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PubMed2004

Influence of polymerization technique and experimental variables on the particle properties and release kinetics of methotrexate from poly(butylcyanoacrylate) nanoparticles.

Poly(butylcyanoacrylate) nanoparticles were prepared by dispersion polymerization (DP) and emulsion polymerization (EP) of n-butyl cyanoacrylate monomer. The particles were characterized by infrared spectroscopy, differential scanning calorimetry, X-ray diffractometry and transmission electron microscopy. Particle properties such as size and zeta potential were determined for nanoparticles prepared by DP and EP techniques and compared. EP technique resulted in a low particle size compared to the DP. A high zeta potential was observed for nanoparticles prepared by the DP method. Incorporation of methotrexate resulted in a decrease in zeta potential in both types of nanoparticles, the decrease being greater in DP nanoparticles. Effect of experimental variables such as monomer concentration, polymerization time and temperature on drug entrapment and particle size was studied. Both types of nanoparticles showed an increase in drug entrapment with increased monomer concentrations. Variable polymerization time did not influence the drug entrapment of EP nanoparticles. Polymerization at 60 +/- 2 degrees C resulted in a decrease of drug entrapment and a great increase in the particle size of both types of nanoparticles. In vitro drug release studies showed a comparatively high release of methotrexate from DP nanoparticles suggesting the channelizing effect of dextran chains incorporated into nanoparticles during polymerization. Though the release profiles of nanoparticles appeared similar, a significant difference in release rates was found for DP and EP nanoparticles in 0.1 mol L(-1) HCl and pH 7.4 phosphate buffer (p < 0.01). Drug release data indicate that the release of methotrexate from DP and EP nanoparticles followed Fickian diffusion in 0.1 mol L(-1) HCl, while the mechanism was found anomalous in pH 7.4 phosphate buffer. An effort was also made to critically correlate the properties of nanoparticles synthesized by the above two techniques, and emphasize the importance of these characteristics in targeted drug delivery.

Enbucrilate↗
PubMedDate not supplied

Effect of polymerization modes and resin composite on the temperature rise of human dentin of different thicknesses: an in vitro study.

This in vitro study evaluated the effect of different polymerization modes and the presence of resin composite on the temperature rise (TR) in human dentin of different thicknesses. For this purpose, 90 specimens were assigned to 30 groups (n=3): five polymerization modes (1-conventional; 2-soft-start; 3-high intensity; 4-ramp cure: progressive and high intensity; 5-high intensity with the tip of the light cure at a distance of 1.3 cm for 10 seconds and the tip leaned in the sample); two levels of resin composite presence (absence or presence of resin composite) and three dentin thicknesses (1, 2, 3 mm). During polymerization, temperature was measured by a digital laser thermometer (CMSS2000-SL/SKF). Three-way ANOVA and Tukey tests were performed. There were statistical differences in TR among polymerization modes, presence of resin composite and dentin thicknesses. Within the limits of this study, it can be concluded that 1) conventional and high intensity polymerization modes presented lower TR means, and it was statistically different from soft start, distanced tip and ramp curing polymerization modes; 2) the presence of resin composite showed a statistically significant reduction TR means and 3) the thicker the dentin, the less the temperature rise.

Analysis of Variance↗
PubMed2005

[Study of transport properties of the polymeric membranous dressing with silver ions].

The transport properties of polymeric membraneous dresing silver ion containing Textus Bioactive were studied. This dressing is made of three types of theromoplastic polymeric fibers, formed into two-layers membrane. In first layer occure the polymeric fiber, which the core is hydrophobic and hydrophilic surfaces contain a silver zeolite. These fibers neighborours with hydrophilic super absorbing polymers. Third type of polymeric fibers occur in the second layer of membrane and is arranged parallel to surface's skin, creating a net preventing stick of membraneous dressing to treated wound. Using of the Kedem-Katchalsky equations the transport model of this membrane and the temporal and concentration characteristics of transport parameters (hydraulic permeability, refection and solute permeability) were determined. Experimental results show that the polymeric membranous dressing contain the silver ions posses non-linear transport properties, which are consequence of structure and physicochemical properties of polymeric membranes.

Bandages↗
PubMedDate not supplied

Principles of emulsion stabilization with special reference to polymeric surfactants.

This overview summarizes the basic principles of emulsion stabilization with particular reference to polymeric surfactants. The main breakdown processes in emulsions are briefly described. A section is devoted to the structure of polymeric surfactants and their conformation at the interface. Particular attention is given to two polymeric surfactants that are suitable for oil-in-water (O/W) and water-in-oil (W/O) emulsions. For O/W emulsions, a hydrophobically modified inulin (HMI), obtained by grafting several alkyl groups on the backbone of the inulin (polyfructose) chain, is the most suitable. For W/O emulsions, an A-B-A block copolymer of polydroxystearic acid (PHS), the A chains, and polyethylene oxide (PEO), the B chain, is the most suitable. The conformation of both polymeric surfactants at the O/W and W/O interfaces is described. A section is devoted to the interaction between emulsion droplets containing adsorbed polymer surfactant molecules. This interaction is referred to as steric stabilization, and it is a combination of two main effects, namely, unfavorable mixing of the A chains, referred to as the mixing interaction, Gmix, and loss of configurational entropy on significant overlap of the stabilizing chains, referred to as elastic interaction, Gel. The criteria for effective steric stabilization are summarized. O/W emulsions based on HMI are described, and their stability in water and in aqueous electrolyte solutions is investigated using optical microscopy. Very stable emulsions can be produced both at room temperature and at 50 degrees C. The reason for this high stability is described in terms of the multipoint anchoring of the polymeric surfactant (by several alkyl groups), the strong hydration of the inulin (polyfructose) chains, and the high concentration of inulin in the adsorbed layer. W/O emulsions using PHS-PEO-PHS block copolymer can be prepared at a high volume fraction of water, varphi, and these emulsions remain fluid up to high varphi values (> 0.6). These emulsions also remain stable for several months at room temperature and at 50 degrees C. The last two sections are concerned with the problems of creaming or sedimentation and phase inversion. Creaming or sedimentation can be prevented by the use of "thickeners" in the continuous phase. These molecules produce non-Newtonian systems that will have a high residual or zero shear viscosity. The latter, which may exceed 1000 Pas, can also be prevented by control of the bulk (or elastic) modulus of the system. Phase inversion in O/W emulsions can also be prevented using HMI, since this polymeric surfactant is not soluble in the oil phase. As long as coalescence and Ostwald ripening are prevented, the emulsions can remain stable for very long times both at room temperature and at 50 degrees C.

Cosmetics↗
PubMed1991

Resistance to antimitotic drugs in Chinese hamster ovary cells correlates with changes in the level of polymerized tubulin.

A sensitive and reproducible method to measure relative levels of polymerized and soluble tubulin in cells has been developed. This method involves metabolically labeling cells with radioactive amino acids followed by lysis in a microtubule-stabilizing buffer, centrifugation to separate soluble from polymerized tubulin, resolution of the proteins in each fraction by two-dimensional gel electrophoresis, and quantitation of the tubulin by liquid scintillation counting of spots excised from the gel. Several buffers were evaluated for their reproducibility and efficacy in preserving the state of in vivo microtubule assembly at the time of cell lysis, and the ability of the technique to measure drug-induced changes in tubulin polymerization was determined. Results using this method indicate that Chinese hamster ovary cells maintain approximately 40% of the cellular tubulin in an assembled form. Dose-dependent decreases in tubulin polymerization could be measured in Colcemid-treated cells, while dose-dependent increases in assembly were measured in taxol-treated cells. The results with taxol indicate that, following the increase in microtubule polymerization, there is a time-dependent bundling of microtubules that occurs without further increases in the extent of tubulin assembly. Examination of drug-resistant Chinese hamster ovary cells reveals that Colcemid-resistant mutants maintain more tubulin in the polymerized state (approximately 50%), while taxol-resistant mutants maintain less assembled tubulin (about 28%). Similar changes occur regardless of whether the mutant cells have an alteration in alpha- or in beta-tubulin. A model to explain these results is discussed.

Alkaloids↗
PubMed2006

One-year clinical evaluation of two resin composites, two polymerization methods, and a resin-modified glass ionomer in non-carious cervical lesions.

AIM: The aim of this study was to examine clinically relevant data on four restorative procedures for non-carious cervical lesions using United States Public Health Service (USPHS)-compatible clinical and photographic criteria and to compare different methods of analyzing clinical data. METHODS AND MATERIALS: Fourteen patients with at least one or two pairs of non-carious lesions under occlusion and a mean age of 50 were enrolled in this study. A total of 56 restorations (14 with each material) were placed by three experienced, calibrated dental practitioners. Two other experienced and calibrated practitioners, under single-blind conditions, followed up on all restorations for a period of one year. Three materials were randomly placed: a micro-hybrid composite with two polymerization methods (G1 and G2), a flowable micro-hydrid composite (G3), and a resin-modified glass ionomer (G4). Statistical analysis was performed using the Kruskall-Wallis test (p<0.05) and a Mann-Whitney U modified test with a corrected significance level. RESULTS: At the one year evaluation time, there were no restorations with secondary caries and the retention rates in G1 (IntenS with a hard polymerization), G2 (IntenS with a soft polymerization), G3 (Filtek flow), and G4 (Fuji II LC) were 85.7% (two losses), 92.8% (one loss), 100%, and 100%, respectively. The total visual comparison of the results at baseline (15 days later) showed significant differences only with the clinical acceptance criterion: G1 was different from G2, with a soft polymerization device (p<0.05). In terms of surface quality at one year, G1, G2, and G3 exhibited a statistically significant difference from G4, p<0.05. The digital analysis at baseline showed significant differences only with the clinical acceptance criterion: G1=G2 was different from G3=G4, p<0.05. At one year, only the microporosity criterion showed any statistical differences: G1=G2=G3 was different from G4, p<0.05. CONCLUSIONS: The resin-modified glass ionomer was easier to use and had a high retention rate, but it failed in terms of surface quality (visual mode) and porosity (digital mode) criteria compared to the others groups. Overall results showed no difference between groups G1 (hard-polymerized) and G2 (soft-polymerized), and only G1 was affected by the marginal edge (p<0.03) and integrity criteria (p<0.02) at one year.

Bisphenol A-Glycidyl Methacrylate↗
PubMed1990

Constant-volume polymerization of composites by addition of ammonia-modified montmorillonite.

Polymerization shrinkage is a major limitation of dental composites. It generates internal stresses within bonded restorations and leads to marginal leakage, sensitivity and recurrent decay in the absence of adequate bonding. A method is presented for formulating composites that cure at constant volume by adding to the resin system small amounts of the hydrated mineral montmorillonite (MMT), which has been modified by replacing part of its hydration water with ammonia (NH3). The polymerization exotherm of composites containing ammonia-modified montmorillonite (NH3/MMT), cured at ambient temperatures, raises their temperature to between 60 and 80 degrees C, which causes the NH3/MMT particles to swell and counteract polymerization shrinkage. The polymerization shrinkage was measured for the BIS-GMA resin system, without filler, to which had been added NH3/MMT. The addition of 4 to 5 weight percent NH3/MMT resulted in zero polymerization shrinkage. An experimental composite was formulated using the BIS-GMA resin system, hydroxyapatite filler and 4 weight percent NH3/MMT. Cured samples developed compressive strength and hardness comparable to reported values for dental composite resins. Porosity was not detected by scanning electron microscopy. The use of NH3/MMT for formulating polymeric systems that cure at constant volume should be explored for application in direct dental composites.

Bentonite↗
PubMed1989

The polymerization of fibrin prepared from fibrinogen Haifa (gamma 275Arg----His).

Fibrinogen Haifa is a congenital heterozygous fibrinogen variant (gamma 275 Arg----His) characterized by prolonged thrombin and reptilase times and normal fibrinopeptide (FPA, FPB) release. We compared the polymerization rate (by turbidity measurements at 350 nm) and the ultrastructure of Haifa alpha-, beta-, and alpha, beta-fibrin with that of normal. Haifa alpha, beta-fibrin polymerized less rapidly than did normal and formed a highly branched matrix with a smaller mean fiber diameter; this network closely resembled that of normal alpha, beta-fibrin with EDTA added. In the presence of CaCl2 (1 to 10 mM), Haifa alpha, beta-fibrin polymerized more rapidly than in buffer alone and possessed a matrix structure closely resembling that of normal fibrin. From these observations it appears that the functional defect in Haifa fibrin can be related to the inability of the abnormal molecule to effectively utilize available calcium. The polymerization profile of Haifa alpha-fibrin differed only modestly from that of normal alpha-fibrin, whereas that of Haifa beta-fibrin was markedly impaired. This finding plus similarities in the ultrastructure of Haifa and normal alpha-fibrin specimens suggests that the defective gamma chain structure of Haifa fibrinogen results in greater impairment of the carboxy terminal "b" polymerization domain reacting with the site exposed by cleavage of FPB ("B" site) than it does that of the carboxy terminal "a" domain reacting with the site exposed by cleavage of FPA ("A" site). Whether this effect is due to absolute differences in the degree of impairment of these two types of polymerization sites, or whether proper utilization of the "B" to "b" site is dependent upon participation of the "A" to "a" site remains to be determined.

Calcium↗
PubMed1978

[Polymerization shrinkage and fitness of denture (author's transl)].

Polymerization shrinkage causes dimensional change of denture during the processing. We can not control the amount of shrinkage but can control the initiation points and the direction of polymerization. Heat conductivities of dough and mould, the distance from heating or cooling place, and amount of MMA etc. have influence on the polymerization. Polymerization shrinkage mainly appears on the point where polymerize last. In the case of heat processing the shrinkage mainly appeared on the point where heat supply was slow. On the other hand in that of self-curing resin, the polymerization starts at the center of the mass when the mould is immersed in an ice water bath and the shrinkage appeared on the surface of mass. Dentures were prepared by heat processing resin conventionally heated in a water bath, heated from mucous side only on a heat plate, and self-curing fluid resin. An amount of filled light body silicone impression materials between the denture and cast was minimum in the case of the one side heat processed denture. That was middle in the self-cured. The maximum case was conventional heat cured denture.

Composite Resins↗
PubMed1988

Facilitation of Hb S polymerization by the substitution of Glu for Gln at beta 121.

In an effort to clarify the role of Glu-beta 121 of Hb S molecules in polymerization, we studied the solubility and kinetics of polymerization of various mixtures of deoxyhemoglobins S (Glu-beta 6----Val) and D Los Angeles (Glu-beta 121----Gln). It is known that patients with Hb S-D Los Angeles have a relatively severe clinical course. Mixtures of Hb S and Hb D Los Angeles polymerized after a distinct delay time, the length of which depended on the initial hemoglobin concentration and the fraction of Hb S in the mixture. There was a linear relationship between the logarithmic plot of delay time and initial hemoglobin concentration. The line for a 1:1 mixture of Hb S and Hb D Los Angeles shifted to the right of that for deoxy-Hb S by 0.08. This shift is much smaller than the shift of 0.32 for 1:1 AS mixtures. From these data, the probability factor for nucleation of S-D Los Angeles hybrid hemoglobin was calculated to be 1.16, which is higher than that of Hb S (1.0) and AS hybrid hemoglobin (0.5). The degree of co-polymerization of Hb D Los Angeles in S-D Los Angeles mixtures was similar to that of Hb A in AS mixtures. The critical concentration for the polymerization of Hb D Los Angeles was between that of Hb A and Hb Machida, which has the same amino acid substitution (Glu----Gln) at the beta 6 position. These results suggest that the protein interaction of Hb S molecules during nucleation involves at least two steps. First, the Val-beta 6 of a Hb S molecule interacts hydrophobically with the Phe-beta 85 and the Leu-beta 88 of an adjacent Hb S molecule. In the second step, Glu-beta 121 weakens the interaction with His-beta 116 and Pro-alpha 114. The substitution of Glu-beta 121----Gln may strengthen this second reaction and facilitate nucleation as well as polymerization.

Glutamates↗
PubMed1985

Characterization of peptides cleaved by plasmin from the C-terminal polymerization domain of human fibrinogen.

The C-terminal region of the fibrinogen gamma chain is known to participate in several functional interactions including fibrin polymerization. This part of the molecule is retained on the gamma chain of fragment D (FgD) when fibrinogen is digested by plasmin in the presence of calcium to produce the fragment D-fragment E (FgD X FgE) complex but is lost if FgD is prepared in the absence of calcium. In an attempt to characterize the C-terminal polymerization domain we have used three techniques to examine this further degradation of FgD following the addition of EDTA and plasmin. Analysis of the digestion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a progressive cleavage of the gamma chain to two small remnants. The polymerization-inhibitory activity of the whole digest was studied using acid-solubilized fibrin. A progressive loss of inhibitory activity was associated with gamma chain shortening, reaching greater than a 120-fold reduction at the end of digestion. The cleavage of peptides was followed by reverse-phase high performance liquid chromatography and the release of a characteristic peptide triplet was associated with gamma chain cleavage. Manual sequencing, amino acid analysis, and fast atom bombardment mass spectrometry established the three peptides as gamma 303-356, 357-373, and 374-405. These peptides have sequences in common with those peptides recently reported by other investigators to be potent polymerization inhibitors. However, when a mixture of the three peptides was added in a 200-fold molar excess to polymerizing fibrin, no inhibitory activity could be demonstrated. It is concluded that the C-terminal polymerization domain of fibrinogen may be an extended region which includes the sequence gamma 303-405, when this is contiguous with the remainder of the gamma chain.

Amino Acid Sequence↗
PubMed1987

Polymeric prodrugs.

Polymeric prodrugs can be defined as latent pharmaceutical agents which must undergo chemical or enzymatic transformation to the active or parent drug in the organism after administration. Polymeric prodrugs may also be considered special types of drug delivery systems where the drug release is realized by cleavage of a chemical bond. The concept of polymeric prodrugs finds application in the design of novel agents when pharmacokinetical modification of a parent drug is necessary, or when the aim is to achieve selective action at a target site, utilizing enzymatic activation specific for that site. The types of polymeric prodrugs synthetized in the last decade are reviewed regarding the chemical structure of the carrier backbone and the drug linkages applied. Relationship between the chemical structure, physicochemical characteristics of polymeric prodrugs, and their physiological behavior is discussed, with special regard to the bioavailability, body distribution, and rate of elimination of the carrier from the living organism. In vitro and in vivo experimental data concerning drug activation processes, as well as potential clinical applications of polymeric prodrugs, are surveyed.

Anti-Infective Agents↗
PubMed1988

Regulation of actin polymerization by membrane fraction of platelets.

We studied the interaction between the purified membrane fraction of human platelets and the polymerization of skeletal actin. The viscosity of actin was measured by the falling ball method. The fraction suppressed the polymerization of actin in the presence of 20 mM KCl and 0.4 mM EGTA. The addition of calcium ion or thrombin to the fraction did not cause suppression. A DNase I affinity column bound the membrane fraction in the presence of calcium ion. The frozen membrane fraction and the vesicles reconstituted with lipids from the platelet membrane enhanced the polymerization of actin. Trypsinized membrane fraction and the membrane fraction treated with phospolipase A2 enhanced the polymerization of actin, but membrane fraction treated with phospholipase C had no effect. The reconstituted membrane vesicles mentioned above lowered the critical concentration for actin polymerization. These findings suggested that the polymerization of intracellular actin is enhanced not only by the mobilization of calcium ion, but also by biochemical changes in the membrane lipids.

Actins↗
PubMed1988

Inhibition of an early stage of actin polymerization by actobindin.

Actobindin, a 25,000-dalton dimeric protein purified from Acanthamoeba castellanii was previously shown to form a 1:1 molar complex with both Acanthamoeba and rabbit muscle G-actin with KD values of about 5 and 7 microM, respectively, and not to interact with F-actin (Lambooy, P. K., and Korn, E. D. (1986) J. Biol. Chem. 261, 17150-17155). We now find that actobindin is a much more potent inhibitor of the early phases of polymerization of both Acanthamoeba and muscle G-actin than can be accounted for by its binding to G-actin. Actobindin inhibits the polymerization of both G-ATP-actin and G-ADP-actin, and has little, if any, effect on the rate of ATP hydrolysis that accompanies polymerization of G-ATP-actin. The kinetics of actin polymerization in the presence of actobindin are qualitatively consistent with the postulation that actobindin binds reversibly to and inhibits the elongation of an intermediate between G-actin and F-actin, perhaps a small oligomer(s) or a species in equilibrium with such an intermediate. This hypothesis implies the, at least transient, existence of an actin species with properties different from those of monomers and filaments. Actobindin may, then, provide a useful experimental tool for investigating the still relatively obscure early steps in actin polymerization. Irrespective of its mechanism of action, actobindin might serve in situ to reduce the rate of actin polymerization de novo while having relatively little effect on the rates of elongation of existing filaments or from actobindin-resistant nucleating sites.

Acanthamoeba↗
PubMed1984

Cellular origins of human polymeric and monomeric IgA: intracellular and secreted forms of IgA.

Intracellular and secreted IgA from pokeweed mitogen (PWM)-stimulated normal peripheral blood lymphocytes, from 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated peripheral blood lymphocytes of a patient with chronic lymphocytic leukemia (CLL), or from an IgA-producing human Epstein Barr virus (EBV)-transformed lymphoblastoid cell line were analyzed by molecular-sieve chromatography, electrophoresis in sodium dodecyl sulfate, and sucrose density ultracentrifugation. Fluorochrome-labeled anti-human IgA and secretory component (SC) were used as probes for the detection of polymeric IgA in individual cells. These methods demonstrated that the majority of intracellular IgA occurred in monomeric form, even when the predominant form of secreted IgA was polymeric. Sequential analyses of the IgA secreted by PWM-stimulated normal peripheral blood lymphocytes revealed that the proportion of polymeric IgA increased with the time of culture and that polymers represented the prevalent form of secreted IgA from the fifth day of culture. Although approximately one-half of TPA-stimulated CLL cells bound fluorochrome-labeled SC, only trace amounts of extracellular and intracellular polymeric IgA were detected in both culture supernatants and lysates. Culture supernatants of an IgA-secreting EBV-transformed cell line contained predominantly polymeric IgA. However, intracellular IgA was largely represented by monomers. The predominance of intracellular monomers in polymeric IgA-secreting cells suggested that the pathway of the assembly of human IgA molecules is analogous to that described for mouse IgA synthesis.

Autoradiography↗
PubMed1983

Kinetics of activation of acetyl-CoA carboxylase by citrate. Relationship to the rate of polymerization of the enzyme.

The kinetics of citrate-induced activation and polymerization (into filaments) of the 450,000-dalton protomeric form of acetyl-CoA carboxylase were compared to assess the concertedness of the two processes. Rapid-quench techniques were employed to measure the time course of activation by citrate of the carboxylase-catalyzed reaction. When enzyme was preincubated with citrate prior to initiating the steady state turnover reaction with acetyl-CoA in the rapid-quench device, the observed rate of carboxylation of acetyl-CoA was apparently linear from the moment of mixing. However, when enzyme was mixed with citrate to initiate the reaction, a lag (t1/2 = 0.7 s) occurred in the approach to steady state carboxylation rate. This lag was independent of enzyme concentration over a 230-fold range and was marginally dependent upon citrate concentration. Over the same range of enzyme concentration, polymerization of carboxylase protomers, as determined by right angle light scattering, was enzyme concentration-dependent in a manner predicted by a single protomer activation step, followed by a rate-limiting dimerization of active protomer and subsequent polymerization. Based on these results, it is concluded that activation of catalysis and the polymerization of carboxylase protomers are not concerted. Furthermore, activation of carboxylation leading to the formation of an active protomer was faster than polymerization under all conditions, and therefore precedes polymerization. It was also shown that the activation constant (Kact) for citrate is altered in a predictable manner by the accumulation of the reaction product, malonyl-CoA, the Kact increasing with increasing malonyl-CoA concentration. Additional evidence is presented indicating that this change in Kact was not caused by autophosphorylation of the enzyme under these conditions and that phosphorylation does not affect the mechanism of activation elucidated above.

Acetyl-CoA Carboxylase↗
PubMed1981

7-Chloro-4-nitrobenzeno-2-oxa-1,3-diazole actin as a probe for actin polymerization.

Lysine 372 of N-ethylmaleimide actin was specifically (60%) labeled by 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole chloride (NBD-Cl), which also reacted with lysines on cyanogen bromide fragment 17 (20%) and other undetermined residues (20%). Isolation of N-ethylmaleimide peptides and two-dimensional peptide mapping demonstrated that 90% of bound N-ethylmaleimide was attached to an adjacent residue, cysteine 373, independent of the polymerization state of actin during the labeling reaction. Formation of NBD cysteine severely inhibited lysine modification. After N-ethylmaleimide blockage of cysteine 373, lysine labeling with NBD was greatly accelerated. The kinetics of formation of fluorescent compounds were biphasic, with fluorescence decreasing upon prolonged incubation of actin in NBD-Cl. Lysine 372 of purified NBD actin reproducibly responded to polymerization by a 2.2- to 2.3-fold enhancement of fluorescence. By contrast, interaction of NBD actin with several actin-binding proteins caused only very small or undetectable changes in fluorescence intensity: 10% enhancement on myosin subfragment 1 binding, about 6% quenching by DNase I, and no change at all by tropomyosin-troponin. Despite its sensitivity to polymerization the probe did not affect it. Native and modified actin polymerized randomly indicating that the rate constants for polymerization remained the same. Labeling actin with NBD did not diminish its cofactor activity for myosin ATPase activity. Contrary to previous reports we observed that myosin subfragment 1 (single myosin heads) caused actin polymerization in the absence of salt.

4-Chloro-7-nitrobenzofurazan↗
PubMed1982

Effects of red cell membrane on the polymerization of sickle hemoglobin.

We have investigated the effect of erythrocyte membrane preparations on the rate of polymerization and the solubility of deoxyhemoglobin S. The kinetics of polymerization were monitored by measuring changes in turbidity of hemoglobin S solutions as a function of time in a temperature-controlled cuvette. The solubility of deoxyhemoglobin S, an equilibrium measurement, was determined by measuring the hemoglobin concentration of the supernatant following centrifugation of the polymerized hemoglobin S solution. The solubility of deoxyhemoglobin S was unaffected by the presence of red cell membrane ghosts, inside-out red cell vesicles, or inside-out vesicles enriched in erythrocyte spectrin and actin (Bands 1, 2, and 5) in the hemoglobin solution. Furthermore, inside-out vesicles had no significant effect on the kinetics of polymerization. The addition of erythrocyte membrane ghosts to the hemoglobin solution, in ratios approximating that of the intact red cell, increased the rate of polymerization up to 8-fold. However, when corrections were made for the excluded volume contributed by the membrane ghosts, the enhancement of the rate of polymerization was reduced to less than 3-fold. These results fail to show significant facilitation of intracellular sickling by nucleation sites on the red cell membrane.

Anemia, Sickle Cell↗