Peptide-protein complex from cattle sclera: Structural aspects and chaperone activity.

The influence of temperature and chaotropic agents on the spatial organization of the peptide-protein complex isolated from cattle sclera at the level of secondary structure was studied by UV, CD spectroscopy, and dynamic light scattering. It is shown that this complex has high conformational thermostability. The point of conformational thermal transition (65 °C) was determined, after which the peptide-protein complex passes into a denatured stable state. It was found that the peptide-protein complex in aqueous solutions forms thermostable nanosized particles. It was shown that the peptide-protein complex isolated from cattle sclera shows the properties of chaperone, an inhibitor of model protein aggregation induced by dithiothreitol.

Extraction of residual heavy metals from commercial chitosan and approach to preparation of oligochitosan hydrochloride.

For biomedical applications, chitosan and oligochitosan must be appropriately characterized and meet pharmacological requirements in terms of contamination by residual heavy metals. In this work, a series of commercial chitosans was analyzed by ICP-MS method, and high concentration of Fe (44-382 ppm), Cr (3.1-35.5 ppm) and Ni (0.33-7.91 ppm) exceeding pharmacologically acceptable level was found. It was shown that as a chelating agent EDTA was an ineffective remedy for solid-phase extraction of residual heavy metals from chitosan. It was proposed that corrosion of stainless steel apparatus in the process of chitin deacetylation contributed to chitosan contamination by heavy metals. A two-step treatment of chitosan with hydrochloric acid allowed remediation of chitosan and preparation of oligochitosan hydrochloride with molecular weight 5-16 kDa and acceptable level of Fe<10, Cr<1 and Ni<1 ppm.

Consequences of chitosan decomposition by nitrous acid: Approach to non-branched oligochitosan oxime.

It is well known that chitosan degradation by nitrous acid leads to oligochitosan (oligoCHIt-ahm) bearing reactive 2,5-anhydromannose (3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-aldehyde) units at the new reducing ends of macromolecules. Standard protocol requires reduction of oligoCHIt-ahm with NaBH4 to corresponding oligoCHIt-hml bearing unreactive hydroxymethyl group instead of reactive aldehyde group. For the first time, HP SEC as well as UV and CD spectroscopy methods have revealed that the reduction leads to an indefinite side modification and the formation of a branched oligoCHIt-hml with increased molecular weight. Here, it is shown that the branching and modification can be prevented by means of the simple and reproducible reaction of oligoCHIt-ahm with hydroxylamine that allows preparation of a stable linear oligochitosan oxime, oligoCHIt-oxm. Cytotoxicity tests show that oligoCHIt-ahm, oligoCHIt-hml and oligoCHIt-oxm are non-toxic at concentration below 2.5 mg/ml, and the cytotoxicity is concentration dependent and decreases in the order oligoCHIt-ahm > oligoCHIt-hml > oligoCHIt-oxm at higher concentrations both before and after long shelf-storage. The elaborated approach and cytotoxicity data give an opportunity to use the non-branched oligoCHIt-oxm for biomedical applications.

Influence of glucosamine on oligochitosan solubility and antibacterial activity.

Light scattering studies indicate that oligochitosan (short-chain chitosan) solutions contain aggregates at pH values below the critical pH of phase separation, while at or above this point the gel phase coexists with the aggregate solution. This work demonstrates for the first time that the presence of D-glucosamine in an oligochitosan solution shifts the critical pH to a higher value and improves the oligochitosan antibacterial activity against Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermis in neutral and slightly alkaline aqueous media. By comparing the results of light scattering studies and antimicrobial assays one can conclude that the antimicrobial activity of oligochitosan is dependent on its unimolecular form, not its supramolecular structures. The widening of the homogeneity region of an oligochitosan solution could lead to promising biomedical applications.

Polyelectrolyte thromboresistant affinity coatings for modification of devices contacting blood.

The modification of hydrophobic polyethylene/polystyrene surfaces of medical devices with bilayer/multilayer coatings (BCs/MCs) based on polyelectrolyte complexes (PEC) of modified poly(N-vinylpyrrolidone-co-maleic acid) copolymer (VPMA) with chitosan, amphiphilic chitosan, or albumin was studied. The VPMA contained l-Lysine as affinity ligand for plasminogen attached through alpha-amino group. The surface properties and chemical composition of the surfaces investigated were analyzed, using sessile-drop water contact angle measurements, attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The specific adsorption of plasminogen (precursor of fibrinolytic enzyme plasmin) from its solutions and from human blood plasma on the modified surfaces was investigated. It was established that polyelectrolyte MCs are more efficient than single-layer BCs and the affine polymer coatings without interlayer. A thrombogenicity decrease for the materials modified with BCs and MCs was shown in in vitro and ex vivo trials.

The mechanism of alpha-proton isotope exchange in amino acids catalysed by tyrosine phenol-lyase. What is the role of quinonoid intermediates?

To shed light on the mechanism of isotopic exchange of alpha-protons in amino acids catalyzed by pyridoxal phosphate (PLP)-dependent enzymes, we studied the kinetics of quinonoid intermediate formation for the reactions of tyrosine phenol-lyase with L-phenylalanine, L-methionine, and their alpha-deuterated analogues in D2O, and we compared the results with the rates of the isotopic exchange under the same conditions. We have found that, in the L-phenylalanine reaction, the internal return of the alpha-proton is operative, and allowing for its effect, the exchange rate is accounted for satisfactorily. Surprisingly, for the reaction with L-methionine, the enzymatic isotope exchange went much faster than might be predicted from the kinetic data for quinonoid intermediate formation. This result allows us to suggest the existence of an alternative, possibly concerted, mechanism of alpha-proton exchange.

Use of the affinity chromatography principle in creating new thromboresistant materials.

The principle of affinity chromatography was used for preparation of thromboresistant bilayer coatings. The outer biospecific layer containing epsilon-aminocaproic acid residues (from 2.2 up to 5.5 nmol/cm2) was synthesized using a copolymer of maleic anhydride with N-vinylpyrrolidone and L-lysine dihydrochloride or N-epsilon-tert-BOC-L-lysine. This surface can selectively adsorb plasminogen (fibrinolytic zymogen) from blood. The biospecific layer (from 2.0 up to 3.6 microg/cm2) was applied for covering chitosan (native or modified) or albumin interlayer. Such bilayer coatings (BCs) were stable and represented the insoluble polyelectrolyte complexes. BCs were proposed for bilayer modification of synthetic vascular grafts, polyethylene, and other materials contacting with blood. This technique allowed us to significantly reduce thrombogenic properties of polyethylene surfaces.