Characterization Of Complex Formation Research Articles

Curcumin complexation with cyclodextrins by the autoclave process: Method development and characterization of complex formation

One approach to enhance curcumin (CUR) aqueous solubility is to use cyclodextrins (CDs) to form inclusion complexes where CUR is encapsulated as a guest molecule within the internal cavity of the water-soluble CD. Several methods have been reported for the complexation of CUR with CDs. Limited information, however, is available on the use of the autoclave process (AU) in complex formation. The aims of this work were therefore to (1) investigate and evaluate the AU cycle as a complex formation method to enhance CUR solubility; (2) compare the efficacy of the AU process with the freeze-drying (FD) and evaporation (EV) processes in complex formation; and (3) confirm CUR stability by characterizing CUR:CD complexes by NMR, Raman spectroscopy, DSC, and XRD. Significant differences were found in the saturation solubility of CUR from its complexes with CD when prepared by the three complexation methods. The AU yielded a complex with expected chemical and physical fingerprints for a CUR:CD inclusion complex that maintained the chemical integrity and stability of CUR and provided the highest solubility of CUR in water. Physical and chemical characterizations of the AU complexes confirmed the encapsulated of CUR inside the CD cavity and the transformation of the crystalline CUR:CD inclusion complex to an amorphous form. It was concluded that the autoclave process with its short processing time could be used as an alternate and efficient methods for drug:CD complexation.

Electrodeposition of Ni–W–B nanocomposite from tartrate electrolyte as alternative to chromium plating

The effect of the bath chemistry and operating conditions on the chemical composition, microstructure and properties of Ni–W–B alloys deposited from tartrate baths on working electrode was studied for the first time by the pulsed current method. The investigations included the measurement of the current efficiencies and determination of the tungsten content in the electrodeposits. UV spectrometry was used for characterisation of complex formation. The grain size of deposits was determined by XRD. Also, the morphology of the deposits was studied by SEM. Amorphous Ni–W–B alloys were successfully obtained by electrodeposition from the tartrate bath. The corrosion behaviour of Ni–W–B coating on mild steel has been evaluated during exposure to 3·5%NaCl solution by electrochemical impedance spectroscopy. The electrochemical and corrosion behaviour of Ni–W–B alloy showed a good corrosion resistance in the 3·5%NaCl medium. The microhardness of nanocrystaline Ni–W–B is in the range between 700 and 850 HV.

Molecular Features Governing the Stability and Specificity of Functional Complex Formation by Mycobacterium tuberculosis CFP-10/ESAT-6 Family Proteins

The Mycobacterium tuberculosis complex CFP-10/ESAT-6 family proteins play essential but poorly defined roles in tuberculosis pathogenesis. In this article we report the results of detailed spectroscopic studies of several members of the CFP-10/ESAT-6 family. This work shows that the CFP-10/ESAT-6 related proteins, Rv0287 and Rv0288, form a tight 1:1 complex, which is predominantly helical in structure and is predicted to closely resemble the complex formed by CFP-10 and ESAT-6. In addition, the Rv0287.Rv0288 complex was found to be significantly more stable to both chemical and temperature induced denaturation than CFP-10.ESAT-6. This approach demonstrated that neither Rv0287.Rv0288 nor the CFP-10.ESAT-6 complexes are destabilized at low pH (4.5), indicating that even in low pH environments, such as the mature phagosome, both Rv0287.Rv0288 and CFP-10.ESAT-6 undoubtedly function as complexes rather than individual proteins. Analysis of the structure of the CFP-10.ESAT-6 complex and optimized amino acid sequence alignments of M. tuberculosis CFP-10/ESAT-6 family proteins revealed that residues involved in the intramolecular contacts between helices are conserved across the CFP-10/ESAT-6 family, but not those involved in primarily intermolecular contacts. This analysis identified the molecular basis for the specificity and stability of complex formation between CFP-10/ESAT-6 family proteins, and indicates that the formation of functional complexes with key roles in pathogenesis will be limited to genome partners, or very closely related family members, such as Rv0287/Rv0288 and Rv3019c/Rv3020c.

Development of a new electroplating process for Ni–W alloy deposits

In the present work, the effect of the complexing agents citrate, glycine and triethanolamine (TEA) on the electrodeposition of Ni–W layers from electrolytes based on NiSO 4 and Na 2WO 4, is investigated. The investigations include measurement of the current efficiencies, determination of the tungsten content in the electrodeposits (by energy dispersive X-ray analysis), voltammetry studies and characterization of complex formation by UV-spectrometry. High W content and current efficiency could be realized by using electrolytes containing all of the three complexing agents. The results show that small amounts of glycine in a citrate–triethanolamine based electrolyte positively influences both the mass-transfer and the deposition potential.

Characterisation of complex formation between members of theMycobacterium tuberculosiscomplex CFP-10/ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility

Characterisation of complex formation between members of the<i>Mycobacterium tuberculosis</i>complex CFP-10/ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility

Characterisation of complex formation between members of the Mycobacterium tuberculosis complex CFP-10/ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility

We have previously shown that the secreted M. tuberculosis complex proteins CFP-10 and ESAT-6 form a tight, 1:1 complex, which may represent their functional form. In the work reported here a combination of yeast two-hybrid and biochemical analysis has been used to characterise complex formation between two other pairs of CFP-10/ESAT-6 family proteins (Rv0287/Rv0288 and Rv3019c/Rv3020c) and to determine whether complexes can be formed between non-genome paired members of the family. The results clearly demonstrate that Rv0287/Rv0288 and Rv3019c/3020c form tight complexes, as initially observed for CFP-10/ESAT-6. The closely related Rv0287/Rv0288 and Rv3019c/Rv3020c proteins are also able to form non-genome paired complexes (Rv0287/Rv3019c and Rv0288/Rv3020c), but are not capable of binding to the more distantly related CFP-10/ESAT-6 proteins.

Cationized human serum albumin as a non-viral vector system for gene delivery? Characterization of complex formation with plasmid DNA and transfection efficiency

Cationized human serum albumin (cHSA) could serve as a potential non-viral vector system for gene delivery. Native human serum albumin was cationized by covalent coupling of hexamethylenediamine to the carboxyl groups resulting in a shift of the isoelectric point from pH 4–5 to 7–9. The cationized albumin underwent spontaneous self-assembly with DNA as demonstrated by retardation of CMV-nlacZ plasmid in agarose gel electrophoresis. Photon correlation spectroscopy showed a decrease of complex size with increasing cHSA/plasmid ratios. Under optimized conditions complexes were formed with 230–260 nm mean diameter and a homogenous, narrow size distribution. At room temperature complexes were stable in 0.9% sodium chloride solution pH 7.4 for 1 h without aggregation. Process parameters such as albumin concentration, incubation time, temperature, pH, order of reagent addition, the presence of bivalent ions and the ionic strength of the complexation medium all influenced the complex size. Confocal laser scanning microscopy showed interactions of a Texas Red labeled cationized albumin with cell membranes of ECV 304 cells and an enhanced endocytic uptake compared to native albumin. The potential for introducing exogeneous DNA into cells was shown using NIH 3T3 fibroblasts. Successful, albeit low reporter gene expression could be achieved in the presence of chloroquine. Under in vitro conditions no toxic effect could be observed. In conclusion, cationized albumin may have promise as a non-toxic vector for gene delivery, especially for DNA vaccination.

Cationic lipid binding to DNA: characterization of complex formation.

We recently demonstrated that cationic lipids, added in monomer or micellar form, bind to DNA, resulting in the formation of a hydrophobic complex. This complex can serve as a well-defined intermediate in the preparation of DNA-lipid particles (DLPs) with many potential applications for delivery of polynucleotides in vitro and in vivo. To develop a better understanding of the factors governing complex formation, we have characterized the cationic lipid/DNA binding reaction. This was evaluated by measuring DNA and cationic lipid (DODAC) complex formation using the Bligh and Dyer extraction procedure. Efficient recovery of DNA (> 95%) in the organic phase was achieved when sufficient monocationic lipids interact with DNA phosphate groups. The rate of binding depends on the amount of DNA or cationic lipid present in the system. The time required to generate the hydrophobic complex was increased when < 10 micrograms of DNA or < 40 nmol of DODAC was present. Surprisingly, the rate of complex formation was contingent on the incubation period after partitioning the DNA/lipid mixture into organic and aqueous phases. These results suggest that the cationic lipid/DNA complex forms at the aqueous/organic interface and that DNA/lipid binding is dependent on multivalent interactions at this interface. A Scatchard analysis of DNA/DODAC binding demonstrated that the binding reaction exhibits a high degree of positive cooperativity. The apparent dissociation constant (Kn), using data obtained under conditions where DODAC binding to DNA approached saturation, indicated a high-affinity reaction (Kn > 10(-11) mol L-1). At this point, approximately 8400 mol of DODAC was bound per mole of DNA, which is equivalent to a charge ratio (+/-) of 0.585 for the 7.2 kb plasmid used and suggests that formation of the hydrophobic complex occurs at a stage prior to charge neutralization. The influence of other lipids on DNA/cationic lipid binding at the aqueous/organic interface was also studied. Cholesterol and DOPC had little effect on DNA/DODAC binding while the anionic lipids LPI, DOPS, and DMPG inhibited complex formation. The zwitterionic lipid DOPE, however, had a concentration-dependent effect on cationic lipid binding that was also dependent on the mixing order. We believe that this approach for evaluating lipid/DNA binding provides an effective procedure for assessing factors which control the dissociation of lipids from DNA and may be beneficial in the selection of lipids for effective use in gene transfection studies.

Characterization of complex formation by humanized anti-IgE monoclonal antibody and monoclonal human IgE.

The interaction of human IgE with high-affinity IgE Fc receptors on cells of the immune system plays an essential role in the type I hypersensitivity reaction. A proposed therapy is to use an anti-IgE monoclonal antibody to block the binding of IgE to its high-affinity receptor on mast cells and basophils, thus preventing subsequent release of the inflammatory agents after exposure to allergen. We report here the solution characteristics of immune complexes formed by a humanized anti-IgE monoclonal antibody (rhuMAb E25) and IgE using sedimentation analysis and size exclusion chromatography. We demonstrate that the rhuMAb E25 is able to form a variety of complexes with IgE at different molar ratios. The largest complex was identified by sedimentation equilibrium analysis as a heterohexamer with very high stability. The intermediate complex formed when one of the interacting components is in large molar excess appears to have a trimeric structure. The high-affinity interaction of rhuMAb E25 and IgE has also been confirmed. Furthermore, by using hydrodynamic modeling, we show that the largest complex may be represented by a cyclic structure.

Characterization of complex formation between G i2 and octyl glucoside solubilized neutrophil N-formyl peptide chemoattractant receptor by sedimentation velocity

The reversible formation of complexes between N-formyl peptide chemoattractant receptor (FPR) and G i2 protein was analyzed by velocity sedimentation in linear sucrose density gradients. FPR complexed with heterotrimeric G i2, sediments at different rate than uncomplexed FPR and the two forms have apparent sedimentation coefficients of 7S and 4S, respectively. The biochemical variables important for the reconstitution of the 7S complex from the 4S receptor and G i2 were studied. The formation of 7S was saturable with G i2 and addition of excess G i did not cause oligomerization. The reconstituted 7S complex was stable under a variety of conditions including octyl glucoside concentrations below and above the critical micellar concentration. The optimum pH for the reconstitution is between 7 and 9, where the 4S and 7S species sedimented reproducibly, at distinct positions in the gradient. Below pH 6 both the 4S and the 7S species appear to undergo denaturation and form precipitates. Magnesium ions have no significant effect on the sedimentation of either forms of FPR. Reconstitution was stable up to a NaCI concentration of 0.2 M. At 1 M NaC1 reconstitution was inhibited and at 3 M salt FPR aggregated. Since guanine nucleotides GTP, GTPγS, GDPβS selectively dissociated the 7S complex in a concentration-dependent manner and adenine nucleotides had no effect, we conclude that the FPR-G i2 system displays a vacant guanyl nucleotide binding site, the hallmark of a functional guanine nucleotide exchange complex. Moreover, our results indicate that the reconstitution of FPR-Gi2 complexes is reproducible at physiologically relevant conditions, shows selectivity, specificity, and biochemically functional properties consistent with a specific and functional interaction between solubilized FPR and G protein.

Characterization of complex formation between lipopolysaccharide and lysozyme

The binding of lysozyme (LZM) to bacterial lipopolysaccharide (LPS) inhibited the biological activities of LPS as well as the enzymic activity of LZM. The mode of binding has been characterized by using dansylated LZM and enzyme inhibition. The binding of LPS to LZM significantly increased the fluorescence intensity (Fl-intensity) of the danyl group and was found to be time-dependent; the complex was produced gradually and became stabilized within 20 min at 37°, 10 min at 50°, and 1 min at 70°. The maximum level of binding was also dependent on the reaction temperature, and more complex was formed at higher temperatures. Complexation was strongly dependent on the salt concentration and was not observed at >0.5 m NaCl. From collected evidence of the Fl-intensities of various dansyl derivatives and amphiphiles, it is concluded that LZM interacts with LPS by multiple binding-modes, the first being strongly related to the enzyme inhibition, the second being close to the Fl-intensity, and the third being dependent on the inhibition of immunopharmacological activities. For the amphiphiles used in this study, sdium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-propanesulfonate (CHAPSO), decansulfonic acid, and cardiolipin have binding modes similar to that of LPS.

Effect of 2-hydroxypropyl-beta-cyclodextrin on the ocular absorption of dexamethasone and dexamethasone acetate.

Complexation of dexamethasone (DX) and dexamethasone acetate (DXA) with 2-hydroxypropyl-beta-cyclodextrin (HPCD) was investigated with an ultimate goal of formulating a topical ophthalmic solution of DXA. Aqueous solubility of DX and DXA was markedly increased due to formation of soluble inclusion complexes with HPCD. Based on characterization of complex formation by phase solubility and UV-spectroscopy methods, a stoichiometry of 1:1 and 1:1, 1:2 was assumed for DX-HPCD and DXA-HPCD complexes, respectively. The stability constants for complex formation estimated by phase solubility and UV-spectroscopy methods, respectively, were as follows: for DX-HPCD complex, K1:1 = 2193 and 2221 M-1; and for DXA-HPCD complex, K1:1 = 2240 and 2445 M-1 and K1:2 = 3 and 17 M-1. K1:1 of 2266 M-1 and K1:2 of 20 M-1 were also estimated for the DXA-HPCD complex by kinetics. The kinetics of DXA degradation in pH 7 phosphate buffer at 25 degrees C followed pseudo first order. The addition of HPCD decreased the rate but the order of reaction remained unchanged. Free DXA degraded at a faster rate than complexed DXA. Ocular bioavailability in conjunctiva, cornea, iris, and aqueous humor postadministration of a 25-microliters dose of formulations containing an equivalent of 0.1% (w/v) DX followed a rank-order of DXA-HPCD solution greater than DXA suspension greater than DX-HPCD solution greater than DX suspension.