scholarly journals Isolation and partial purification of anticoagulant fractions from the venom of the Iranian snake Echis carinatus.

2012 ◽  
Vol 60 (1) ◽  
Author(s):  
Mahdi Babaie ◽  
Hossein Zolfagharian ◽  
Hossein Salmanizadeh ◽  
Abbas Zare Mirakabadi ◽  
Hafezeh Alizadeh
Keyword(s):  
Blood Coagulation ◽  
Gel Filtration ◽  
Partial Purification ◽  
Crude Venom ◽  
Molecular Weights ◽  
Time Value ◽  
Sds Page ◽  
Echis Carinatus ◽  
The Many ◽  
Coagulation Pathway

Many snake venoms comprise different factors, which can either promote or inhibit the blood coagulation pathway. Coagulation disorders and hemorrhage belong to the most prominent features of bites of the many vipers. A number of these factors interact with components of the human blood coagulation. This study is focused on the effect of Echis carinatus snake venom on blood coagulation pathway. Anticoagulant factors were purified from the Iranian Echis carinatus venom by two steps: gel filtration (Sephadex G-75) and ion-exchange (DEAE-Sephadex) chromatography, in order to study the anticoagulant effect of crude venom and their fractions. The prothrombin time was estimated on human plasma for each fraction. Our results showed that protrombin time value was increase from 13.4 s to 170 s for F2C and to 280 s for F2D. Our study showed that these fractions of the venom delay the prothrombine time and thus can be considered as anticoagulant factors. They were shown to exhibit proteolytic activity. The molecular weights of these anticoagulants (F2C, F2D) were estimated by SDS/PAGE electrophoresis. F2C comprises two protein bands with molecular weights of 50 and 79 kDa and F2D a single band with a molecular weight of 42 kDa.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

Affinity Chromatography of Human Factor VIII Using Human and Rabbit Antibodies to Factor VIII

1979 ◽  
Vol 42 (04) ◽  
pp. 1306-1315 ◽  
Author(s):  
Janet L Lane ◽  
H Ekert ◽  
A Vafiadis
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Affinity Chromatography ◽  
Protein Concentration ◽  
Gel Filtration ◽  
Subunit Structure ◽  
Molecular Weights ◽  
Human Factor Viii ◽  
Sds Page ◽  
Normal Human

SummaryFactor VIII, purified by gel filtration on Sepharose 2B, has an 8 band multiple subunit structure, with molecular weights ranging from 30,000 to 230,000, on reduction and SDS-PAGE at a protein concentration of 400 μg/gel. Affinity chromatography of this factor VIII preparation with insolubilized haemophilic antibody to factor VIII showed that 45-81% VIII:C and 0-33% VIILRag were attached to the column. Elution of the column with 0.25 M CaCl2 did not show VIII:C or VIILRag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed that 95 % of the protein bound by haemophilic antibody had a molecular weight similar to the low molecular weight subunits of the reduced factor VIII.In control experiments with normal Human IgG, 3% of VIII:C and 5% of VIILRag were attached to the column. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the protein, showed 2 faint bands with molecular weight consistent with heavy and light chains of IgG.Similar experiments with antibody to factor VIII showed that 67-83% of VIILC and 61-76% of VIII:Rag were attached to the column. Elution of the column with 0.25 M CaCl2 showed 10% of the applied VIII:C, but no VIII:Rag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed an 8 band subunit structure similar to the reduced factor VIII.

Purification and Some Chemical Properties of Thrombin-Like Enzyme from Trimeresurus Flavoviridis Venom

1986 ◽  
Vol 55 (01) ◽  
pp. 024-030 ◽  
Author(s):  
T Kosugi ◽  
Y Ariga ◽  
M Nakamura ◽  
K Kinjo
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Fold Increase ◽  
Crude Venom ◽  
Fibrin Gel ◽  
Bovine Thrombin ◽  
Trimeresurus Flavoviridis ◽  
Sds Page ◽  
Ammonium Sulphate Fractionation

SummaryThere has been no previous report indicating whether thrombin-like enzyme is contained in the venomof Trimeresurus flavoviridis whichhas the strongest toxic effect in the case of Habu bite. The presentstudy was undertaken to clarify the existence of thrombin-like enzyme in Trimeresurus flavoviridis venom. As a starting material, lyophilized crude venom of Trimeresurus flavoviridis was used, and ammonium sulphate fractionation, gel filtration using Sephadex G-25, Sephadex G-150 and arginine-Sepharose affinity chromatography were carried out to separate and purify a thrombin-like enzyme from the crude venom. The enzyme was purified to a 137-fold increase in specific activity and the purified preparation revealed a single band on SDS-PAGE. The molecular weight of the enzyme was estimated to be 65,000-70,000 daltons by means of SDS-PAGE and gel filtration, and its isoelectric point was pH 4.5-5.5. Furthermore, the optimal pH of the enzyme was in the range of pH 8.0 to 8.5. Some of the differences in enzymatic properties between this enzyme and bovine thrombin were studied. The snake enzyme could coagulate only rabbit plasma and convert only purified rabbit fibrinogen to fibrin gel. In addition, this thrombin-like enzyme released only fibrinopeptide A from purified rabbit fibrinogen and did not release fibrinopeptide B.

Homologues Of Fibrin X Oligomers

1981 ◽  
Author(s):  
R Hafter ◽  
H Graeff ◽  
R v Hugo
Keyword(s):  
Gel Filtration ◽  
Advanced Ovarian Cancer ◽  
Simultaneous Action ◽  
Coagulation Disorder ◽  
Subunit Structure ◽  
Molecular Weights ◽  
Sds Page ◽  
D Dimer

Crosslinked fibrin derivatives signalize intravascular coagulation. D-dimer, Y-D and X oligomers are observed in plasma from obstetric patients with severe coagulation disorder. They are also found in ascitic fluid from patients with advanced ovarian cancer and can be produced in vitro by simultaneous action of thrombin, plasmin and factor XIII with fibrinogen. The study was aimed to evaluate the subunit structure of separated molecular entities. The derivatives were separated by 4% SDS-PAGE preceded in case of the in vivo products by gel filtration and/or by immunoabsorption technique. The gels were sliced at the respective migration positions and derivatives therein reelectrophoresed on 7,5% gels after reduction. Subunit characterisation revealed that D-dimer is composed of the chain remnants γ1-γ1, β2, α2, while Y-D is composed of γ-γ1, β2, α3, α2, besides αE, βE and γE Crosslinked X oligomers are composed of γ-γ, γ-γ1, β, β1, γ2, α1 and α2 besides αE, βE and γE Three possible combinations of plasmin degraded and undegraded dimeric γ-chains were observed in vivo and in vitro: γ-γ γ-γ1 and γ1-γ1. The ratio of degraded (γ1) to undegraded γ-chains in dimeric γ-chain patterns indicates the mol. structure of the respective derivative. Two X oligomers could be demonstrated in which the ratio of γ-γ to γ-γ1 in terms of stain intensity was either 1:1 or 2:1. Their subunit compositions are in accordance with structures describ- able as D-X-Y and D-X-X-Y. Their molecular weights, calculated from the subunit compositions are 476,000 and 716,000 respectively. - It is proposed that crosslinked X oligomers exist as a homologous family with increasing X fragment content.

ISOLATION AND FUNCTIONAL PROPERTIES OF MONOMERIC BLOOD COAGULATION FACTOR XI

1987 ◽  
Author(s):  
M Berrettini ◽  
M J Heeb ◽  
J H Griffin
Keyword(s):  
Blood Coagulation ◽  
Gel Filtration ◽  
Coagulation Factor ◽  
Fluid Phase ◽  
Factor Ix ◽  
Contact Activation ◽  
Factor Xi ◽  
Dimeric Structure ◽  
Sds Page ◽  
Mild Reduction

To evaluate the significance of the normal dimeric structure (160,000 MW) of blood coagulation Factor XI (F.XI), a monomeric form (80,000 MW) was produced by mild reduction and alkylation of native F.XI. Since initial efforts to reduce and alkylate F.XI in solution inactivated the molecule, F.XI was bound to high MW kininogen (HMWK) to stabilize the native structure. Purified F.XI was bound to HMWK-Sepharose, and the column was washed for 2 h with 40 μM dithiothreitol in 4mM acetate buffer, 2mM EDTA, 1mM benzamidine, pH 7.8, and then for 2 h with 50 μM iodoacetamide in the same buffer. Elution with 0.5 M NaCl gave a preparation containing ∼ 85% F.XI monomer and ∼ 15% dimer, as judged by nonreduced SDS-PAGE and by gel filtration of the radiolabeled preparation. The monomeric F.XI preparation had only 10% of the clotting activity of dimeric F.XI (per mole of enzymatic site) as measured in APTT clotting assays using F.XI deficient plasma. After activation with β-Factor XIIa in solution, the monomer F.XIa preparation exhibited 85% of the clotting activity of native F.XIa in unactivated PTT assays using F.XI deficient plasma. In addition, when compared to native F.XIa, monomeric F.XIa gave 65% amidolytic activity against the substrate, S-2366, and 75% activity against Factor IX in assays of the release of the activation peptide from 3H-Factor IX. Polystyrene tubes were coated with HMWK then blocked with 1% BSA to study the binding of 125I-F.XI to HWMK. When the binding of the 125I-labeled preparations of monomeric and dimeric forms of F.XI to HMWK was studied, two distinct components were identified in the association of dimeric F.XI, one with high affinity (Kd ∼ 2.5 X 10-9M) and one with less affinity (Kd ∼ 1.7 X 10-8M), while the binding of monomeric F.XI occurred with a single low affinity component (Kd ∼ 1.1 X 10-8M). These observations suggest that the dimeric structure of F.XI is required for efficient binding of the molecule to HMWK and for normal activation by the contact activation system in plasma, but that the dimeric structure of F.XIa does not play a role in the expression of the enzymatic activity against Factor IX in fluid phase.

scholarly journals Partial purification and characterization of collagen galactosyltransferase from chick embryos

1976 ◽  
Vol 155 (1) ◽  
pp. 145-153 ◽  
Author(s):  
L Risteli ◽  
R Myllyä ◽  
K I Kivirikko
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Helical Conformation ◽  
Tissue Homogenate ◽  
Partial Purification ◽  
Molecular Weights ◽  
Soluble Collagen ◽  
Insoluble Collagen ◽  
Chick Embryo Extract ◽  
Triton X

Collagen galactosyltransferase was purified 50-150-fold from chick-embryo extract. The tissue homogenate was prepared in the presence of Triton X-100, since the addition of the detergent doubled the enzyme activity in the homogenate and the extract. Three species of the enzyme activity with different molecular weights were recovered on gel filtration, the mol.wts. being about 450000, 200000 and 50000. Collagen galactosyltransferase activity was strongly inhibited by p-mercuribenzoate, and stimulated by the addition of dithiothreitol to the incubation system. Studies on substrate requirements indicated that denatured citrate-soluble collagen is a more effective substrate than gelatinized insoluble collagen, as judged from their Km values. Experiments on three peptide fractions prepared from citrate-soluble collagen indicated that a fraction with an average mol.wt. of 500-600 contained peptides large enough to meet a minimun requirement for interaction with the enzyme. However, longer peptides were clearly better substrates. When native and heat-denatured citrate-soluble collagens were compared as substrates, practically no synthesis of galactosylhydroxylysine was found with native collagen. This finding suggests that the triple-helical conformation of collagen prevents the galactosylation of hydroxylysine residues.

PURIFICATION AND CHARACTERIZATION OF THE PROCOAGULANT OF THE VENOM OF TROPIDECHIS CARINATUS

1987 ◽  
Author(s):  
P P Masci ◽  
A N Whitaker ◽  
J J Morrison ◽  
E A Bennett
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Factor Xa ◽  
Factor V ◽  
Crude Venom ◽  
Adult Rats ◽  
Sds Page ◽  
Sepharose 4B ◽  
Blue Sepharose

Tropidechis carinatus is a venomous elapid snake distributed throughout Eastern Queensland. It has been considered as a tropical relative of Notechis scutatus and, similarly, the crude venom contains an indirect prothrombin activator, which will clot plasma provided that Factor V is present. Myotoxins and neurotoxins are also present. Envenomated patients regularly develop disseminated intravascular coagulation. The crude whole venom of T.carinatus was shown to have five major components by gel filtration, SDS PAGE and HPLC, and even more components by isoelectric focusing. The procoagulant eluted with a molecular weight of 55,000, being found in peak II on gel filtration on Sephadex-G150. The procoagulant was purified using a combination of Sephadex-G150 chromatography and ion-exchange on DEAE Sephadex-A50 and shown to migrate as a single band of molecular weight 55.000 by SDS PAGE. On reduction by β -mercaptoethanol this component was resolvec into u heavy chain of molecular weight 30.000 and a light chain of 25,000. The procoagulant was shown to bind to con A-Sepharose 4B and Blue Sepharose 4B. Coagulation studies using this purified procoagulant confirmed a factor Xa-like activity activating prothrombin in the presence of factor V. The purified fraction is unstable in buffer solutions at 4°C, probably because of trypsin - like autodigestion. Ouchterlony studies of the procoagulants of T.carinatus and N.scutatus show both lines of homogeneity and spurring, indicating similarities but also significant differences between the two proteins. The purified procoagulant was lethal to adult rats, an IV injection of 10 μg killing in 1 - 2 minutes. Death was prevented by prior heparinization, suggesting that the procoagulant is toxic in the absence of neurotoxin and other components.

Purification of PRL receptors from toad kidney: comparisons with rabbit mammary PRL receptors

1988 ◽  
Vol 254 (3) ◽  
pp. C372-C382 ◽  
Author(s):  
M. Dunand ◽  
J. P. Kraehenbuhl ◽  
B. C. Rossier ◽  
M. L. Aubert
Keyword(s):  
Mammalian Species ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Structural Features ◽  
Polyclonal Antiserum ◽  
Partial Purification ◽  
Molecular Characteristics ◽  
Binding Characteristics ◽  
Sds Page

The binding characteristics of the prolactin (PRL) receptors present in toad (Bufo marinus) kidneys were investigated and compared to those of PRL receptors present in rabbit mammary glands. The molecular characteristics of the Triton X-100 solubilized renal and mammary PRL receptors were assessed by gel filtration and by migration analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after affinity labeling of the binding sites with 125I-human growth hormone. Similar results were obtained for both receptors. Partial purification of the toad PRL receptor could be achieved by affinity chromatography. The molecular weight of this purified receptor could be determined by analysis on SDS-PAGE. With the use of a polyclonal antiserum raised against a purified preparation of rabbit mammary PRL receptor, one or several antigenic epitope(s) could be identified on the core of the toad renal PRL receptor. In conclusion, although the structure and the biological role(s) of PRL have substantially changed during evolution, the receptor for this hormone has retained many of its structural features as could be assessed between an amphibian and a mammalian species on functionally different target tissues.

scholarly journals Identification of cytosolic protein tyrosine kinases of human prostate by renaturation after SDS/PAGE

1992 ◽  
Vol 284 (3) ◽  
pp. 653-658 ◽  
Author(s):  
Y Durocher ◽  
A Chapdelaine ◽  
S Chevalier
Keyword(s):  
Tyrosine Kinases ◽  
Cell Function ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Protein Tyrosine Kinases ◽  
Partial Purification ◽  
Major Band ◽  
Protein Tyrosine ◽  
Sds Page

The identification of protein tyrosine kinases (PTKs) was successfully achieved by renaturation in gels after SDS/PAGE. To this effect, samples were mixed with a PTK substrate, namely the polydispersed co-polymer of glutamic acid and tyrosine [poly(Glu, Tyr), M(r) from 30,000 to 94,000], and were simultaneously submitted to electrophoresis. Following guanidine hydrochloride denaturation, renaturation and phosphorylation with [gamma-32P]ATP, kinase activity was detected by autoradiography. When applied to cytosol from human hyperplastic prostate, eleven protein kinases were detected, among which one major (M(r) 50,000) and two minor proteins (M(r) 40,000 and 38,000) were identified as PTKs by the presence of phosphotyrosine. Incubation of the gel in hot alkali after glutaraldehyde cross-linking almost completely eliminated the detection of non-PTK enzymes. On the other hand, in the absence of poly(Glu,Tyr), no PTK activity was detected. Partial purification of cytosolic PTKs indicates that the native M(r) of the major phosphotransferase was 44,000, as estimated by gel filtration following ammonium sulphate precipitation and anion-exchange chromatography. Upon renaturation after electrophoresis, this fraction showed only one major band active on poly(Glu,Tyr) which was associated with the polypeptide of M(r) 50,000. This enzyme was also identified following two-dimensional electrophoresis and renaturation in the presence of poly(Glu,Tyr), allowing the determination of a pI in the range 7.5-7.8. Thus PTKs can be easily renatured following electrophoresis and rapidly identified on the basis of their M(r) and pI in both crude or partially purified preparations. With the crucial role played by PTKs in the activation of cell function and carcinogenesis, this procedure could be useful in the identification of such enzymes and in distinguishing them from their substrates in gels.

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