Structure and functional properties of temperature-sensitive derivatives of immobilized proteins

Polydiethylacrylamides (degree of polymerization, 13-470) containing a terminal carboxyl group were obtained by the method of radical polymerization of N,N-diethylacrylamide in the presence of mercaptoacetic acid. In the presence of 1-ethyl-(3,3-dimethylaminopropyl)-carbodiimide, these polymers reacted with ovomucoid to produce its polymeric derivatives. The values of the lower critical mixing temperature of these derivatives and the inhibitory activities of immobilized ovomucoid were determined by the length and amount of polydiethylacrylamide macromolecules bound to the molecule of ovomucoid.     

Structural and functional characteristics of various forms of red pigment of yeast Saccharomyces cerevisiae and its synthetic analog

Structural and functional characteristics of the yeast red pigment (product of polymerization of N¹-(β-D-ribofuranosyl)-5-aminoimidazole), isolated from ade1 mutant cells of Saccharomyces cerevisiae and its deribosylated derivatives (obtained by acid hydrolysis) and its synthetic pigment analogue (product of polymerization of N¹-methyl-5-aminoimidazole in vitro) were obtained. Products of in vitro polymerization were identified using mass spectrometry. The ability of these pigments to inhibit amyloid formation using insulin fibrils was compared. All the studied compounds are able to interact with amyloids and inhibit their growth. Electron and atomic force microscopy revealed a common feature inherent in the insulin fibrils formed in the presence of these compounds—they are merged into conglomerates more stable and resistant to the effects of ultrasound than are insulin aggregates grown without pigments. We suggest that all these compounds can cause coalescence of fibrils partially blocking the loose ends and, thereby, inhibit attachment of monomers and formation of new fibrils.     

Analysis of histidine-containing dipeptides, polyamines, and related amino acids by high-performance liquid chromatography: application to guinea pig brain.

A procedure is described for coupling wheat germ agglutinin to cyanogen bromide-activated Sepharose to yield a lectin affinity column of high capacity. Covalent linkage of the lectin to the insoluble matrix is carried out in the presence of a mixture of β-1,4-linked N-acetylglucosamine oligosaccharides prepared from chitin. The lectin-affinity column specifically recognizes glycoproteins containing N-acetylglucosamine residues with the capacity of binding 0.6–1.0 mg of ovomucoid per milliliter of gel. The affinity column is stable (as determined by ovomucoid binding) and shows little loss in binding capacity or specificity after repeated usage. Important characteristics for the use of this column to purify glycoproteins are described.     

Light-scattering measurement of lipid-glycoprotein interaction

Light-scattering measurements of DHPC (1,2 dihehadecyl-sn-glycero-3-phosphatidylcholine) vesicles and complex DHPC-ovomucoid were used to follow the time course of the osmotic response after mixing with solutions containing various salts and neurtral molecules. The osmotic effects of these salts and neutral molecules. on pure DHPC vesicles and vesicles formed by the complex DHPC-ovomucoid can be used to characterize the membrane permeability under the conditions of the experiment. The half-permeation times were determined in the presence and absence of the ovomucoid. The presence of ovomucoid on the vesicles appears to increase the half-permeation times of positively charged salt ions in comparison to the negatively charged dipicrylamine, (DPA^-) lipophilic ion. This phenomenon is explained in terms of complex formation thereby diminishing the available negatively charged ionic groups which presumably are involved in the transition process across the lipid vesicles. The half-permeation times for neutral molecules did not change in the presence of ovomucoid. This indicates a mechanism of transition quite different from the one characteristic for the salt ions. On the basis of this evidence and the electrostatic bonding it is concluded that the ovomucoid is bound on the polar surface of the lipid vesicles.     

Analytical chiral separation of a new quinolone compound in biological fluids by high-performance liquid chromatography

Two methods for the separation of a new racemic quinolone compound, temafloxacin (TMFX), in biological fluids by high-performance liquid chromatography (HPLC) were studied. The first method was coupling of TMFX to S-(−)-N-1-(2-naphthyl sulfonyl)-2-pyrrolidine carbonylchloride (L-NSPC). The diastereomeric derivatives were separated on a silica gel column. The second method was separation on a chiral stationary phase with an ovomucoid conjugated to aminopropyl silica gel. Two enantiospecific methods gave a satisfactory result concerning both accuracy and precision, and the second method was superior to the first one for chromatographic separation. Furthermore, the pharmacokinetics of the enantiomers after oral administration of racemic TMFX to healthy volunteers was investigated by the second method.     

Electrophoretic properties of ovomucoid

1. The nature of the electrophoretic heterogeneity of ovomucoid was investigated. Optimum resolution of the fractions on starch-gel electrophoresis occurred over a narrow range of pH and ionic strength. The pattern was not altered in the presence of 8m-urea but the bands were sharper. Ovomucoid–trypsin complex is stable at pH4·6 but dissociated in 6m-urea. 2. The two major fractions of ovomucoid were eluted from the gels. One of these was virtually free of sialic acid and the other, which contained 0·4mole of sialic acid/mole of protein, split into two components on electrophoresis after neuraminidase treatment. It was concluded that these two components, and likewise the two major fractions of ovomucoid, differ by a unit charge/mol. Differences in sialic acid content account for only part of the electrophoretic heterogeneity of ovomucoid.     

Unfolding-refolding behaviour of chicken egg white ovomucoid and its correlation with the three domain structure of the protein

The urea and heat-induced unfolding-refolding behaviours of chicken egg white ovomucoid and its four fragments representing domains I, II + III, I + II and III were systematically investigated in 0.06 M sodium phosphate buffer (pH 7.0) by difference spectral measurements. The effect of temperature on ovomucoid and its fragments was also studied in 0.05 M sodium acetate buffer (pH 5.0) and in presence of 2 M urea at pH 7.0. Intrinsic viscosity data showed that ovomucoid and its different fragments did not lose any significant amount of their structure under mild acidic conditions (pH 4.6). Difference spectral results showed extensive disruption of the native structure by urea or temperature. Isothermal transitions showed single-step for domain I, domain I + II and domain III, and two-step having one stable intermediate, for ovomucoid and its fragment representing domain II + III. However, the presence of intermediate was not detected when the transitions were studied with temperature at pH 7.0. Strikingly, the single-step thermal transitions of ovomucoid and its fragment representing domain II + III, became two-step when measured either at pH 5.0 or in presence of 2 M urea at pH 7.0. Analysis of the equilibrium data on urea and heat denaturation showed that the second transition observed with ovomucoid or domain II + III represent the unfolding of domain III. The kinetic results of ovomucoid and its fragments indicate that the protein unfolds with three kinetic phases. A comparison of three rate constants for the unfolding of intact ovomucoid with that of its various fragments revealed that domain I, II and III of the protein correspond to the three kinetic phases having rate constants 0.456, 0.120 and 0.054 min-1, respectively. These data have led us to conclude: (i) the unusual stability of ovomucoid towards various denaturants, including temperature, is due to its domain III, (ii) initiation of the folding of the ovomucoid molecule starts from its NH2-terminal region which probably provides the nucleation site for the formation of the subsequent structure and (iii) domains I and II have greater mutual recognition between them as compared to the recognition either of them have with domain III.     

Occurrence and characterization of stable intermediate state(s) in the unfolding of ovomucoid by guanidine hydrochloride

Reversible unfolding of ovomucoid by guanidine hydrochloride, as followed by viscosity and difference-spectral measurements at 25°C, pH6, occurred in two distinct steps involving at least three major conformational states, namely the native, intermediate and completely denatured states, occurring respectively in 60mm-sodium phosphate buffer, 3.5m-guanidine hydrochloride and 6m-guanidine hydrochloride. The overall native conformation of ovomucoid, as indicated by its intrinsic viscosity (5.24ml/g) and gel-filtration behaviour, differs significantly from that of a typical globular protein. Exposures of tyrosine residues in native ovomucoid measured by difference spectroscopy following perturbation with glycerol, ethylene glycol and dimethyl sulphoxide were, respectively, 0.42, 0.56 and 0.57. Of the exposed phenolic groups only one titrated normally (pK_int., 9.91, electrostatic-interaction factor, w, 0.04). Results on difference spectra, solvent perturbation, phenolic titration and intrinsic viscosity (7.4ml/g) taken together showed that, although ovomucoid in 3.5m-guanidine hydrochloride was significantly unfolded, it retained a degree of native structure, removable with 6m-guanidine hydrochloride. In the latter, all the six tyrosine residues were available for titration, and the intrinsic viscosity of ovomucoid increased to 9.4ml/g. Furthermore, the characteristic fine structures in circular-dichrosim spectra of ovomucoid, associated with the elements of native structure, were abolished in 6m-guanidine hydrochloride, suggesting that the completely denatured state is structureless and presumably behaves as a cross-linked random coil. The latter state has been shown by analysis of the results on guanidine hydrochloride-dependence of the transition, intermediatedenatured, to be less stable than the intermediate state under native conditions by about 46kJ/mol at 25°C. Attempts have been made to interpret the above results in the light of available information on the amino acid sequence of ovomucoid.     

Synthesis,cytotoxic evaluation and molecular docking study of 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazoles as tubulin polymerization inhibitors

A series of cis-restricted 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazole analogues of combretastatin A-4 were synthesized and investigated for inhibition of cell proliferation against three cancer cell lines, HT-29, MCF-7, and AGS, and a normal mouse fibroblastic cell line, NIH-3T3, using an MTT assay. The biological study showed that 2-(methylthio) substituted compounds showed little cytotoxic activity against the four cell lines. In contrast, the presence of the 2-(benzylthio) group on the thiazole ring resulted in a significant improvement in cytotoxic activity relative to the 2-(methylthio) substituted derivatives. Furthermore, the inhibition of tubulin polymerization by some potent compounds was evaluated. All the compounds studied were moderate tubulin polymerization inhibitors. The flow cytometry analysis confirmed that the synthesized compounds led to cell cycle arrest at the G₂/M phase. Docking simulation was performed to insert these compounds into the crystal structure of tubulin at the colchicine binding site to determine a probable binding model.     

A combined approach of experiments and computational docking simulation to the Coprinus cinereus peroxidase-catalyzed oxidative polymerization of alkyl phenols

The characteristics of the oxidative polymerization of alkyl phenol derivatives catalyzed by Coprinus cinereus peroxidase (CIP) were studied qualitatively and quantitatively using a combined approach of experiments and computational docking simulations. As determined by docking study of CIP and alkyl phenols, the binding interaction was found to be important for the determination of substrate specificity. The distant binding and indirect orientation of o-isopropyl phenol and o-tertiary butyl phenol to the catalytic residue (56His) could explain the inability of CIP to polymerize these substrates. Three hydrophobic residues (156Pro, 192Leu, and 230Phe) at the entrance of the binding pocket were also found to be crucial in binding and orientation of alkyl phenols. A two-parameter QSAR equation with the binding distance and the molecular volume of the substrates was proposed and the polymerization yield was accurately predicted by two-parameter QSAR equation.     

Ovomucoid domains: preparation and physico-chemical characterization

Four fragments of ovomucoid representing its individual domains and their different combinations were prepared by peptic and cyanogen bromide cleavages of the protein. The fragments corresponding to domains I + II, II + III, I and III of the parent ovomucoid molecule, were found to be homogeneous by gel filtration and polyacrylamide gel electrophoresis in presence and absence of SDS. Various physico-chemical properties of these proteins, such as molecular weight, NH2- and COOH-terminal amino acid residues, sugar content, isoionic pH, specific extinction coefficient, fluorescence emission spectra, intrinsic viscosity, frictional coefficient, Stokes radius, diffusion coefficient and geometrical mean radius were determined. Analysis of the results on trypsin inhibitory activity of ovomucoid and its different fragments suggested that only domain II is involved in the antitryptic activity of the inhibitor. Optical characteristics of these fragments indicate that they are devoid of tryptophan residues. The hydrodynamic properties suggest that intact ovomucoid and two of its fragments (domain I + II and domain II + III) are significantly different from those of typical globular proteins and are asymmetric in nature. However, the shape of the two remaining fragments representing domains I and III of the intact protein appeared to be compact and globular. Furthermore, domain II of ovomucoid has been suggested to primarily contribute towards the apparent asymmetry in the intact protein.     

Intracellular routing of GLcNAc-bearing molecules in thyrocytes: selective recycling through the Golgi apparatus

Previous experiments led us to speculate that thyrocytes contain a recycling system for GlcNAc-bearing immature thyroglobulin molecules which prevents these molecules from lysosomal degradation (Miquelis, R., C. Alquier, and M. Monsigny. 1987. J. Biol. Chem. 262:15291-15298). To confirm this hypothesis, the fate of GlcNAc-bearing proteins after internalization by thyrocytes was monitored and compared to that of fluid phase markers. Kinetic internalization studies were performed using 125I-GlcNAc-BSA and 131I-Man-BSA. We observed that the apparent intake rate as well as the amount of hydrolyzed GlcNAc-BSA are smaller than the corresponding values for Man-BSA. These differences were reduced by GlcNAc competitors (thyroglobulin and ovomucoid) or a weak base (chloroquine). Part of the internalized GlcNAc-BSA was released into the extracellular milieu at a higher rate and shorter half life (t1/2 = approximately 30 min) than the Man-BSA (t1/2 = approximately 8 h). Subcellular homing was first studied by cell fractionation after internalization using 125I-ovomucoid and 131I-BSA. During Percoll density gradient fractionation, endogenous thyroperoxidase was used to separate subsets of organelles involved in the biosynthetic exocytotic pathway. Incubation of the cell homogenate in the presence of DAB and H2O2 before cell fractionation give rise to a shift in the density of organelles containing 3.5 times more ovomucoid than BSA. Discontinuous sucrose gradient showed that: (a) thyroperoxidase was colocalized with galactosyltransferase-contraining organelles in Golgi-rich subfractions; and (b) that at every time studied from 10 to 100 min, the ovomucoid/BSA ratio was higher in these organelles than in other subfractions. Finally we also observed that: (a) ovomucoid sequestered in the Golgi-rich subfraction incorporated [3H]galactose; and (b) that part of internalized ovomucoid was localized on the Golgi stacks as well as elements of the trans-Golgi, as revealed by immunogold labeling on ultrathin cryosections. These data prove that in thyrocytes GlcNAc accessible sugar moieties on soluble internalized molecules are sufficient to trigger their recycling via the Golgi apparatus.     

Design,synthesis and antiplasmodial activity of novel imidazole derivatives based on 7-chloro-4-aminoquinoline

A series of short chain 4-aminoquinoline-imidazole derivatives have been synthesized in one pot two step multicomponent reaction using van leusen standard protocol. The diethylamine function of chloroquine is replaced by substituted imidazole derivatives containing tertiary terminal nitrogen. All the synthesized compounds were screened against the chloroquine sensitive (3D7) and chloroquine resistant (K1) strains of Plasmodium falciparum. Some of the compounds (6, 8, 9 and 17) in the series exhibited comparable activity to CQ against K1 strain of P. falciparum. All the compounds displayed resistance factor between 0.09 and 4.57 as against 51 for CQ. Further, these analogues were found to form a strong complex with hematin and inhibit the β-hematin formation, therefore these compounds act via heme polymerization target.     

Substituted 1,6-diphenylnaphthalenes as FtsZ-targeting antibacterial agents

Bacterial cell division occurs in conjunction with the formation of a cytokinetic Z-ring structure comprised of FtsZ subunits. Agents that disrupt Z-ring formation have the potential, through this unique mechanism, to be effective against several of the newly emerging multidrug-resistant strains of infectious bacteria. Several 1-phenylbenzo[c]phenanthridines exhibit notable antibacterial activity. Based upon their structural similarity to these compounds, a distinct series of substituted 1,6-diphenylnaphthalenes were synthesized and evaluated for antibacterial activity against Staphylococcus aureus and Enterococcus faecalis. In addition, the effect of select 1,6-diphenylnaphthalenes on the polymerization dynamics of S. aureus FtsZ and mammalian β-tubulin was also assessed. The presence of a basic functional group or a quaternary ammonium substituent on the 6-phenylnaphthalene was required for significant antibacterial activity. Diphenylnaphthalene derivatives that were active as antibiotics, did exert a pronounced effect on bacterial FtsZ polymerization and do not appear to cross-react with mammalian tubulin to any significant degree.     

Trypsin inhibition activity of heat-denatured ovomucoid: a kinetic study

A kinetic study was conducted on the effect of heating in the temperature range of 75-110 degrees C on the trypsin inhibition activity of ovomucoid. Heat treatment of isolated ovomucoid resulted in a time-dependent decrease in trypsin inhibition activity that could accurately be described by a first-order kinetic model. The magnitude and the temperature dependence of the rate constants was affected by the pH during heat treatment. The heat stability of ovomucoid was the lowest at pH 7.6. Heat treatments intended to decrease the trypsin inhibition activity should therefore be carried out as soon as possible after laying, because the ovomucoid was inactivated faster at the pH of fresh egg white (pH 7.6). The presence of the other egg white constituents decreased the heat stability of ovomucoid compared to that of the model system of ovomucoid in buffer, presumably by the formation of ovomucoid-lysozyme complexes in the former.     

Preparation of polysaccharide supramolecular films by vine-twining polymerization approach

In this study, we investigated the preparation of polysaccharide supramolecular films through the formation of inclusion complexes by amylose in vine-twining polymerization using carboxymethyl cellulose-graft-poly(?-caprolactone) (CMC-g-PCL) as a new guest polymer. First, hydrogels were prepared by phosphorylase-catalyzed enzymatic polymerization in the presence of CMC-g-PCL according to the vine-twining polymerization manner. The XRD result of a powdered sample obtained by lyophilization of the resulting hydrogel indicated the presence of inclusion complexes of amylose with the PCL graft-chains between intermolecular (CMC-g-PCL)s, which acted as supramolecular cross-linking points for the hydrogelation. Then, the supramolecular films were obtained by adding water to the powdered samples, followed by drying. The mechanical properties of the selected films examined by tensile testing were superior to those of a CMC film. The effect of the supramolecular cross-linking structures on the mechanical properties of the films was evaluated further by several investigations.     

Galactosylated derivatives of low-molecular-weight chitosan: Obtaining and properties

Chitosan, a binary heteropolysaccharide consisting of 2-acetamide-2-deoxy-β-D-glucopyranose and 2-amino-2-deoxy-β-D-glucopyranose residues linked in different proportions via β-glycosidic bonds. The presence of a primary amino group in the chitosan structure allows for the synthesis of various derivatives. The procedure of obtaining activated N-hydroxysuccinimide esters with the use of lactobionic acid was applied to obtain galactosylated derivatives of low-molecular-weight chitosan with a substitution degree varying from 8 to 23%. The properties of these derivatives (viscosity, solubility, and biodegradability) were studied. These derivatives are well soluble at pH values greater than the acidity constant of amino groups of chitosan (6.5). Broadening the pH range towards increase and the presence of galactose residues allows these derivatives to be used in working with biological objects.     

Blocking effect of trypsin associated with ovomucoid on the antibody binding to ovomucoid

Ovomucoid-trypsin association complex was prepared by incubating chicken egg white ovomucoid with bovine trypsin. The reactivity of ovomucoid-trypsin complex was investigated by immunodiffusion, quantitative precipitation and enzyme-linked immunosorbent assay (ELISA). It was demonstrated that the association of trypsin with ovomucoid hindered the binding of the specific antibody at some antigenic sites of ovomucoid by lowering the antibody-binding affinity of these sites. The anti-ovomucoid antiserum was absorbed with ovomucoid-trypsin complex, and non-absorbed antibody was collected by immunoaffinity chromatography of ovomucoid-coupled Sepharose 4B. The antibody blocked the trypsin-inhibitory activity of ovomucoid in a molar ratio (antibody/ovomucoid) of about 1.2:1. The findings suggested that at least one antigenic site is located near the reactive site of trypsin inhibition (Arg89 decreases Ala90) of ovomucoid.     

Isolation and characterization of the chicken ovomucoid gene.

The chicken ovomucoid gene has been isolated by screening a chicken DNA library with a plasmid containing ovomucoid mRNA sequences. Twelve recombinant phages carrying ovomucoid mRNA sequences were isolated. Two of them, extending farthest into the 5' and 3' direction respectively, were characterized by restriction mapping and Southern hybridization as well as by electron microscopic analysis of hybrids between the cloned DNA and ovomucoid mRNA. Seven intervening sequences interrupt the ovomucoid mRNA sequence in chromosomal DNA. From these data a minimal size of 5.6 kb can be estimated for the length of the ovomucoid gene.