Characterization of a fibrinogen-clotting enzyme from Trimeresurus stejnegeri venom, and comparative study with other venom proteases

Trimeresurus stejnegeri venom, which contains TSV-PA (a specific plasminogen activator sharing 60-70% sequence homology with venom fibrinogen-clotting enzymes), also possesses fibrinogen-clotting activity in vitro. A fibrinogen-clotting enzyme (stejnobin) has been purified to homogeneity by gel filtration and ion-exchange chromatography on a Mono-Q column. It is a single-chain glycoprotein with a mol. wt of 44,000. The NH2-terminal amino acid sequence of stejnobin shows great homology with venom fibrinogen-clotting enzymes and TSV-PA. Like TSV-PA, stejnobin was able to hydrolyse several chromogenic substrates. Comparative study of substrate specificities of stejnobin and other venom proteases purified in our laboratory was carried out on five chromogenic substrates. Stejnobin clotted human fibrinogen with a specific activity of 122 NIH thrombin-equivalent units/mg protein. However, stejnobin did not act on other blood coagulation factors, such as factor X, prothrombin and plasminogen. Diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride inhibited its activity, whereas ethylenediamine tetracetic acid had no effect on it, indicating that it is a serine protease. Although stejnobin showed strong immunological cross-reaction with polyclonal antibodies raised against TSV-PA, it was interesting to observe that, unlike the case of TSV-PA, these antibodies did not inhibit the amidolytic and fibrinogen-clotting activities of stejnobin. (C) 1998 Elsevier Science Ltd. All rights reserved.

Purification and characterization of an irreversible serine protease inhibitor from skin secretions of Bufo andrewsi

Amphibian skin secretions contain many bioactive compounds. In the present work, an irreversible serine protease inhibitor, termed baserpin, was purified for the first time from the skin secretions of toad Bufo andrewsi by Successive ion-exchange and gelfiltration chromatography. Baserpin is a single chain glycoprotein, with an apparent molecular weight of about 60 kDa in SDS-PAGE. Baserpin is an irreversible inhibitor and effectively inhibits the catalytic activity of trypsin, chymotrypsin and elastase. SDS-stable baserpin-trypsin complex could be seen in SDS-PAGE indicates that it possibly belongs to the serpin superfamily. According to the association rates determined, baserpin is a potent inhibitor of bovine trypsin (4.6 X 10(6) M-1 S-1), bovine chymotrypsin (8.9 X 10(6) M-1 s(-1)) and porcine elastase (6.8 X 10(6) M-1 s(-1)), whereas it shows no inhibitory effect on thrombin. The N-terminal sequence of baserpin is HTQYPDILIAKPXDK, which shows no similarity with other known serine protease inhibitors. (c) 2005 Elsevier Ltd. All rights reserved.

Frog albumin is expressed in skin and characterized as a novel potent trypsin inhibitor

A novel potent trypsin inhibitor was purified and characterized from frog Bombina maxima skin. A full-length cDNA encoding the protein was obtained from a cDNA library constructed from the skin. Sequence analysis established that the protein actually comprises three conserved albumin domains. B. maxima serum albumin was subsequently purified, and its coding cDNA was further obtained by PCR-based cloning from the frog liver. Only two amino acid variations were found in the albumin sequences from the skin and the serum. However, the skin protein is distinct from the serum protein by binding of a haem b (0.95 mol/mol protein). Different from bovine serum albumin, B. maxima albumin potently inhibited trypsin. It bound tightly with trypsin in a 1: 1 molar ratio. The equilibrium dissociation constants (K-D) obtained for the skin and the serum proteins were 1.92 x 10(-9) M and 1.55 x 10(-9) M, respectively. B. maxima albumin formed a noncovalent complex with trypsin through an exposed loop formed by a disulfide bond (Cys(53)-Cys(62)), which comprises the scissile bond Arg(58)(P-1)-His(59)(P-1'). No inhibitory effects on thrombin, chymotrypsin, elastase, and subtilisin were observed under the assay conditions. Immunohistochemical study showed that B. maxima albumin is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in the skin, suggesting that it plays important roles in skin physiological functions, such as water economy, metabolite exchange, and osmoregulation.

Isolation and preliminary characterization of a 22-kDa protein with trypsin inhibitory activity from toad Bufo andrewsi skin

A novel trypsin inhibitor termed BATI was purified to homogeneity from the skin extracts of toad Bufo andrewsi by successive ion-exchange, gel-filtration and reverse-phase chromatography. BATI is basic single chain glycoprotein, with apparent molecular weight of 22 kDa in SDS-PAGE. BATI is a thermal stable competitive inhibitor and effectively inhibits trypsin's catalytic activity on peptide substrate with the inhibitor constant (K-i) value of 14 nM and shows no inhibitory effect on chymotrypsin, thrombin and elastase. The N-terminal sequence of BATI is EKDSITD, which shows no similarity with other known trypsin inhibitors. (c) 2005 Elsevier Ltd. All rights reserved.

Antihemostatic molecules from saliva of blood-feeding arthropods

The ability to feed on vertebrate blood has evolved many times in various arthropod clades. Consequently, saliva of blood-feeding arthropods has proven to be a rich source of antihemostatic molecules. A variety of platelet aggregation inhibitors antagonize platelet responses to wound-generated signals, including ADP, thrombin, and collagen. Anticoagulants disrupt elements of both the intrinsic and extrinsic pathways. Vasodilators include nitrophorins (nitric oxide storage and transport heme proteins), a variety of peptides that mimic endogenous vasodilatory neuropeptides, and proteins that catabolize or sequester endogenous vasoconstrictors. Multiple salivary proteins may be directed against each component of hemostasis, resulting in both redundancy and in some cases cooperative interactions between antihemostatic proteins. The complexity and redundancy of saliva ensures an efficient blood meal for the arthropod, but it also provides a diverse array of novel antihemostatic molecules for the pharmacologist.

Two serine protease inhibitors from the skin secretions of the toad, Bombina microdeladigitora

Two serine protease inhibitors (named BMSI 1 and BMSI 2, respectively) were identified from the skin secretions of the toad, Bombina microdeladigitora. The cDNAs encoding BMSIs were cloned from a cDNA library prepared from the toad skin. The deduced complete amino acid sequences of BMSIs indicate that mature BMSI1 and BMSI2 are composed of 60 amino acids including 10 half-cystines to form 5 disulfide bridges. A FASTA search in the databanks revealed that BMSIs exhibit sequence similarity with other serine protease inhibitors from amphibians of the genus Bombina. BMSI1 potently inhibited trypsin and thrombin with a K(i) value of 0.02 mu M and 0.15 mu M, respectively. Sequence analysis revealed that all serine protease inhibitors from five amphibians of the genus Bombina share highly conserved primary structures. (c) 2007 Elsevier Inc. All rights reserved.

Biochemical and biological properties of Trimeresurus jerdonii venom and characterization of a platelet aggregation-inhibiting acidic phospholipase A(2)

Several biochemical and biological activities such as phospholipase A(2), arginine esterase, proteolytic, L-amino acid oxidase, 5'nucleotidase, acetylcholinesterase, thrombin-like, anticoagulant, and hemorrhagic activities were determined for whole desiccated venom of Trimeresurus jerdonii. An acidic phospholipase (named TJ-PLA(2)) was purified by anionic exchange chromatography, gel filtration, and reverse phase HPLC. TJ-PLA(2) had a molecular weight of 16,000 and a pI of 4.8. TJ-PLA(2) was non-lethal to mice up to an i.p. dose of 15 mg/kg body weight and lacked neurotoxicity and myotoxicity. It induced edema in the footpads of mice. The purified enzyme inhibited ADP- and collagen-induced human platelet aggregation in a manner which was both dose- and time-dependent.

Molecular characterization of a weak fibrinogen-clotting enzyme from Trimeresurus jerdonii venom

A fibrinogen-clotting enzyme designed as jerdonobin-II was isolated from the venom of Trimeresurus jerdonii. It differed in molecular weight and N-terminal sequence with the previously isolated jerdonobin, a thrombin-like enzyme from the same venom. The enzyme consists of a single polypeptide chain with molecular weights of 30,000 and 32,000 under non-reducing and reducing conditions, respectively. Jerdonobin-II showed weak fibrinogen clotting activity and its activity unit on fibrinogen was calculated to be less than one unit using human thrombin as standard. The precursor protein sequence of jerodonobin-II was deduced from cloned cDNA sequence. The sequence shows high similarity (identity = 89%) to TSV-PA, a specific plasminogen activator from venom of T stejnegeri. Despite of the sequence similarity, jerdonobin-II was found devoid of plasminogen activating effect. Sequence alignment analysis suggested that the replacement of Lys(239) in TSV-PA to Gln(239) in jerdonobin-II might play an important role on their plasminogen activating activity difference. (C) 2005 Elsevier Ltd. All rights reserved.

大蹼铃蟾（Bombina maxima）白蛋白的皮肤表达、蛋白酶抑制及细胞调亡诱导活性

两栖类动物的皮肤是其得以生存的重要器官，它担负着许多生理功能，如呼吸、水份调节、温度控制、排泄、繁殖、抵抗微生物、防御天敌等。存在于两栖动物皮肤分泌物中的生物活性成分已成为研究热点。目前，己分离鉴定出许多具有各种生物活性的蛋白质及多肤。通过三步分离纯化过程：DEAE-SephadexA-50离子交换，SephadeX075凝胶过滤，和DEAE-SephadexA-50离子交换层析，我们从大蹼铃蟾（Bombinamaxima）皮肤匀浆物中分离得到纯化的大蹼铃蟾皮肤白蛋白（Bm-A-skin）。SDS-PAGE电泳表明，该蛋白为单链蛋白质，在还原状态下表观分子量为67kDa。非还原状态下至少存在三条带，分子量分别为50，55和110kDa。经N-端氨基酸序列分析，其序列均相同，故该蛋白在SDS存在下有同分异构体及多聚体形式。根据所测得的N端氨基酸序列及内肤序列设计引物，通过筛选已构建的大蹼铃蟾皮肤cDNA文库，获得了编码该蛋白的全长cDNA。经序列分析发现该蛋白由三个保守的血清白蛋白结构域组成，并且同人血清白蛋白及牛血清白蛋白的序列相似度分别为39％和38％。随后，我们从血清中也分离纯化到血清白蛋白（BmA-serum），并通过RT-PCR，从大蹼铃蟾肝脏中扩增得到其全长cDNA序列。对由cDNA序列推导的两个大蹼铃蟾白蛋白的氨基酸序列进行比较，发现二者基本相同，只有两个氨基酸的差异，即BmA-skin的Gly417，Ser569，在BmA-serum中均为Asn。造成这两个氨基酸差异的只有一个碱基的突变，即编码BmA-skinGly417，Ser569密码子的第二位碱基A在BmA-serum中变为G。另外，从肝脏获得的BmA-serum的cDNA3'非翻译区还有8个碱基的插入。经扫描光谱分析，BmA-skin含有大量的血红素b，含量为0．95moFinol蛋白，而BmA-serum中含量较少，为0.05mol／mol蛋白。经schiff试剂染色发现，BmA-skin及BmA-serum均为非糖蛋白质。两者均具有抑制trypsin水解小肚底物的活性，但对其它丝氨酸蛋白酶的活性则无抑制，如thrombin、chyomotryPSin、elastase及substilisin。利用表面等离子共振技术研究BmA-skin及BmA-serum与trysin的相互作用，分别得到它们与trrpsin结合的动力学常数，解离平衡常数KD为两者均通过由一对二硫键cys53-Cys62形成的一个暴露的活性位点环，以1：1分子摩尔比同tryrsin形成稳定的非共价结合的复合物，其反应活性位点为Arg58（P1）-His59（P1'）。利用免疫组织化学方法研究发现，BmA-skin广泛地分布于成年大蹼铃蟾上皮细胞的细胞膜及真皮的疏松结缔组织层。表明其在蛙皮肤的生理功能中发挥重要作用，如水及代谢物质交换，渗透压的维持，皮肤呼吸等。另外，我们还从非洲爪蟾（xenopus勿即is）的血清及皮肤中分离到其68扔a的血清白蛋白，经初步鉴定也具有trtPsin抑制活性，但其抑制机制与B．maxima白蛋白不同，还有待于进一步研究。通过MTT法研究发现，BmA-skin对人T淋巴细胞H9、C8166及hemin处理的红白血病细胞K562具有细胞毒活性。三种细胞经BmA-skin处理72h，CC50A片段化，细胞核形态变化及流式细胞仪分析，结果显示，BmA-skin具有选择性诱导细胞调亡的特性。而BmA-serum对三种细胞均无毒性作用，单独的hemin对三种细胞的，胜也很弱。实验结果表明，BmA-skin结合的血红素b可能对其细胞毒及诱导细胞调亡的活性具有较大的贡献。用Cy3标记的BmA-skin与Hg和C8166细胞保温后，发现其进入细胞内发挥作用，这可能是其诱导细胞调亡机制之一。

Amplified electrochemical aptasensor taking AuNPs based sandwich sensing platform as a model

Here, we report a sensitive amplified electrochemical impedimetric aptasensor for thrombin, a kind of serine protease that plays important role in thrombosis and haemostasis. For improving detection sensitivity, a sandwich sensing platform is fabricated, in which the thiolated aptamers are firstly immobilized on a gold substrate to capture the thrombin molecules, and then the aptamer functionalized Au nanoparticles (AuNPs) are used to amplify the impedimetric signals.

Multifunctional label-free electrochemical biosensor based on an integrated aptamer

Aptamers, which are in vitro selected functional oligonucleotides, have been employed to design novel biosensors (i.e., aptasensors) due to their inherent selectivity, affinity, and their multifarious advantages over traditional recognition elements. In this work, we reported a multifunctional reusable label-free electrochemical biosensor based on an integrated aptamer for parallel detection of adenosine triphosphate (ATP) and alpha-thrombin, by using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). A An electrode as the sensing surface was modified with a part DNA duplex which contained a 5'-thiolated partly complementary strand (PCS) and a mixed aptamer (MBA).

Ultrasensitive colorimetric detection of protein by aptamer - Au nanoparticles conjugates based on a dot-blot assay

A simple, rapid and ultrasensitive colorimetric detection of protein using aptamer-Au nanoparticles (AuNPs) conjugates based on a dot-blot array has been developed, which was combined with the unique optical properties of AuNPs, enabling the visual detection of protein within minutes without any instrument.

DNAzyme-based Colorimetric Sensing of Lead (Pb2+) Using Unmodified Gold Nanoparticle Probes

Novel functional oligonucleotides, especially DNAzymes with RNA-cleavage activity, have been intensively studied due to their potential applications in therapeutics and sensors. Taking advantage of the high specificity of 17E DNAzyme for Pb2+, highly sensitive and selective fluorescent, electrochemical and colorimetric sensors have been developed for Pb2+. In this work, we report a simple, sensitive and label-free 17E DNAzyme-based sensor for Pb2+ detection using unmodified gold nanoparticles (GNPs) based on the fact that unfolded single-stranded DNA could be adsorbed on the citrate protected GNPs while double-stranded DNA could not. By our method the substrate cleavage by the 17E DNAzyme in the presence of Pb2+ could be monitored by color change of GNPs, thereby Pb2+ detection was realized.

Multifunctional G-Quadruplex Aptamers and Their Application to Protein Detection

Two significant G-quadruplex aptamers named AGRO100 and T30695 are identified as multi functional aptamers that can bind the protein ligands nucleolin or HIV-1 integrase and hemin. Besides their strong binding to target proteins, both AGRO100 and T30695 exhibit high hemin-binding affinities comparable to that of the known aptamer (termed PS2M) selected by the in vitro evolution process. Most importantly, their corresponding hemin-DNA complexes reveal excellent peroxidase-like activities. higher than that of the reported hemin-PS2M DNAzyme. This enables these multifunctional aptamers to be applied to the sensitive detection of proteins. which is demonstrated by applying AGRO100 to the chemiluminescence detection of nucleolin expressed at the surface of HeLa cells. Based on the specific AGRO100-nucleolin interaction, the surface-expressed nucleolin of HeLa cells is labeled in situ with the hemin-AGRO100 DNAzyme, and then determined in the luminol-H2O2 system.