The bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor): a milestone protein

The pancreatic Kunitz inhibitor, also known as aprotinin, bovine basic pancreatic trypsin inhibitor (BPTI), and trypsin-kallikrein inhibitor, is one of the most extensively studied globular proteins. It has proved to be a particularly attractive and powerful tool for studying protein conformation as well as molecular bases of protein/protein interaction(s) and (macro)molecular recognition. BPTI has a relatively broad specificity, inhibiting trypsin- as well as chymotrypsin- and elastase-like serine (pro)enzymes endowed with very different primary specificity. BPTI reacts rapidly with serine proteases to form stable complexes, but the enzyme: inhibitor complex formation may involve several intermediates corresponding to discrete reaction steps. Moreover, BPTI inhibits the nitric oxide synthase type-I and -II action and impairs K+ transport by Ca2+-activated K+ channels. Clinically, the use of BPTI in selected surgical interventions, such as cardiopulmonary surgery and orthotopic liver transplantation, is advised, as it significantly reduces hemorrhagic complications and thus blood-transfusion requirements. Here, the structural, inhibition, and bio-medical aspects of BPTI are reported.     

Biochemical aspects and structural characterization of sulphur bonded oxovanadium(V) and manganese(II) complexes

Summary The preparation, characterisation and biological activities of some oxovanadium(V) and manganese(II) complexes of ligands having nitrogen-sulphur donor sets are described. Reaction of VOCl₃ and MnCl₂ with HTSCZ (HTSCZ = thiosemicarbazone) resulted in the formation of complexes of the type [VOCl₂(TSCZ)], [MnCl(TSCZ)H₂O], [VOCl(TSCZ)₂] and [Mn(TSCZ)₂]. These have been characterized by elemental analyses, molecular weight determinations and conductivity measurements and found to be monomeric and nonelectrolytes. Distorted trigonal bipyramidal (11) and octahedral (12) geometries for the vanadium(V) complexes and a tetrahedral geometry for the manganese(II) complexes are proposed. All the free ligands and their complexes have been tested in vitro against a number of pathogenic fungi and bacteria and were found to possess good fungicidal and bactericidal properties.     

Preparation and characterization of plasma polymerized (ethylene + oxygen) thin films

This report describes the preparation and characterization of plasma polymerized (ethylene + oxygen) (PPEO) thin films. The PPEO films described herein represent a unique class of materials from the standpoint of stoichiometry, chemical functionality, and crosslink density. It will be shown that PPEO deposition rate and structure, evidenced both at the surface and in the bulk, are strongly dependent upon the flow rate of O₂ in the ethylene/O₂ feed gas mixture. High O₂ flow rates produce films with relatively high oxygen concentrations. Furthermore, increasing O₂ flow rates lead to film structures which increasingly favor the incorporation of highly oxidized carbon functionalities. These effects (increasing film concentrations of oxygen and highly oxidized carbon functionalities) appear to show no further increase for O₂ flow rates greater than or equal to ca. 10% of the ethylene flow rate.     

Novel multifunctional chitosan-GMA-IDA-Cu(II) nanospheres for high dynamic range characterization of the human plasma proteome

In this study, we describe characterization of the human plasma proteome based on analysis with multifunctional chitosan-GMA-IDA-Cu(II) nanospheres. Chitosan-GMA-IDA-Cu(II) nanospheres with diameters of 20 to 100?nm have unique properties due to multifunctional chemical moieties, high surface area, high capacity, good dispersibility in buffer solution as well as good biocompatibility and chemical stability which improves their specific interaction with peptides and proteins of the human plasma using different binding buffers. Combining these chitosan-GMA-IDA-Cu(II) nanospheres with MS spectrometry results in a novel strategy which should make it possible to characterize the plasma proteome in a single test. Peptides and proteins adsorbed on the nanosphere can be directly detected by MALDI-TOF-MS. The eluted lower molecular weight peptides and proteins are identified by nano-LC-ESI-MS/MS. A total of 842 unique LMW peptides and 1,682 human unredundant proteins IDs were identified in two different binding buffers, which included relatively low-level proteins (e.g., pg/mL of IL3 Interleukin-3) co-distributed with high-abundance proteins (e.g., 35?C55?mg/mL level serum albumin). As such, this nanosphere technique selectively enabled the identification of proteins over a dynamic range of greater than 8 orders of magnitude. Considering this capacity for selective enrichment of peptides and proteins in human plasma, and the large number of LMW peptides and proteins which can be identified, this method promises to accelerate discovery of biomarkers for clinical application.

Purification and electrophoretic characterization of bovine conglutinin

In this study a two-stage procedure for purification of conglutinin using affinity and ion-exchange chromatography was developed. To isolate conglutinin from bovine serum, its unique ability to bind to complement component iC3b was exploited. Incubation of bovine serum with chromatographic beads (TSK, Toyopearl HW-75 F) at 37 °C allows for iC3b deposition and subsequent binding of conglutinin. A single protein fraction eluted with ethylenediaminetetraacetic acid (EDTA) was then separated on an ion-exchange column in an NaCl gradient. The purification was evaluated by SDS-PAGE and western blotting. Conglutinin analyzed by SDS-PAGE under reducing conditions showed two main bands at 41 and 47 kDa and eight weaker bands. Nonreduced conglutinin appeared as a ladder pattern composed of many fractions ranging from 34 to 630 kDa. The bands at 34, 153, 174, 247, 338 and 387 kDa displayed the highest optical density. In the native conglutinin profile four fractions were observed, and the pI of this protein was below 8.5. The presence of sugar residues in the conglutinin molecule was detected using Schiff's reagent.     

Balancing conformational and oxidative kinetic traps during the folding of bovine pancreatic trypsin inhibitor (BPTI) with glutathione and glutathione disulfide

The oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) has served as a paradigm for the folding of disulfide-containing proteins from their reduced form, as well as for protein folding in general. Many extracellular proteins and most pharmaceutically important proteins contain disulfide bonds. Under traditional conditions, 0.125 mM glutathione disulfide (GSSG) and no glutathione (GSH), the folding pathway of BPTI proceeds through a nonproductive route via N* (a two disulfide intermediate), or a productive route via N' (and other two disulfide intermediates which are in rapid equilibrium with N'). Both routes have the rearrangement of disulfide bonds as their rate-determining steps. However, the effects of the composition of the redox buffer, GSSG and GSH, on folding has not been extensively investigated. Interestingly, BPTI folds more efficiently in the presence of 5 mM GSSG and 5 mM GSH than it does under traditional conditions. These conditions, which are similar to those found in vivo, result in a doubly mixed disulfide between N' and glutathione, which acts as an oxidative kinetic trap as it has no free thiols. However, with 5 mM GSSG and 5 mM GSH the formation of the double mixed disulfide is compensated for by N* being less kinetically stable and the more rapid conversion of the singly mixed disulfides between N' and glutathione to native protein (N). Thus a major rate-determining step becomes the direct conversion of a singly mixed disulfide to N, a growth-type pathway. Balancing the formation of N* and its stability versus the formation of the doubly mixed disulfide and its stability results in more efficient folding. Such balancing acts may prove to be general for other disulfide-containing proteins.     

Synthesis, characterization of Ag(I), Pd(II) and Pt(II) complexes of a triazine-3-thione and their interactions with bovine serum albumin

Ag(I), Pd(II) and Pt(II) complexes of 5-methoxy-5,6-diphenyl-4,5-dihydro-2H-[1,2,4]triazine-3-thione (LH(2)OCH(3)) have been synthesized and characterized by elemental analysis, molar conductance, (1)H NMR, IR spectra, UV spectra and thermal analysis (TG-DTA). The components of the three complexes are [Ag(C(15)H(10)N(3)S)](6), Pd(C(15)H(10)N(3)S)(2) and Pt(C(15)H(10)N(3)S)(2).C(3)H(6)O.2H(2)O, respectively. All the complexes are nonelectrolyte and have high thermodynamic stability. The ligand may act as bidentate NS donor for Pd(II) and Pt(II) complexes, while it seems to be bidentate NS bridging via sulphur atom for Ag(I) complex. A planar quadrangular structure is proposed for Pd(II) and Pt(II) complexes and Ag(I) complex may be a hexanuclear cluster. Their interactions with bovine serum albumin (BSA) are investigated using steady state fluorescence technology. It is observed that all of them can quench the intrinsic fluorescence of BSA through static quenching procedure. The binding constants (K(A)) at different temperatures, thermodynamic parameters enthalpy changes (DeltaH) and entropy changes (DeltaS) between BSA and the compounds are calculated. Based on the values of DeltaH and DeltaS, it is judged that the main acting force of PtL(2).C(3)H(6)O.2H(2)O with BSA may be electrostatic interaction, and for the LH(2)OCH(3), Ag(6)L(6) and PdL(2), hydrophobic and electrostatic interactions may be involved in their binding processes.     

Biochemical characterization of peptidyl carrier protein (PCP), the thiolation domain of multifunctional peptide synthetases

Background: A structurally diverse group of bioactive peptides is synthesized by peptide synthetases which act as templates for a growing peptide chain, attached to the enzyme via a thloester bond. The protein templates are composed of distinctive substrate-activating modules, whose order dictates the primary structure of the corresponding peptide product. Each module contains defined domains that catalyze adenylation, thioester and peptide bond formation, as well as substrate modifications. To show that a putative thiolation domain (PCP) is involved in covalent binding and transfer of amino aryl residues during non-ribosomal peptide synthesis, we have cloned and biochemically characterized that region of tyrocidine synthetase 1, TycA.Results: The 327-bp gene fragment encoding PCP was cloned using its homology to the genes for the acyl carrier proteins of fatty acid and polyketide biosynthesis. The protein was expressed as a His₆, fusion protein, and purified in a single step by affinity chromatography. Incorporation of β-[³H]alanine, a precursor of coenzyme A, demonstrated the modification of PCP with the cofactor 4′-phosphopantetheine. When an adenylation domain is present to supply the amino adenylate moiety, PCP can be acylated in vitro.Conclusions: PCP can bind covalently to the cofactor phosphopantetheine and can subsequently be acylated, strongly supporting the multiple carrier model of non-ribosomal peptide synthesis. The adenylation and thiolation domains can each act as independent multifunctional enzymes, further confirming the modular structure of peptide synthees, and can also perform sequential steps in trans, as do multienzyme complexes.     

Identification and partial characterization of an unusual distribution of the photosensitizer meta-tetrahydroxyphenyl chlorin (temoporfin) in human plasma

Temoporfin (m-THPC) is an extremely powerful photosensitizing drug, more than 100-fold more photocytotoxic than Photofrin and many other drugs. The reasons for this are not yet known but are likely to be associated with the mechanism of uptake of the drug and its intratumoral and intracellular localization. Uptake itself is likely to be dependent upon the plasma binding of the drug following administration. In the current work, we have shown that the addition of m-THPC to human plasma in vitro at clinically relevant doses of sensitizer and administration solvent (diluant) gives rise to a protein-binding pattern quite different to that of Photofrin and other hydrophobic drugs as judged by density-gradient ultracentrifugation. Analysis of the binding immediately after addition to human plasma has shown that lipoprotein binding accounts for only a minor proportion of the sensitizer, which is mainly associated with a high-density protein fraction that is not coincident with serum albumin. The m-THPC protein complex does not fluoresce significantly even on dilution. This binding pattern is highly dependent on administration conditions and storage. Over a period of 6-8 h at 37 degrees C the m-THPC that is associated with this unidentified fraction redistributes to the plasma lipoproteins. Plasma collected from rats after intravenous administration of m-THPC also contains this low fluorescent complex, showing that this phenomenon is not limited to human plasma and also occurs in vivo. It is postulated that the m-THPC bound to the unknown protein fraction is highly aggregated and that it is likely to be taken up into tissues in this form. This unusual uptake may possibly be associated with the very high activity of m-THPC and also to the recent finding of a second peak in the plasma pharmacokinetics of the drug.     

Biochemical characterization of malate synthase G of P. aeruginosa

Background  

Malate synthase catalyzes the second step of the glyoxylate bypass, the condensation of acetyl coenzyme A and glyoxylate to form malate and coenzyme A (CoA). In several microorganisms, the glyoxylate bypass is of general importance to microbial pathogenesis. The predicted malate synthase G of Pseudomonas aeruginosa has also been implicated in virulence of this opportunistic pathogen.     

Laser-based characterization of a flame-assisted plasma

A well-characterized flame-assisted plasma was developed to understand the role of flow nonuniformities and plasma/wall interactions in plasma devices for use in validation of laser-based Doppler shift spectroscopic methods. A hydrogen/oxygen capillary diffusion flame burner was used as a plasma source, with barium seeded into the reactants to provide a source of ions and electrons. For analysis the plasma was assumed to be a stationary, partially ionized, collision dominated, thermal plasma consisting of barium ions, electrons, and neutrals between two parallel-plate electrodes. The plasma was examined in terms of the continuum equations for ions and electrons, together with Poisson's equation to predict spatial profiles of electron and positive ion density and potential as functions of applied potential. First an analytic solution based on constant plasma properties and negligible difusion was introduced. The model was then extended by including effects of diffusion and variable plasma properties. Experimentally, current/voltage characteristics of the plasma were measured conventionally, relative ion concentration and temperature were measured with laser-induced fluorescence, and local potential distribution was measured using an electrostatic probe. The diffusionless theory predicted well the bulk behavior of the plasma, but not the correct spatial distributions of ion concentration and potential. The extended model produced a more satisfactory fit to the data. At conditions of 1.4 equivalence ratio, 70 torn pressure, 300 ppm seed concentration, and 100–400 V applied potentials, electric fields of the order of 10², 10³ V/cm were observed near the powered electrode, and of few tens of V/cm in the hulk of tire plasma. The field strength in the sheath ensures the operation of the Doppler shift diagnostics, once the recommendations tor LIF signal detectability are fulfilled.     

石墨烯量子点在生化分析中的应用研究进展

介绍了石墨烯量子点（GQDs）在生化分析领域如生物分子检测、金属离子检测、细菌检测、细胞成像、组织成像以及活体成像等方面的最新研究动态。     

Thermal characterization of mercury(II) bis(dialkyldithiocarbamate) complexes

Summary Solid-state M-4-MeO-Bz compounds, where M stands for bivalent Mn, Co, Ni, Cu and Zn and 4-MeO-Bz is 4-methoxybenzoate, have been synthesized. Simultaneous thermogravimetry-differential thermal analysis (TG-DTA), differential scanning calorimetry (DSC), X-ray powder diffractometry, infrared spectroscopy, elemental analysis and complexometry were used to characterize and to study the thermal behaviour of these compounds. The results led to have information about the composition, dehydration, thermal stability and thermal decomposition of the isolated compounds.