Svetla Petrova

An effective method for bioconversion of delignified waste-cellulose fibers from the paper industry with a cellulase complex.

Abstract An effective method for production of glucose was developed using enzymatic hydrolysis of waste-cellulose fibers by the cellulase complex from Trichoderma reesei . However, these cellulosic materials are strongly resistant to direct enzymatic hydrolysis (the degree of degradation after 48 h of enzyme treatment did not exceed 14%) apparently due to the presence of various chemical substances used in the process of paper production. This adhesive “envelope” around the cellulose fibers was effectively pretreated with 0.25% H 3 PO 4 and after that the free cellulose mass was extensively hydrolyzed by the cellulase complex (degree of degradation more than 80%). The HPLC analyses of the enzyme hydrolysates revealed glucose as the main component as well as some cellobiose and xylose.

Biochemical and catalytic properties of endo-1,4-β-xylanases from Thermomyces lanuginosus (wild and mutant strains)

Endoxylanases from the thermophilic fungus, Thermomyces lanuginosus ATCC 44008 (cellulase free wild and mutant strains), were purified to homogeneity by anion-exchange and molecular-sieve chromatographic methods. The purified enzymes were monomers with molecular masses of 22 kDa (wild type) and 24 kDa (mutant), estimated by SDS-PAGE and gel filtration. As glycoproteins, the purified enzymes had 0.74% (wild type) and 11.8% (mutant) carbohydrate contents, and pI values of 5.8 and 6, respectively. The optimal pH and temperature values of wild type xylanase were determined to be pH 7 and 60 °C, whereas pH 6.7 and 70 °C, were optimal for the purified mutant enzyme (Km and Vmax values of 3.7 mg ml−1 and 670 μmol min−1 xylose compared to the kinetic values of the purified wild type xylanase −5.1 mg ml−1 and 385 μmol min−1 xylose). Inhibition studies suggested the possible involvement of histidine, tryptophan residues and carboxylic groups in the binding or catalysis.

Hemolytic and anticoagulant study of the neurotoxin vipoxin and its components—basic phospholipase A2 and an acidic inhibitor

In the present study, we demonstrate for the first time that the potent neurotoxin vipoxin from the venom of Vipera ammodytes meridionalis exhibits hemolytic and anticoagulant properties. By investigating the effects of phospholipids and calcium ions on hemolysis, we established that the phospholipase A2 (PLA2) enzyme activity is responsible for the hemolytic properties. This was confirmed by chemical modification of the PLA2 active-site histidine residue with p-bromophenacylbromide. Applying different clotting assays, we show that the PLA2 is a weakly anticoagulant enzyme, which affects intrinsic tenase complex by the hydrolysis of procoagulant phospholipids, rather than by nonenzymatic mechanisms (binding to specific coagulation factors). The whole complex—vipoxin—does not affect the coagulation system.

Recognition of Vipera ammodytes meridionalis neurotoxin vipoxin and its components using phage-displayed scFv and polyclonal antivenom sera

Vipoxin is a potent postsynaptic heterodimeric neurotoxin isolated from the venom of the Bulgarian snake Vipera ammodytes meridionalis, whose snakebites cause different and strongly manifested pathophysiological effects (neurotoxic, hemolytic, anticoagulant, convulsant, hypotensive, hyperglycemic etc.). The neutralization of snake toxins calls for extensive research through the application of different approaches: antibodies, non-immunologic inhibitors, natural products derived from plants and animals, as well as synthetic drugs. In this study, we applied naive Tomlinson I + J (Cambridge, UK) libraries to obtain recombinant human scFv antibodies against the vipoxins two subunits--basic and toxic phospholipase A₂ (PLA₂) and acidic, non-toxic component. We found that 33 of more than hundred tested clones were positive and recognized vipoxin and its subunits. Enriched scFv-phage samples (1.2 × 10⁹ pfu/ml) were analyzed for their binding (ELISA) and enzyme-inhibiting abilities. Single chain Fv-phage clones--D₁₂, E₃, F₆, D₁₀ and G₅ exhihest binding affinity for the toxic component. Clones A₁, D₁₂ and C₁₂ recognized preferentially vipoxins acidic component. Clones E₃, G₅ and H₄ inhibited the enzymatic activity of both vipoxin and its purified and separated toxic subunit to the highest extent. Six of the selected clones (E₃, G₅, H₄, C₁₂, D₁₀ and A₁₁) inhibited direct hemolytic activity of vipoxin and its pure PLA₂ subunit. The obtained specific scFv antibodies will be used for epitope mapping studies required to shed light on the role of the phospholipase A₂ activity for the vipoxin toxicity and its effective neutralization.

New insight into the autoimmunogenicity of the complement protein C1q

C1q along with its physiological role in maintenance of homeostasis and normal function of the immune system is involved in pathological conditions associated with repetitive generation of anti-C1q autoantibodies. The time and events that cause their first appearance are still unknown. We addressed this issue by analyzing the immunogenicity of C1q in two target groups-one of non-diseased humans and the other of lupus nephritis (LN) patients whose autoimmune disorder is associated with high titers of anti-C1q autoantibodies. The non-diseased humans were represented by pregnant women because the sex hormones are thought to be involved in triggering autoimmune pathologies by their ability to tip the balance of female adaptive immune response to production of antibodies. We screened, using ELISA, 31 sera from healthy pregnant women for the presence of IgM and IgG classes of autoantibodies, recognizing epitopes within the native C1q molecule, its collagen-like region (CLR) and globular head fragment (gC1q). The latter was represented by recombinant analogs of the three globular fragments of A, B and C chains, comprising C1q-ghA, ghB and ghC. We did not find IgM antibodies for all test-antigens which suggest that the natural IgM antibodies are not involved in triggering autoimmunity to C1q. Still more, we did not detect anti-CLR antibodies which have been proved pathogenic in already manifested LN. We completed the analysis with comparative epitope mapping of gC1q and we found similar immunogenic behavior in both target groups-ghA and ghC contained the immunodominant epitopes. This implies that the initial immune response to C1q might occur when the molecule has interacted with its ligands via ghB as part of gC1q. The presence of anti-gC1q in both healthy and diseased humans also implies that these antibodies, unlike anti-CLR, may have a contribution to an onset of autoimmunity.

Kinetics of degradation of dipalmitoylphosphatidylcholine (DPPC) bilayers as a result of vipoxin phospholipase A2 activity: an atomic force microscopy (AFM) approach.

In this paper we used AFM as an analytical tool to visualize the degradation of a phospholipid bilayer undergoing hydrolysis of the vipoxins PLA(2). We obtained time series images during the degradation process of supported 1, 2-dipalmitoylphosphatidylcholine (DPPC) bilayers and evaluated the occurrence and the growth rate of the bilayer defects. The special resolution of the AFM images allowed us to measure the area and the perimeter length of these defects and to draw conclusions about the kinetics of the enzyme reaction. Moreover, we also report for some unique characteristics discovered during the vipoxins PLA(2) action. Experimentally for the first time, we observed the appearance and the growth of three-dimensional (3D), crystal-like structures within the formed defects of the degraded bilayer. In an effort to explain their nature, we applied bearing image analysis to estimate the volume of these crystals and we found that their growth rate follows a similar kinetic pattern as the degradation rate of the supported bilayer.

HPLC Assay of Phospholipase A2 Activity Using Low-Temperature Derivatization of Fatty Acids

Abstract A modified isocratic reverse phase high-performance liquid-chromatographic (RP-HPLC) method for phospholipase (PLA) activity assay is developed. Natural lecithins and synthetic phospholipids are used as substrates, and released fatty acids are analyzed after single-phase derivatization (with p-bromophenacyl bromide) at low temperature. The procedure allows simultaneous determination of the total and specific phospholipase activity. This method was successfully applied to snake venom PLA2 activity assay using natural (soybean and egg yolk lecithins) and synthetic (dipalmytoylphosphatydylcholine) substrates for quantitative determination of the enzyme activity.

Severe coagulopathy after Vipera ammodytes ammodytes snakebite in Bulgaria: a case report.

The case report presents a severe coagulopathy in a 56-year-old man following envenomation by the snake (Vipera ammodytes ammodytes) on his left hand. Initially the man was in shock, with an extremely low blood pressure and tachycardia. Local signs included a painful blister formation on the envenomation site. Twenty-four hours later, the man developed acute thrombocytopenia (platelets number 10 x 10(9)/l) and ecchimoses formation on the affected limb and on the left side of his body due to a disseminated intravascular coagulation syndrome, which lasted 13 days and required repeated administration of blood products, antivenin and supportive treatment. The patient was discharged from the hospital after 18 days in a good condition. The case report indicates that the coagulopathy may be a serious life-threatening complication after V. ammodytes ammodytes snakebite.

Vipoxin and Its Components

The neurotoxin Vipoxin has been of growing research interest since the time of its isolation from the venom of the Bulgarian viper Vipera ammodytes meridionalis. Vipoxin is a heterodimeric postsynaptic ionic complex composed of two protein subunits-a basic and strongly toxic His48 secretory phospholipase A(2) (sPLA(2)) enzyme and an acidic, enzymatically inactive and nontoxic component, originally named Inhibitor. When separated, sPLA(2) enzyme loses its toxicity in 3-4 days and catalytic activity in 2 weeks. After the establishment of the high degree of sequence homology (62%) and crystal structure of the subunits, Vipoxin was served as an example of molecular evolution of a toxic but unstable sPLA(2) into an inhibitor subunit which stabilizes the enzyme and preserves its pharmacological activity. Beginning our research on Vipoxin, intrigued by the unique relationship-structure-function and based on the previous experience, we were more than surprised to establish the lack of so-called inhibitory function of the acidic subunit on the toxicity and catalytic activity of basic sPLA(2). On the contrary, the acidic subunit activated the sPLA(2) enzyme in vitro. Our studies undoubtedly proved that is more correctly to present Vipoxin as a heterodimeric complex composed of one basic catalytic subunit and one acidic regulatory subunit. Their interaction in a common quaternary protein structure is more than a noncovalent association between the two subunits. It allows pharmacological sites to be targeted and biological functions to be potentiated. We attempt to present the previous studies and new findings on Vipoxin and its components.

Vipoxin and its components: structure-function relationship.

The neurotoxin Vipoxin has been of growing research interest since the time of its isolation from the venom of the Bulgarian viper Vipera ammodytes meridionalis. Vipoxin is a heterodimeric postsynaptic ionic complex composed of two protein subunits-a basic and strongly toxic His48 secretory phospholipase A(2) (sPLA(2)) enzyme and an acidic, enzymatically inactive and nontoxic component, originally named Inhibitor. When separated, sPLA(2) enzyme loses its toxicity in 3-4 days and catalytic activity in 2 weeks. After the establishment of the high degree of sequence homology (62%) and crystal structure of the subunits, Vipoxin was served as an example of molecular evolution of a toxic but unstable sPLA(2) into an inhibitor subunit which stabilizes the enzyme and preserves its pharmacological activity. Beginning our research on Vipoxin, intrigued by the unique relationship-structure-function and based on the previous experience, we were more than surprised to establish the lack of so-called inhibitory function of the acidic subunit on the toxicity and catalytic activity of basic sPLA(2). On the contrary, the acidic subunit activated the sPLA(2) enzyme in vitro. Our studies undoubtedly proved that is more correctly to present Vipoxin as a heterodimeric complex composed of one basic catalytic subunit and one acidic regulatory subunit. Their interaction in a common quaternary protein structure is more than a noncovalent association between the two subunits. It allows pharmacological sites to be targeted and biological functions to be potentiated. We attempt to present the previous studies and new findings on Vipoxin and its components.