Stabilization of proteases by entrapment in a new composite hydrogel

A new one-step procedure for entrapping proteases into a polymeric composite calcium alginate-poly(N-vinyl caproladam) hydrogel was developed that provided 75–90% retention of the activity of entrapped enzymes compared to soluble ones. Properties of entrapped carboxypeptidase B, trypsin, and thrombin were investigated. The immobilized enzymes were active within a wide pH range. The temperature optima of entrapped trypsin and carboxypeptidase B were approx 25°C higher than that of the soluble enzymes, and the resistance to heating was also increased. The effects of various polar and nonpolar organic solvents on the entrapped proteases were investigated. The immobilized enzymes retained their activity within a wide concentration range (up to 90%) of organic solvents. Gel-entrapped trypsin and carboxypeptidase (CPB) were successfully used for obtaining human insulin from recombinant proinsulin. The developed stabilization method can be used to catalyze various reactions proceeding within wide pH and temperature ranges.     

Combinatorial docking and combinatorial chemistry: Design of potent non-peptide thrombin inhibitors

A computational algorithm was used to design automatically novel thrombin inhibitors that are available from a single-step chemical reaction. The compounds do not contain amide bonds, are achiral and have a molecular weight below 400. Of the 10 compounds that were synthesized, five bind to thrombin with a Ki in the nanomolar range. Subsequent X-ray structure determination of the thrombin-inhibitor complex for the best compound (Ki=95 nM) confirms the predicted binding mode. The novel algorithm is applicable to a broad range of chemical reactions.     

Complete 1H and 13C assignments of (21R) and (21S) diastereomers of argatroban

The complete 1H and 13C NMR assignments are reported for the antithrombotic (21R)- and (21S)-argatroban by 1D and 2D NMR experiments (HSQC, HMBC, NOESY and 1H--1H COSY). Some well-resolved signals could be used for an accurate measurement of the diastereomeric composition of argatroban.     

Selectivity of neutral/weakly basic P1 group inhibitors of thrombin and trypsin by a molecular dynamics study

Molecular dynamics (MD) simulations followed by molecular mechanics generalized Born surface area (MM-GBSA) analyses have been carried out to study the selectivity of two neutral and weakly basic P1 group inhibitors (177 and CDA) to thrombin and trypsin. Detailed binding free energies between these inhibitors and individual protein residues are calculated by using a per-residue basis decomposition method. The analysis of the detailed interaction energies provides insight on the protein-inhibitor-binding mechanism and helps to elucidate the basis for achieving selectivity through interpretation of the structural and energetic results from the simulations. The study shows that the dominant factor of selectivity for both inhibitors is van der Waals energy, which suggests better shape complementarity and packing with thrombin. Nonpolar solvation free energy and total entropy contribution are also in favor of selectivity, but the contributions are much smaller. Binding mode and structural analysis show that 177 binds to thrombin and trypsin in a similar binding mode. In contrast, the CDA binds to thrombin and trypsin in very different modes.     

Evaluating the relative free energy of hydration of new thrombin inhibitor candidates using the finite difference thermodynamic integration (FDTI) method

Thrombin is a serine protease involved in blood coagulation. Since thrombin inhibitors appear to be effective in the treatment and prevention of thrombotic and embolic disorders, considerable attention has been focused on the structure and interactions of the enzyme. In this work, we calculated the relative free energies of hydration of the new thrombin inhibitor candidates, p‐substituted derivatives of benzamidine, a well‐known noncovalent thrombin inhibitor. We used molecular dynamics and the finite difference thermodynamic integration (FDTI) algorithm within the Discover program of MSI. We have shown that the orthogonality problem that occurs in the calculation of intraperturbed‐group contributions to the free energy is treated adequately by the FDTI method. We have also shown that problems of singularity and convergence in free energy calculations can be properly solved using this method. To conclude, the calculated free energies of hydration gave the following order of solvation for the candidates: p‐(2‐oxo‐1‐propyl)benzamidine > p‐methylbenzamidine > p‐ethylbenzamidine > p‐(1‐propyl)benzamidine > benzamidine. © 2001 John Wiley & Sons, Inc. Int J Quantum Chem, 2001     

Direct Measurement of Through-Bond Effects in Molecular Multivalent Interactions

Multivalent interactions occur throughout biology, and have a number of characteristics that monovalent interactions do not. However, it remains challenging to directly measure the binding force of molecular multivalent interactions and identify the mechanism of interactions. In this study, the specific interaction between bivalent aptamer and thrombin has been measured directly and quantitatively by force-induced remnant magnetization spectroscopy to investigate the binding force and through-bond effects of the multivalent interactions. The measured differential binding forces enable through-bond effects in thrombin–aptamer complexes to be identified, where aptamer binding at exosite II produces visible effects on their binding at exosite I and vice versa. This method might be suitable for practical applications in the design of high-performance ligands.     

Ion‐Responsive Hemin–G‐Quadruplexes for Switchable DNAzyme and Enzyme Functions

Programmed nucleic acid sequences undergo K⁺ ion‐induced self‐assembly into G‐quadruplexes and separation of the supramolecular structures by the elimination of K⁺ ions by crown ether or cryptand ion‐receptors. This process allows the switchable formation and dissociation of the respective G‐quadruplexes. The different G‐quadruplex structures bind hemin, and the resulting hemin–G‐quadruplex structures reveal horseradish peroxidase DNAzyme catalytic activities. The following K⁺ ion/receptor switchable systems are described: 1) The K⁺‐induced self‐assembly of the Mg²⁺‐dependent DNAzyme subunits into a catalytic nanostructure using the assembly of G‐quadruplexes as bridging unit. 2) The K⁺‐induced stabilization of the anti‐thrombin G‐quadruplex nanostructure that inhibits the hydrolytic functions of thrombin. 3) The K⁺‐induced opening of DNA tweezers through the stabilization of G‐quadruplexes on the “tweezers’ arms" and the release of a strand bridging the tweezers into a closed structure. In all of the systems reversible, switchable, functions are demonstrated. For all systems two different signals are used to follow the switchable functions (fluorescence and the catalytic functions of the derived hemin–G‐quadruplex DNAzyme).     

Fractal Dimension of Microbead Assemblies Used for Protein Detection

We use fractal analysis to calculate the protein concentration in a rotating magnetic assembly of microbeads of size 1 μm, which has optimized parameters of sedimentation, binding sites and magnetic volume. We utilize the original Forrest–Witten method, but due to the relatively small number of bead particles, which is of the order of 500, we use a large number of origins and also a large number of algorithm iterations. We find a value of the fractal dimension in the range 1.70–1.90, as a function of the thrombin concentration, which plays the role of binding the microbeads together. This is in good agreement with previous results from magnetorotation studies. The calculation of the fractal dimension using multiple points of reference can be used for any assembly with a relatively small number of particles.     

基于聚苯乙烯微球放大的凝血酶化学发光检测新技术

本文以羧基96孔板为分离载体,核酸适配体作为分子特异性识别元件,聚苯乙烯微球作为放大载体,辣根过氧化物酶为标记物,构建了化学发光（CL）高灵敏度凝血酶检测新技术.实验结果表明：该放大技术不但灵敏度高,且抗干扰能力强,其他蛋白质如IgG、IgM、IgA、IgE、IFN均无明显干扰.聚苯乙烯微球放大体系中凝血酶的线性范围为7.8～250pmol/L,最低检测浓度可达3.9pmol/L;而不放大检测技术的线性范围为0.94～30nmol/L,最低检测浓度为0.46nmol/L,放大体系将检测灵敏度提高100多倍.综合而言,基于适配体识别和聚苯乙烯微球放大的凝血酶CL检测新技术具有通量大、简单快速和灵敏度高的特点,有望在凝血酶高通量检测领域获得应用.     

酶促-化学法合成(R)-N-苄基吗啉-3-酮-2-乙酰(4-脒基)苄胺

报道了一种酶促-化学法合成光学纯吗啉酮凝血酶抑制剂的新途径.以马来酸酐和氨基醇为起始原料,一锅法合成N-苄基吗啉-3-酮-2-乙酸甲酯;再用脂肪酶CAL-B在甲基叔丁基醚/水（体积分数=1/9）混合溶剂中将其选择性水解,制备光学纯（R）-N-苄基吗啉-3-酮-2-乙酸;经过酰胺化、还原/胺解,得到（R）-N-苄基吗啉-3-酮-2-乙酰（4-脒基）苄胺;所得化合物结构经IR,1H NMR,13C NMR表征确证.文中还讨论了酶源、溶剂、含水量、反应时间等因素对水解拆分的影响.