of xanthan. Using a chitosan polymer instead of tri-glucosamine gave rise to two different stages in the interaction process. A model where the first stage of the ITC curve represent an initial polyelectrolyte complexation stage followed by aggregation on further titration of chitosan to the xanthan is suggested. Ultrastructure images by applying atomic force microscopy at some selected extents of titration are consistent with the two-stage interpretation of the thermodynamic data.     

One-Stage Resection of Seven Arterial Aneurysms

Coagulation times and protamine titration values for heparin are apt to be lower than average in patients with allergic disease, particularly asthma. Severe asthma is commonly treated with gluco-corticoids or ACTH. Prolonged high dosage of gluco-corticoids, either endogenous or exogenous, depresses mast cell function and heparin production. Thrombo-embolic phenomena occur frequently in severe cases of asthma so treated, particularly if predisposing vascular disease is present. Heparin can be used to treat and prevent these complications. Adjuvant antiasthmatic effects under these conditions are noted in addition to the desired anticoagulation.