Arginine Ester Hydrolase Research Articles

SNAKE BITE PRESENTING AS INTRACRANIAL BLEED WITH NORMAL COAGULATION PARAMETERS

Snakebite being commonly encountered emergency in our country and most dreaded one too. It has been estimated that as many as 2.8 million people are bitten by snakes, and 45 900 people die from snakebite every year in India1. The most common coagulopathy associated with snake-bite envenoming is Venom Induced Consumptive Coagulopathy. Venom contain enzymes like proteases, phospholipase A2, hyaluronidase and arginine ester hydrolase. Phospholipase A2 is the factor responsible for hemolysis secondary to the esterlytic effect on the red cell membranes The hyaluronidase causes spread of the venom in the subcutaneous tissue by disrupting mucopolysaccharides. In majority of cases there is disruption in coagulation profile causing increase in PT, INR, aPTT, thrombocytopenia and increase in FDP, which suggests DIC as the probable cause for intracerebral hemorrhage . But always it is not true there are some cases in which there is hemorrhagic risk without alteration in coagulation profile. All my 3 cases present to us with normal coagulation profile , One 26 year old male present within one hour of snake bite and died within 3 days of the bite, while other two presented lately that is 2and 6 day of the snake bite ,of which both survived and had no residual focal deficit at time of discharge. This delayed clinical, laboratory manifestation of vasculotoxic snake due to the delayed seepage of venom from deeper reservoirs in the bite site or due to disassembly of the antigen-antibody complex with reinstitution of circulating unbound venom constituents. Intracranial hemorrhages are poorly understood in case of snake bite and can occur later complication also even after treatment with ASV. Still use of FFP is not advocated in much studies, there is immense need to investigate this area. Use of ASV and FFP without increased WBCT to avoid later complication is to be studied.

Isolation and characterization of the thrombin-like enzyme from Cryptelytrops albolabris (white-lipped tree viper) venom

A thrombin-like enzyme (termed albolabrase) was isolated in purified form from the venom of Cryptelytrops albolabris (white-lipped tree viper) using high performance anion ion exchange and gel filtration chromatography. The molecular mass of albolabrase was 33.7 kDa as determined by SDS-PAGE and 35.8 kDa as determined by Superose gel filtration chromatography. The N-terminal sequence was determined to be VVGGDECNINE which is homologous to many snake venom thrombin-like enzymes. Albolabrase exhibits both arginine ester hydrolase and arginine amidase activities and the enzyme is fastidious towards tripeptide chromogenic anilide substrates. The fibrinogen clotting activity was optimum at 3mg/mL bovine fibrinogen, and showed distinct species differences in the following decreasing order: bovine fibrinogen>dog fibrinogen≈human fibrinogen>goat fibrinogen. The enzyme failed to clot both rabbit and cat fibrinogens. Reversed-phase HPLC analysis on the breakdown products of fibrinogenolytic action of albolabrase indicated that the enzyme belongs to the AB class of snake venom thrombin-like enzyme. In the indirect ELISA, IgG anti-albolabrase reacted extensively with most crotalid venoms, except with Tropidolaemus wagleri and Calloselasma rhodostoma venoms. The double sandwich ELISA, however, showed that anti-albolabrase reacted strongly only with venoms from the Trimeresurus complex, and that the results support the proposed new taxonomy changes concerning the Trimeresurus complex.

ChemInform Abstract: Studies on Hemorrhagic Toxins from the Venoms of Trimeresurus mucrosquamatus, Crotalus ruber ruber, Vipera aspis aspis, and Agkistrodon acutus and Arginine Ester Hydrolases from Trimeresurus mucrosquamatus Venom

Venom samples were corrected from several poisonous snakes, such as Bungarus multicinctus, Trimeresurus mucrosquamatus, T. gramineus, T. flavoviridis, and Agkistrodon acutus, and stored in a desiccator at room temperature for 25 to 31 years. Then they were compared with fresh venoms as to their biological activities. The characteristic local symptoms produced by the bite of venomous snakes of Crotalidae and Viperidae are hemorrhage, necrosis and muscular degeneration. Hemorrhagic toxins were purified from Trimeresurus mucrosquamatus, Crotalus ruber ruber, Vipera aspis aspis, and Agkistrodon acutus venoms and their biological, biochemical, and pathological properties were investigated. Arginine ester hydrolases are present in the venoms of Crotalidae and Viperidae, but are not found in the venoms of Elapidae and Hydrophiidae. In this paper we describe the enzymatic and biological activities of arginine ester hydrolases from a Trimeresurus mucrosquamatus venom.

Isolation and characterization of the thrombin-like enzyme from Cryptelytrops purpureomaculatus venom

A thrombin-like enzyme, purpurase, was purified from the Cryptelytrops purpureomaculatus (mangrove pit viper) venom using high performance ion-exchange and gel filtration chromatography. The purified sample (termed purpurase) yielded a homogeneous band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 35,000. The N-terminal sequence of purpurase was determined to be VVGGDECNINDHRSLVRIF and is homologous to many other venom thrombin-like enzymes. Purpurase exhibits both arginine ester hydrolase and amidase activities. Kinetic studies using tripeptide chromogenic anilide substrates showed that purpurase is not fastidious towards its substrate. The clotting times of fibrinogen by purpurase were concentration dependent, with optimum clotting activity at 3 mg fibronogen/mL. The clotting activity by purpurase was in the following decreasing order: cat fibrinogen > human fibrinogen > dog fibrinogen > goat fibrinogen >> rabbit fibrinogen. Reversed-phase HPLC analysis of the products of action of purpurase on bovine fibrinogen showed that only fibrinopeptide A was released. Indirect ELISA studies showed that anti-purpurase cross-reacted strongly with venoms of most crotalid venoms, indicating the snake venom thrombin-like enzymes generally possess similar epitopes. In the more specific double-sandwich ELISA, however, anti-purpurase cross-reacted only with venoms of certain species of the Trimeresurus complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex.

Effects of Russell's viper venom fractions on systemic and renal hemodynamics

Changes in systemic and renal hemodynamics induced by Russell's viper venom are well established. The component of the venom responsible for hemodynamic alteration has not been identified. By Sephadex column chromatography five fractions of Russell's viper ( Daboia russellii siamensis) venom were isolated. Each venom fraction consisted of phospholipase A 2, proteolytic enzyme, phosphomonoesterase, phosphodiesterase, arginine ester hydrolase and hyaluronidase of varying activities. Hemodynamic effects of each venom fraction including mean arterial pressure, cardiac output, systemic and renal vascular resistance, renal blood flow and glomerular filtration rate were studied in five groups of dogs; each group had four dogs. Minimal hemodynamic changes were observed in dogs receiving venom fraction I. Increased renal vascular resistance with diminution of renal blood flow and glomerular filtration rate was observed in dogs receiving venom fractions II, III, IV and V. A markedly increased renal vascular resistance with maximal decrease in renal blood flow and glomerular filtration rate was caused by fraction III of the venom with highest PLA 2 and proteolytic enzyme activities. However, renal hemodynamic changes appeared to correlate better with proteolytic enzyme activity than PLA 2 activity. The findings suggested the proteolytic enzyme as an important determinant of hemodynamic alteration. Fractional excretion of Na was increased in dogs injected with venom fraction IV, and is presumed to be due to the inhibition of tubular reabsorption of Na by a natriuretic factor in this venom fraction.

Purification and Characterization of a Growth Factor-like Which Increases Capillary Permeability from Vipera lebetina Venom

We have investigated the effect of Vipera lebetina venom on capillary permeability and isolated an increasing capillary permeability protein (ICPP) which is devoid of arginine ester hydrolase and phospholipase A2 activities. This protein was purified with a yield of about 0.2% by fast protein liquid chromatography (FPLC) using successively Superose 12, Mono Q, and Mono S columns and by high-pressure liquid chromatography (HPLC) on a C8 reverse-phase column. The purified protein migrated on SDS–PAGE as a band of about 27 kDa under nonreducing conditions and as a band of about 16 kDa under reducing conditions. Chromatography on a C8 column of reduced and alkylated protein yielded a single peak suggesting that this protein is homodimeric. This protein was refractory to Edman degradation chemistry. We used successfully a chemical unblocking involving the incubation of the protein with HCl in anhydrous methanol. The N-terminal amino acid sequence clearly shows considerable similarity to that of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF).

Studies on hemorrhagic toxins from the venoms of Trimeresurus mucrosquamatus, Crotalus ruber ruber, Vipera aspis aspis and Agkistrodon acutus and arginine ester hydrolases from Trimeresurus mucrosquamatus venom

Venom samples were corrected from several poisonous snakes, such as Bungarus multicinctus, Trimeresurus mucrosquamatus, T. gramineus, T. flavoviridis, and Agkistrodon acutus, and stored in a desiccator at room temperature for 25 to 31 years. Then they were compared with fresh venoms as to their biological activities. The characteristic local symptoms produced by the bite of venomous snakes of Crotalidae and Viperidae are hemorrhage, necrosis and muscular degeneration. Hemorrhagic toxins were purified from Trimeresurus mucrosquamatus, Crotalus ruber ruber, Vipera aspis aspis, and Agkistrodon acutus venoms and their biological, biochemical, and pathological properties were investigated. Arginine ester hydrolases are present in the venoms of Crotalidae and Viperidae, but are not found in the venoms of Elapidae and Hydrophiidae. In this paper we describe the enzymatic and biological activities of arginine ester hydrolases from a Trimeresurus mucrosquamatus venom.

Isolation and characterization of an arginine ester hydrolase from Bothrops jararacussu venom which induces contractions of the isolated rat uterus.

The isolation and partial characterization of a serine protease with arginine ester hydrolase activity from Bothrops jararacussu snake venom are described. The purification procedure consisted of a gel filtration of the crude venom on Sephadex G-75 followed by an ion-exchange chromatography of the active fraction on DEAE-cellulose and a rechromatography on Bio-Rex 70 resin. The esterase fraction (DI-III), M(r) = 25,000 by SDS-PAGE, showed proteolytic activity on fibrinogen and casein. After 2 hr incubation, the A alpha and B beta chains of fibrinogen were intensely hydrolysed, while the gamma chain kept apparently intact, even after 20 hr of incubation. In spite of that, DI-III did not clot fibrinogen. DI-III induced edema in the rat paw. Although unable to release bradykinin, it induced contractions of the isolated rat uterus. DI-III did not catalyse the hydrolysis of bradykinin. Its arginine ester hydrolase activity was completely inhibited by diisopropyl fluorophosphate after 1 hr incubation, but not by phenylmethylsulfonyl fluoride under the same conditions.

Isolation, properties and N-terminal amino acid sequence of a factor V activator from Vipera lebetina (Levantine viper) snake venom

A factor V activator (VLFVA) was separated from Vipera lebetina venom by gel filtration on Sephadex G-100 superfine, followed by chromatography on CM-cellulose and on heparin-agarose. This enzyme (VLFVA) with a molecular mass of 28.4 kDa, as determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry, is a single-chain glycoprotein containing seven residues of neutral sugars, seven residues of hexosamines and three residues of neuraminic acid per molecule. The treatment with N-glycosidase F lowered the molecular mass approximately 6%. The N-terminal sequencing of VLFVA up to the 30th residue evidenced a high homology with Vipera russelli factor V activator RVV-Vγ (90% identity). Aside from factor V, no other protein substrate for VLFVA has yet been identified. VLFVA hydrolyzes several synthetic arginine ester substrates, such as benzoylarginine ethyl ester (BAEE), tosylarginine methyl ester (TAME) and amide substrates such as Pro-Phe-Arg-MCA. The arginine ester hydrolase activity of the enzyme is markedly lower than that of the crude venom. The ability of VLFVA to activate factor V and its activity to BAEE and TAME were inhibited by the serine proteinase inhibitor, diisopropylfluorophosphate. VLFVA is thermostable protein, heating for 20 min at 70°C does not alter the arginine esterase activity of the enzyme.

Electrophoretic profiles and biological activities: Intraspecific variation in the venom of the malayan pit viper ( Calloselasma rhodostoma)

The Malayan pit viper ( Calloselasma rhodostoma) is of major clinical significance both as a leading cause of snakebite and as the source of ancrod (Arvin ™). Although its venom has been extensively studied, the degree to which venom composition varies between individuals is poorly known. We individually analysed the venoms of over 100 C. rhodostoma using isoelectric focusing. In all populations, females produced an intense band that was absent from all males, and significant ontogenetic variation was detected. Principal components analysis of the banding profiles also revealed strong geographic variation, which was significantly congruent with variation in the biological activities of the venom (phosphodiesterase, alkalinephosphoesterase, l-amino acid oxidase, arginine ester hydrolase, 5′-nucleotidase, thrombin-like enzyme, haemorrhagic activity). Studies of captive-bred snakes indicate that the intraspecific variation in venom is genetically inherited rather than environmentally induced. The intraspecific variation in venom composition and biological activity could be of applied importance to snakebite therapy, both in correct diagnosis of the source of envenomation and in the development of a more effective antivenom. Greater attention should be given to the source of C. rhodostoma venom used in research to ensure reproducibility of results.

Comparative study of fibrinogen degradation by four arginine ester hydrolases from the venom of Agkistrodon caliginosus (Kankoku-Mamushi)

When purified capillary permeability-increasing (CPI)-enzyme-1, CPI-enzyme-2, kininogenase-1 or kininogenase-2 was incubated with human fibrinogen at a ratio of 1:100 by weight, a loss of fibrinogen coagulability was seen at prolonged incubation times. CPI-enzyme-2 and kininogenase-1 caused a rapid loss of coagulability, while CPI-enzyme-1 and kininogenase-2 acted more slowly. When human fibrinogen and each enzyme were incubated at 37 °C, CPI-enzyme-2 first cleaved the Aα-chain then the Bβ-chain. The action of CPI-enzyme-1 was similar, but slower. Kininogenase-1 initially caused slow degradation of the Bβ-chain than the Aα-chain, while kininogenase-2 only caused slow cleavage of the Aα-chain. Although these enzymes did not show thrombin-like activity, CPI-enzyme-2 was able to release fibrinopeptide B faster than fibrinopeptide A, while kininogenase-1 only released fibrinopeptide A. These results indicate that these enzymes differ in their ability to degrade human fibrinogen.

A fibrinogen-clotting serine proteinase from Cerastes cerastes (horned viper) venom with arginine-esterase and amidase activities. Purification, characterization and kinetic parameter determination

An enzyme displaying proteolytic activity toward the natural substrate casein as well as clotting activity on fibrinogen was purified to homogeneity from Cerastes cerastes (horned viper) venom and characterized. The enzyme is constituted of two identical subunits of mol. wt 48,500 as determined by SDS-polyacrylamide gel electrophoresis, and has an isoelectric point of 3.75. N-terminal sequencing up to the 33rd residue evidenced a high homology with other snake venom proteinases. The proteinase is of serine-type as indicated by high sensitivity to DFP and shows both arginine-ester hydrolase and amidase activities on synthetic substrates. Both specific activities were 30-fold higher than the respective activities found in the crude venom. The Km value determined for arginine-containing substrate BAEE was 3.0×10−4M and the Km for chromogenic substrate CBS 34–47 0.65×10−4M. The VmKm ratio, however, was two-fold higher for BAEE than for CBS 34–47; the arginine-esterase activity of this enzyme is thus slightly higher than its amidase activity.

Comparative study of the enzymatic, hemorrhagic, procoagulant and anticoagulant activities of some animal venoms

1. The enzymatic, hemorrhagic, procoagulant and anticoagulant activities of venoms of some animals including snakes, lizards, toads, scorpions, spider, wasps, bees and ants were compared. 2. Snake venom was the richest source of enzymes among the animal venoms. Most other animal venoms were devoid of phosphodiesterase, l-amino acid oxidase, alkaline phosphomonoesterase and acetylcholinesterase activities and only a few exhibited arginine ester hydrolase activity. These venoms, however, exhibited wide ranges of protease, 5'-nucleotidase and hyaluronidase activities. Most of the animal venoms examined exhibited some phospholipase A activity. 3. Other than snake venoms, only venoms of the toad Bufo calamita and the lizards were hemorrhagic, and only venoms of the social wasps, social bees and harvester ant exhibited strong anticoagulant activity. Procoagulant activity occurs only in snake venoms.

Isolation and characterization of arginine ester hydrolase from Heloderma horridum (beaded lizard) venom

1. I. An arginine ester hydrolase was isolated from Heloderma horridum (beaded lizard) venom by Sephadex G-75, DEAE-Sephacel and Q-Sepharose column chromatography, resulting in 5.4 mg of purified enzyme from 320.0 mg of crude venom. 2. 2. The enzyme was shown to be homogeneous by both SDS and non-SDS disc electrophoresis on polyacrylamide gel at pH 8.3. 3. 3. The enzyme possesses arginine ester hydrolase and transglutaminase-like activities, but did not exhibit clotting activity. 4. 4. Molecular weight was determined to be ca 29 kDa, with an isoelectric point of 4.4. 5. 5. The enzyme was stable to heat treatment (95°C, 10 min) and to pH changes over the range 2–11. 6. 6. The arginine ester hydrolase was inactivated by diisopropylfluorophosphate (DFP), β-mercaptoethanol and N-bromosuccinimide, suggesting that serine, disulfide bonds and tryptophan are involved in enzymatic activity. 7. 7. Amino terminal sequences were determined and appear to be similar to porcine pancreatic kallikrein.

The biological properties of venoms of some american coral snakes (Genus Micrurus)

1. 1. The biological properties of nine venom samples from six taxa of Micrurus were investigated. The venoms exhibited low protease, phosphodiesterase and 5′-nucleotidase activities, moderate to strong phospholipase A and hyaluronidase activities, variable l-amino acid oxidase activity and were devoid of arginine ester hydrolase and thrombin-like activities. Some venom samples exhibited strong acetylcholinesterase activity. Venoms of M. c. dumerili and M. frontalis exhibited exceptionally high alkaline phosphomonesterase activity while two of the M. f. fulvius venom samples tested exhibited strong hemmorrhagic activity in mice. 2. 2. The polyacrylamide gel electrophoretic patterns of the venoms indicate that most of the Micrurus venom proteins are basic proteins. All Micrurus venoms tested exhibited similar SDS-polyacrylamide gel electrophoretic patterns, with an intense low mol.wt protein band. 3. 3. The Micrurus venoms appear to exhibit biological properties similar to other elapid venoms found in Asia and Africa. There are, however, no common characteristics in the biological properties of the venoms examined at the generic level.

A comparative study of the biological properties of venoms of some old world vipers (Subfamily viperinae)

1. 1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, hyaluronidase, alkaline phosphomonoesterase, 5'-nucleotidase, arginine ester hydrolase, phospholipase A, l-amino acid oxidase and protease activities of 30 samples of venoms from nine species (12 taxa) of the old world vipers (Subfamily Viperinae) including snakes from the genera Bitis, Causus, Cerastes, Echis, Eristicophis and Pseudocerastes, were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. 2. Examination of the biological properties of the venoms of the Viperinae tested indicates the presence of common venom biological characteristics at the various phylogenic levels. 3. 3. Venoms of most species of the Viperinae examined exhibited characteristic biological properties at the species level, and this allows the differentiation of the Viperinae species by differences in their biological properties. 4. 4. Particularly useful for this purpose, are the effects of venom on kaolin-cephalin clotting time of platelet poor rabbit plasma and the Sephadex G-75 gel filtration pattern and arginine ester hydrolase activity of the venom.

A comparative study of the biological activities of rattlesnake (genera Crotalus and Sistrurus) venoms

1. The hemorrhagic, procoagulant, anticoagulant, protease, arginine ester hydrolase, phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, hyaluronidase, phospholipase A and l-amino acid oxidase activities of 50 venom samples from 20 taxa of rattlesnake (genera Crotalus and Sistrurus) were examined. 2. The results show that notwithstanding individual variations in the biological activities of Crotalus venoms and the wide ranges of certain biological activities observed, there are some common characteristics at the genus and species levels. 3. The differences in biological activities of the venoms compared can be used for differentiation of the species. Particularly useful for this purpose are the thrombin-like enzyme, protease, arginine ester hydrolase, hemorrhagic and phospholipase A activities and kaolin-cephalin clotting time measurements.

A comparative study of the biological properties of some sea snake venoms

1. 1. The protease, phosphodiesterase, alkaline phosphomonoesterase, l-amino acid oxidase, acetylcholinesterase, phospholipase A, 5′-nucleotidase, hyaluronidase, arginine ester hydrolase, procoagulant, anticoagulant and hemorrhagic activities of ten samples of venoms from seven taxa of sea snakes were examined. 2. 2. The results show that venoms of sea snakes of both subfamilies of Hydrophiinae and Laticaudinae are characterized by a very low level of enzymatic activities, except phospholipase A activity and, for some species, hyaluronidase activity. 3. 3. Because of the low levels of enzymatic activities and the total lack of procoagulant and hemorrhagic activities, venom biological properties are not useful for the differentiation of species of sea snakes. Nevertherless, the unusually low levels of enzymatic activities of sea snake venoms may be used to distinguish sea snake venoms from other elapid or viperid venoms.

A comparative study of the biological properties of some venoms of snakes of the genus Bothrops (American lance-headed viper)

1. 1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, alkaline phosphomonoesterase, 5′-nucleotidase, hyaluronidase, arginine ester hydrolase, phospholipase A, l-amino acid oxidase and protease activities of 26 samples of venoms from 13 species of Bothrops were determined, and the Sephadex G-75 gel filtration patterns for some of the venoms also examined. 2. 2. The results show that while there are considerable individual variations in the biological activities of many of the Bothrops venoms tested, there are some common characteristics at the genus and species levels. 3. 3. The differences in the biological properties of the Bothrops venoms tested can be used for the differentiation of most Bothrops species examined.

A comparative study of the biological properties of Dendroaspis (mamba) snake venoms

1. The biological properties of twelve samples of venoms from all four species of Dendroaspis (mamba) were investigated. 2. Dendroaspis venoms generally exhibited very low levels of protease, phosphodiesterase and alkaline phosphomonoesterase; low to moderately low level of 5'-nucleotidase and very high hyaluronidase activities, but were devoid of l-amino acid oxidase, phospholipase A, acetylcholinesterase and arginine ester hydrolase activities. The ususual feature in venom enzyme content can be used to distinguish Dendroaspis venoms from other snake venoms. 3. All Dendroaspis venoms did not exhibit hemorrhagic or procoagulant activity. Some Dendroaspis venoms, however, exhibited strong anticoagulant activity. The intravenous median lethal dose of the venoms ranged from 0.5 μg g mouse to 4.2 μg g mouse. 4. Venom biological activities are not very useful for the differentiation of the Dendroaspis species. The four Dendroaspis venoms, however, can be differentiated by their venom SDS-polyacrylamide gel electrophoretic patterns.