Cross-linking and gel formation of water-soluble lysine polypeptides. An insolubilization model reaction for adhesive proteins.

Insolubilizing studies of water-soluble synthetic polypeptides containing lysine residues were examined using organic aliphatic and aromatic cross-linking agents such as dialdehydes, diacyl chlorides and diactive ester, together with an enzyme tyrosinase, in water and simulated seawater systems. The cross-linking reaction was characterized by the viscosity and turbidity changes. Among the organic cross-linking agents used aliphatic glutaraldehyde and aromatic o-phthalaldehyde were the most effective. When excess organic cross-linking agents were added to the lysine polypeptide systems, the corresponding solid gels were formed. As a whole, the molecular weight of the samples, the amino acid compositions, the cross-linking agent used, the molar ratios between cross-linking agents and functional residues and system pH were found to have roles in the insolubilizing reaction and the gel formation. The cross-linking results obtained were compared with those of the polypeptide-tyrosinase systems, whose deep brownish red colour was decolorized by the addition of L-ascorbic acid.     

Polymerization of lignin fragments contained in a model effluent by polyphenoloxidases and horseradish peroxidase/hydrogen peroxide system*

An effluent containing soluble lignin fragments was treated with potato-polyphenoloxidases (PPO) or horseradish peroxidase/hydrogen peroxide system (HRP/H(2)O(2)). In both cases the reaction was evidenced by the formation of a brown precipitate that was a consequence of the polymerization of lignin fragments. The effect of reaction time, pH, and amount of soluble lignin per unit of enzyme activity on the insolubilization yield was evaluated for PPO-initiated reactions. For HRP-initiated reactions, the amount of H(2)O(2) per unit of enzyme activity was also evaluated. Mathematical models were calculated to predict the insolubilization yield as a function of the significant variables. Based on these models, the insolubilization reaction was optimized and reached maximal values of ca. 50% in both reaction systems. Higher insolubilization yields were not achieved. Chemical characterization of the soluble lignin fragments indicated that the insolubilization yield could not be improved, because the lignin fragments had limited amounts of free phenolic substructures available for the initial steps of the polymerization reaction.     

Rapid cross-linking of elastin-like polypeptides with (hydroxymethyl)phosphines in aqueous solution

In situ gelation of injectable polypeptide-based materials is attractive for minimally invasive in vivo implantation of biomaterials and tissue engineering scaffolds. We demonstrate that chemically cross-linked elastin-like polypeptide (ELP) hydrogels can be rapidly formed in aqueous solution by reacting lysine-containing ELPs with an organophosphorous cross-linker, beta-[tris(hydroxymethyl)phosphino]propionic acid (THPP) under physiological conditions. The mechanical properties of the cross-linked ELP hydrogels were largely modulated by the molar concentration of lysine residues in the ELP and the pH at which the cross-linking reaction was carried out. Fibroblasts embedded in ELP hydrogels survived the cross-linking process and were viable after in vitro culture for 3 days. DNA quantification of ELP hydrogels with encapsulated fibroblasts indicated that there was no significant difference in DNA content between day 0 and day 3 when ELP hydrogels were formed with an equimolar ratio of THPP and lysine residues of the ELPs. These results suggest that THPP cross-linking may be a biocompatible strategy for the in situ formation of cross-linked hydrogels.     

Comparison of helix stabilities of poly-L-lysine, poly-L-ornithine, and poly (L-diaminobutyric acid)

The helix–coil transitions of aqueous solutions of poly-α-L -lysine (PLL), poly-α-L -ornithine (PLO), and poly(α,γ-L -diaminobutyric acid) (PLDBA) have been investigated as functions of pH at 25°C and of temperature at pH 11.75, where these polymers are uncharged; in the cases of the latter two polyamino acids, the transitions have also been studied as functions of apparent pH in methanol-water solution (50/50 by volume). The helix stability of the polypeptides is shown to be a direct function of the number of methylene groups on the side chain. From an analysis of potentiometric titration data, we find that the difference between the helix stability of PLL and that of PLO is due to a difference of about 1 e.u. in the ΔS° of the transition. Combining the “melting curves” obtained from optical rotatory dispersion studies with the potentiometric titration data permits evaluation of the initiation parameter Z (or 1/σ^½) of the statistical mechanical theories for these transitions. The value obtained for Z in the case of uncharged aqueous PLO is ca. 35.     

Effect of chain length and concentration of anionic surfactants on the conformational transitions of poly(L-ornithine) and poly(L-lysine) in aqueous solution

The conformation of poly(L-ornithine) (PLO) and poly(L-lysine) (PLL) in solutions of sodium alkyl sulfates, CH₃(CH₂)_nSO₄Na with n = 7, 9, 11, 13 and 15 was studied by circular dichroism. PLO adopts a helical conformation in all 5 homologs and PLL a β-form in only 4 of the homologs. With octyl sulfate PLL has a helical conformation instead. These conformations were observed in solution of surfactants both below and above the critical micelle concentration.     

Tunable DNA release from cross-linked ultrathin DNA/PLL multilayered films

A novel ultrathin enzymatically degradable multilayered film using DNA as building blocks was fabricated by the layer-by-layer (LbL) technique. The UV-vis spectrometry and AFM experiments showed that the buildup of DNA and poly-L-lysine (PLL) was a kind of "exponentially growing films". The dye adsorption experiment suggested that the DNA molecules in the multilayered films were effectively protected by PLL. The films were further cross-linked by glutaraldehyde (GA). The cross-linking density of the films was modulated through the simple controlling of the time of the GA incubation process. An in vitro enzymatic degradation was carried out to investigate the DNA release profiles. The UV-vis spectrometry and fluorescence measurements indicated that the DNA release profiles were accordingly changed with the cross-linking density of the films. The nanoscale, easily processed enzymatically biodegradable PLL/DNA film with the ability to precisely control DNA release profiles may serve as a novel DNA delivery system, which may have great potential for gene therapy applications in implantable materials and biomedical devices.     

DNA cross-linking and sequence selectivity of aziridinylbenzoquinones: a unique reaction at 5'-GC-3' sequences with 2,5-diaziridinyl-1,4-benzoquinone upon reduction.

Several bifunctional alkylating agents of the aziridinylbenzoquinone class have been evaluated as potential antitumor agents. 3,6-Bis[(2-hydroxyethyl)amino]-2,5- diaziridinyl-1,4-benzoquinone (BZQ), 2,5-diaziridinyl-1,4-benzoquinone (DZQ), 3,6-bis(carboxyamino)-2,5-diaziridinyl- 1,4-benzoquinone (AZQ), and six analogues of AZQ have been studied for their ability to induce DNA interstrand cross-linking, as measured by an agarose gel technique, and to determine whether they react with DNA in a sequence-selective manner, as determined by a modified DNA sequencing technique. At an equimolar concentration (10 microM), only DZQ and BZQ showed any detectable cross-linking at pH 7 without reduction. Cross-linking was enhanced in both cases at low pH (4). Reduction by ascorbic acid at both pH's increased the cross-linking, which was particularly striking in the case of DZQ. In contrast, AZQ and its analogues only produced a significant level of cross-linking under both low-pH and reducing conditions, the extent of cross-linking decreasing as the size of the alkyl end group increased. The compounds reacted with all guanine-N7 positions in DNA with a sequence selectivity similar to other chemotherapeutic alkylating agents, such as the nitrogen mustards, although some small differences were observed with BZQ. Nonreduced DZQ showed a qualitatively similar pattern of reactivity to the other compounds, but on reduction (at pH 4 or 7) was found to react almost exclusively with 5'-GC-3' sequences, and in particular, at 5'-TGC-3' sites. A model to explain this unique reaction is proposed.     

Photodynamic protein cross-linking

Exposure of spectrin to visible light in the presence of a photosensitizer results in photo-oxidation of sensitive amino acid residues and covalent cross-linking of the polypeptides. In a previous paper the cross-linking was ascribed to a secondary reaction between photo-oxidized histidine residues and amino groups. The following observations, described in this paper, are in accordance with this supposition. (1) During illumination of spectrin in the presence of a photosensitizer a pronounced photo-oxidation of histidine residues takes place. (2) Simultaneously a decrease of free amino groups is observed. (3) Semicarbazide protects against cross-linking and is bound to a histidine photo-oxidation product in spectrin. (4) The pH profile of histidine photo-oxidation and subsequent reaction with amino groups is similar to the pH profile of spectrin cross-linking. Amidination of NH₂ groups in spectrin does not inhibit cross-linking, as visualized by gel electrophoresis. On the other hand aminidation of denatured myoglobin causes a 50% inhibition of cross-linking. These observations support the notion of NH₂-involvement in cross-linking but also demonstrate, that other photodynamic cross-linking mechanisms exist.     

Effect of transformation of chicken cells by Rous sarcoma virus on in vitro phosphorylation of nuclear non-histone proteins

Mouse macrophages were treated with 42 different glycans in vitro. Macrophages were stimulated—as judged by morphology, cell size, 5′-nucleotidase activity and the incorporation of [¹⁴C]glucosamine by some insoluble glycans, e.g. yeast glucan. Not all insoluble glycans were stimulatory (e.g. chitin). Laminaran, with a monosaccharide content and bonding pattern similar to that of yeast glucan, could be rendered stimulatory by cross-linking and insolubilization. There was a clear correlation between stimulatory effect and the ability to convert complement factor C3. Preincubation of sera with yeast—presumably producing complement cleavage products—potentiated the stimulatory effect of yeast glucan. It is suggested that endocytosis of glycans with subsequent intracellular triggering of a complement reaction is the underlying mechanism of glycan stimulation.     

油瓜油的甘油三酯组成研究

油瓜的种仁油可供食用。应用高效液相色谱技术,从油中分离分析出12种甘油三酯,其主要甘油二醋是甘油二亚油酸—棕榈酸酯(PLL)、甘油二棕榈酸—亚油酸酯(PLP)、甘油—棕榈酸—亚油酸—油酸酯(PLO)和甘油二棕榈酸—油酸酯(POP)。     

Water soluble polyacrylamide as embedding medium for electron microscopy

We have developed a method for embedding biological materials for electron microscopy in the water soluble polymer polyacrylamide (PAM). The fixed samples are infiltrated with an aqueous solution of acrylamide, which is then polymerized to form a hydrated gel. The gel is ready for sectioning after insolubilization by cross-linking with glutaraldehyde. Water is retained throughout the whole embedding process and contact of the samples with organic solvents is completely avoided. Sections are best stained with silicotungstic acid. The potential of this technique has been evaluated by detailed examination of several representative tissues. Observation of the native periodicity of 17 nm in PAM embedded myelin indicates a very good structural preservation. The method gives optimal results with samples which are easily infiltrated, such as pellets of cells.     

Refolding of Taiwan cobra neurotoxin: intramolecular cross-link affects its refolding reaction

In order to explore the effect of intramolecular cross-linking in the folding reaction of cobrotoxin from Naja naja atra (Taiwan cobra) venom, the toxin molecule was modified with glutaraldehyde (GA). The monomeric GA-modified cobrotoxin (mGA-cobrotoxin) was separated from the dimeric and trimeric derivatives using gel filtration. The results of electrophoretic and chromatographic analyses revealed that mGA-cobrotoxin comprised two modified derivatives, which contained modified Lys residues at positions 26 and 27 and at positions 26, 27, and 47, respectively. Moreover, an intramolecular cross-linking of loops II and III by Lys residues was noted with the monomeric derivative containing three modified Lys residues. In sharp contrast to cobrotoxin observations, the folding rate of mGA-cobrotoxin decreased in the presence of GSH/ GSSG, but notably increased in the absence of thiol compounds. Particularly, the accelerated effect of GSH/GSSG on the refolding reaction was affected by the presence of the intramolecular cross-link. Comparative analyses on cobrotoxin and mGA-cobrotoxin CD spectra revealed that modification with the GA reagent caused a change in the gross conformation of cobrotoxin. Fluorescence measurement revealed that the stability of the microenvironment around the single Trp-29 in mGA-cobrotoxin and unfolded mGA-cobrotoxin was appreciably higher than in cobrotoxin and unfolded toxin. Moreover, the ordered structure formation around Trp-29 in refolded mGA-cobrotoxin was faster than in refolded cobrotoxin as evidenced by fluorescence quenching studies. Taken together, these results suggest that the structural flexibility of unfolded cobrotoxin should be favorable for the thiol catalyst to exert its action in the refolding reaction after modification with GA.     

Triacylglycerol Profiles of Tilletia controversa and Tilletia tritici

The triacylglycerol (TG) profiles of teliospores of Tilletia controversa and Tilletia tritici were examined by high-performance liquid chromatography (HPLC) and gas-liquid chromatography. Boiling isopropanol was used to ensure enzyme inactivation during homogenization. The largest lipid component as determined by thin-layer chromatography was TGs. On the basis of thin-layer chromatography of crude lipid extracts, T. controversa and T. tritici do not contain a large amount of free fatty acids. TG profiles of T. controversa and T. tritici were very similar, with 18 species of TGs resolved by HPLC and gas-liquid chromatography. In both organisms, PLL (palmitic, linoleic, linoleic) was the major component, followed by LLL (trilinolein) and PLO (palmitic, linoleic, oleic). The ratio of PLO to PLL was 1:6 and 1:4 in T. tritici and T. controversa, respectively. The TGs of both organisms contain long-chain (>22 carbons) mono- and dienoic acids. Linoleic acid was the major fatty acid found in TGs from both organisms. The differences of TGs were not considered significant for differentiation purposes.     

Ornithine decarboxylase activity associated with a particulate fraction of brain

The enzymic decarboxylation of ornithine by adult rat brain largely occurs in the particulate fraction. The activity is primarily due to ornithine decarboxylase (ODC) as evidenced by several criteria: i) the concurrent production of equimolar amounts of CO₂ and putrescine, ii) the sensitivity of the reaction to difluoromethylornithine (DFMO), a specific inhibitor of ODC, iii) the lack of major effect of two inhibitors of ornithine-2-oxo-acid transaminase, upon the DFMO-sensitive component of decarboxylation, iv) the failure to profoundly reduce decarboxylation activity in the presence of a large excess of many aminoacids which could compete for non-specific decarboxylases. The insoluble ODC activity appears largely within synaptosomal and mitochondrial-enriched morphological fractions, yet cannot be attributed to trapped soluble ODC. Particulate ODC has a pH optimum and kinetic parameters that differ from those of soluble cerebral ODC.     

Preparation and characterization of glutaraldehyde cross-linked O-carboxymethylchitosan microspheres for controlled delivery of pazufloxacin mesilate

O-carboxymethylchitosan (OCMC) microspheres containing an antibiotic drug pazufloxacin mesilate (PM) have been successfully prepared by emulsion cross-linking using glutaraldehyde (GA). Various manufacturing parameters, including amount of cross-linking agent and OCMC:PM ratios were altered to optimize process variables during the microspheres production. The structure and morphology were characterized by Fourier transform infrared (FT-IR), wide-angle X-ray diffraction (WXRD) and scanning electron microscopy (SEM). The swelling and releasing behaviors of the microspheres at pH 1.2 and 7.4 media were investigated. The results revealed that the microspheres had a spherical, rough morphology and with a narrow size distribution. The degree of swelling of microspheres at pH 7.4 media was higher than that at pH 1.2 media. The microspheres proved to be successful in prolonging drug release. The release of PM was found to depend upon the extent of matrix cross-linking and drug loading. The release profiles of PM from OCMC microspheres were found to be biphasic with a burst release followed by a gradual release phase, and followed the Higuchi matrix model.     

Improvement of stability and cell adhesion properties of polyelectrolyte multilayer films by chemical cross-linking

Poly(L-lysine)/hyaluronan (PLL/HA) films were chemically cross-linked with a water soluble carbodiimide (EDC) in combination with a N-hydroxysulfo-succinimide (NHS) to induce amide formation. Fourier transform infrared spectroscopy confirms the conversion of carboxylate and ammonium groups into amide bonds. Quartz crystal microbalance-dissipation reveals that the cross linking reaction is accompanied by a change in the viscoelastic properties of the films leading to more rigid films. After the cross-linking reaction, both positively and negatively ending films exhibit a negative zeta potential. It is shown by fluorescence recovery after photobleaching measured by confocal laser scanning microscopy that cross-linking dramatically reduces the diffusion of the PLL chains in the network. Cross linking also renders the films highly resistant to hyaluronidase, an enzyme that naturally degrades hyaluronan. Finally, the adhesion of chondrosarcoma cells on the films terminating either with PLL or HA is also investigated. Whereas the non cross-linked films are highly resistant to cell adhesion, the cells adhere and spread well on the cross-linked films.     

The arrangement and role of some of the amino acid residues in the beta-ketoacyl synthetase site of chicken liver fatty acid synthetase

The beta-ketoacyl synthetase site of eukaryotic fatty acid synthetases is comprised in part of a pantetheinyl residue on one subunit juxtapositioned with a cysteinyl residue on the adjacent subunit. The present study has confirmed this arrangement and has identified 2 additional residues in the site. The active site residues were identified as summarized below. Sodium borohydride reduction of the keto derivatives of the dibromopropanone cross-linked residues yielded the alcohol derivatives which were amenable to isolation in good yields. The active enzyme yielded primarily a cysteinecysteamine derivative of 2-propanol, demonstrating that a cystyl and the pantetheinyl residues were cross-linked by dibromopropanone. However, in the cold-inactivated enzyme, the primary product of the cross-linking reaction was the dicystyl derivative. In addition, cross-linking between the cystyl and pantetheinyl residues, but not the two cystyl residues, resulted in the cross-linking of the two subunits. Therefore, it is proposed that there are two cystyl residues on one subunit juxtapositioned with the pantetheinyl residue on the adjacent subunit. The cystyl residues are highly reactive toward alkylating agents at pH 6.5, suggesting the presence of a cationic residue interacting with the thiolate anion. This proposal was supported using the bifunctional reagent o-phthalaldehyde which was found to cross-link the epsilon-amino group of lysine with the pantetheinyl-SH or the cystyl-SH in the beta-ketoacyl synthetase site to form a thioisoindole ring. The dialdehyde inhibited the enzyme by inactivating the beta-ketoacyl synthetase activity, and the inhibition could be prevented by malonyl-CoA and to a lesser extent by acetyl-CoA. Blocking the reactive thiol groups with dibromopropanone or 5,5'-dithiobis(2-nitrobenzoic acid) reduced the formation of the fluorescent thioisoindole ring. The close arrangement of a cystyl-SH, the pantetheinyl-SH, and the epsilon-amino group of lysine led us to propose that the positive epsilon-amino group may serve as an electron sink in a general acid-catalyzed decarboxylation reaction.     

Circular dichroism of the non-enzymatic browning products of poly-L-lysine and albumin

Poly-L-lysine (PLL) and bovine serum albumin were used as models in the investigation of non-enzymatic glycation and browning reaction by circular dichroism measurements. Different sugars (glucose, glucose-6-P and ribose) induced different extents of reaction and produced different chromophores that absorbed light in the near u.v. region. All glycated samples gave a common spectral feature in absorption and blue fluorescence, but they gave distinctly different near u.v. c.d. features, indicating that the browning products containing yellow chromophores are different for glycated samples and may have rigid moieties. The ribocated PLL (at pH 11.0, alpha-helix) and albumin were unable to convert completely to beta-conformation upon heating, indicating that browning reaction may have changed the tertiary structure.     

Multiple oxidation products of sulfhydryl groups near the active site of thiolase I from porcine heart

The inactivation of porcine heart thiolase I with the disulfide reagents 5,5'-dithiobis(2-nitrobenzoate) (DTNB) and 2,2- and 4,4-dithiopyridine in 0.2 M phosphate buffer, pH 7.5, follows second-order kinetics with rate constants of 2.2 X 10(2), 25 X 10(2), and 5.8 X 10(2) M-1 min-1, respectively. Stoichiometric concentrations of the thiol-oxidizing reagent diethyl azodicarboxylate inactivate thiolase in less than 1 min at pH 7.5. The presence of saturating concentrations of the substrate acetoacetyl coenzyme A or the formation of the acetyl enzyme (a normal catalytic intermediate) results in a significant protection against the inactivation of thiolase by DTNB, 2,2-dithiopyridine, and diethyl azodicarboxylate. All five sulfhydryl residues of native thiolase react with either of the dipyridyl disulfides, but only the equivalent of 3.2 residues react with DTNB even at high concentrations and prolonged incubation times. The reaction of thiolase with DTNB leads to the formation of 1.0-1.4 mol of intrachain disulfide and 0.65 mol of mixed disulfides. After inactivation of thiolase with an equimolar concentration of diethyl azodicarboxylate, 1.2 mol of intrachain disulfide per subunit is found. No cross-linking between the subunits occurs as a result of the reaction of thiolase with DTNB or diethyl azodicarboxylate. The DTNB-inactivated enzyme can be reactivated with excess dithiothreitol while the diethyl azodicarboxylate inactivated enzyme is totally resistant to reactivation by dithiothreitol. There appear to be at least two different ways of forming inactive, oxidized enzyme products depending on the oxidant used, suggesting the possibility of multiple sulfhydryl groups at or near the active site.