scholarly journals A New Procedure for Purification of Crotalase From Crotalus Adamanteus Venom

1977 ◽  
Author(s):  
F.S. Markland ◽  
J. Chou ◽  
Y. Shih ◽  
H. Pirkle
Keyword(s):  
Sodium Chloride ◽  
Large Scale ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fold Increase ◽  
Tris Buffer ◽  
High Yield ◽  
Crude Venom ◽  
Diamondback Rattlesnake

A new procedure has been developed for large scale, rapid purification of crotalase, the thrombin-1ike enzyme from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus). The three step procedure involves: (1) molecular sieve chromatography on Sephadex G-100 in 0.04 M Tris buffer containing 0.10 M sodium chloride, pH 7.1; (2) gradient elution from DEAE-cellulose with sodium acetate buffer, pH 7.0; and (3) affinity chromatography on p-aminobenzamidine Sepharose using a spacer of 6-aminohexanoic acid. Crotalase was eluted from the affinity resin by 0.05 M Tris buffer containing 0.10 M sodium chloride and 0.15 M benzamidine-hydrochloride, pH 9.0, after first washing with the Tris buffer containing 0.40 M sodium chloride. From the crude venom, pure enzyme was obtained with an overall recovery of 40-60% of clotting activity and a 90-100 fold increase in specific activity. Crotalase was shown to be pure by Polyacrylamide disk gel electrophoresis which gave one band. The molecular weight was estimated to be approximately 31,000 by gel filtration on a calibrated Sephadex G-100 column. Amino acid analysis was performed and the composition was shown to be very similar to that reported earlier (F.S. Markland and P.S. Damus, J. Biol. Chem. 246: 6460, 1971). Clotting activity of the enzyme was not inhibited by heparin, either with or without plasma, whereas, thrombin was rapidly inactivated by heparin in the presence of plasma. In conclusion, we have developed a rapid and reproducible procedure for isolation in high yield of large quantities of the thrombin-like enzyme from the venom of the eastern diamondback rattlesnake. Studies are continuing on the primary structure and possible clinical applications of this enzyme.

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.

I. Isolierung, Reinigung und Charakterisierung der Polynucleotid-Phosphorylase aus der Blaualge Anacystis nidulans

1967 ◽  
Vol 22 (2) ◽  
pp. 204-215 ◽  
Author(s):  
Ingrid Capesius ◽  
Gerhard Richter
Keyword(s):  
Rna Polymerase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fold Increase ◽  
Anacystis Nidulans ◽  
Lag Phase ◽  
Blue Green Alga ◽  
Polynucleotide Phosphorylase ◽  
Isolation And Purification

A method for the isolation and purification of polynucleotide phosphorylase from the blue-green alga Anacystis nidulans is described. It consists in an initial fractional centrifugation which yields up to 70% of the enzyme associated with the ribosomal fraction while the RNA-polymerase together with the bulk DNA is mainly confined to the supernatant. The ribosomal fraction is then subjected to column chromatography (DEAE-cellulose) and gel filtration (Sephadex G-200) to obtain a 120 — 150 fold increase in specific activity over the crude extract. The enzyme is essentially free from nucleic acids, phosphatases, kinases and RNA-polymerase. With ADP, UDP or CDP as substrate the corresponding homopolymers poly-A, poly-U and poly-C, respectively, are synthesized with good yields; no lag phase occurs. Equimolar mixtures of these nucleoside diphosphates give rise to heteropolymers. The optimal formation of poly-G and of AGUC polymer, both of which have rather low yields under standard conditions is achieved by incubating at higher temperatures (45° and 60°) and by replacing Mg2⊕ by Mg2⊕. In the presence of inorganic phosphate the highly purified enzyme also catalyzes the cleavage, to nucleoside diphosphates, of synthetic polynucleotides and of native high molecular RNA from various organisms. The possible association of polynucleotide phosphorylase with the ribosomes and its function is discussed.

Enzyme der Polynucleotid-Synthese in pflanzlichen Organismen

1967 ◽  
Vol 22 (8) ◽  
pp. 876-885 ◽  
Author(s):  
Ingrid Capesius ◽  
Gerhard Richter
Keyword(s):  
Column Chromatography ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fold Increase ◽  
Chlorella Pyrenoidosa ◽  
Stable Complex ◽  
Isolation And Purification ◽  
Sperm Dna ◽  
Salmon Sperm Dna

The isolation and purification of RNA polymerase from the blue-green alga Anacystis nidulans is described. The method consists in an initial fractional centrifugation which yields 60 — 70% of the enzyme associated with a DNA-rich fraction. It is subjected to column chromatography (DEAE-cellulose) and gel filtration (Sephadex G-200) to obtain a 110 —120 fold increase in specific activity over the crude extract. The preparation is free from polynucleotide phosphorylase, kinases, phosphatases and nucleic acids. The enzyme requires DNA, a primer, as well as Mg2⊕, Mn2⊕ and the 4 ribonucleoside triphosphates for the net synthesis of polyribonucleotides. Salmon sperm DNA and native DNA from Chlorella pyrenoidosa can replace Anacystis DNA as primer. The properties of the product, however, depend to a certain extent on the primer DNA used as revealed by column chromatography of the former on methylated serum albumin. After isolation by this method, the synthesized polyribonucleotides were found to be partially stable against the action of RNase. This finding suggests a possible association of the newly synthesized RNA with the primer DNA in form of a stable complex which may function as an intermediate of the DNA directed RNA synthesis.

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.

scholarly journals Hyaluronidase in ram semen. Quantitative determination, and isolation of multiple forms

1988 ◽  
Vol 252 (3) ◽  
pp. 865-874 ◽  
Author(s):  
R A Harrison
Keyword(s):  
Ionic Strength ◽  
Quantitative Determination ◽  
Specific Activity ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
High Yield ◽  
Poly Vinyl Alcohol ◽  
The Media ◽  
Low Ionic Strength

A study was made of hyaluronidase in ram semen. The end-group assay conditions used to determine activity quantitatively were chosen to ensure reliability as well as sensitivity [Gacesa, Savitsky, Dodgson & Olavesen (1981) Anal. Biochem. 118, 76-84]; they led to 1 W.H.O. Standard International Hyaluronidase Unit displaying 0.1263 EC munit (1 EC unit of activity releases 1 mumol equivalent of N-acetylglucosamine end groups/min at 37 degrees C). All the activity in the semen was shown to be sperm-derived, and intact spermatozoa were estimated to contain 1.23 EC units per 10(9) cells. In a low-ionic-strength medium, only some 20% of the hyaluronidase was extractable, although up to 80% of the activity could be extracted as the ionic strength was increased; further addition of detergent extracted the remainder. During purification of the enzyme, it was found that inclusion of poly(vinyl alcohol) in the media stabilized the activity; detergent inclusion also improved the yield, especially during early stages. As a consequence both of reliable quantitative determination and of stabilization, a number of forms of hyaluronidase could be isolated in high yield, by using anion-exchange chromatography, cation-exchange chromatography, affinity chromatography and gel filtration. The existence of all these forms was confirmed by electrophoresis and immunoblotting with the use of a monoclonal anti-(ram hyaluronidase) antibody, and their presence in very freshly prepared sperm extracts was demonstrated. The specific activity of the isolated major hyaluronidase form was 15.0 EC units/mg; this was equivalent to 119,000 W.H.O. units/mg, higher than any other previously reported values.

Purification and characterization of rat epididymal neutral β-galactosidase and its changes during in vivo development

1993 ◽  
Vol 71 (1-2) ◽  
pp. 22-26 ◽  
Author(s):  
Pratima Dutta ◽  
Gopal C. Majumder
Keyword(s):  
Concanavalin A ◽  
Sexual Maturity ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Tissue Homogenate ◽  
Affinity Column ◽  
Ph Optimum

A neutral β-D-galactosidase has been partially purified from rat epididymis and characterized. The enzyme having molecular mass of approximately 50 kilodaltons has been purified 400-fold by using calcium phosphate gel adsorption, DEAE-cellulose chromatography, Sephadex G-100 gel filtration, and concanavalin A - agarose affinity chromatography. Although the neutral enzyme binds to the concanavalin A affinity column, the activity could be eluted with α-methyl mannoside only if the buffer contained salt (NaCl) at a concentration as high as 0.3 M. The enzyme was of cytosolic origin, since 90% of the total enzymic activity of the tissue homogenate was recovered in the soluble fraction of these cells. The neutral β-galactosidase was not dependent on metal ions for its activity and it had a pH optimum of 7.0. Zn2+, p-chloromercuribenzoate, Hg2+, and Pb2+ served as potent inhibitors of the enzyme. There was a marked increase (approximately fourfold) in the specific activity of the neutral β-galactosidase during sexual maturity of epididymis in vivo.Key words: neutral β-galactosidase, rat epididymal, cytosolic, developmental, sexual maturity.

Diacetyl reductase of Lactobacillus casei

1970 ◽  
Vol 16 (10) ◽  
pp. 947-951 ◽  
Author(s):  
A. L. Branen ◽  
T. W. Keenan
Keyword(s):  
Lactobacillus Casei ◽  
Nadh Oxidase ◽  
Reductase Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fold Increase ◽  
Cell Extracts ◽  
Alumina Gel ◽  
Nadh Oxidase Activity ◽  
Nadh Oxidoreductase

Diacetyl reductase (diacetyl:reduced nicotinamide adenine dinucleotide (NADH) oxidoreductase, EC. 1.1.1.5) has been isolated from Lactobacillus casei. Cell sonication, ammonium sulfate fractionation, Sephadex gel filtration, DEAE-cellulose chromatography, and alumina gel adsorption were used to obtain the partially purified enzyme. Both NADH oxidase and diacetyl reductase activity were associated with the same fraction at all stages in purification. Growth in media containing added pyruvate resulted in a 10-fold increase in the NADH oxidase activity and a 3-fold increase in the diacetyl reductase activity of crude cell extracts on a protein basis. Purified preparations showed maximal reductase and oxidase activities at pH 4.5 and 5.0, respectively. Lineweaver–Burke plots yielded intersecting lines when NADH and diacetyl concentrations were varied, suggesting a flavin-linked reaction. The absorption spectrum of the purified preparation was characteristic of that of a flavoprotein. The product of the reduction of diacetyl was identified as acetoin. Acetoin and methylene blue were inactive as acceptors.

scholarly journals Purification and Characterization ofα -l-Arabinopyranosidase andα -l-Arabinofuranosidase from Bifidobacterium breve K-110, a Human Intestinal Anaerobic Bacterium Metabolizing Ginsenoside Rb2 and Rc

2003 ◽  
Vol 69 (12) ◽  
pp. 7116-7123 ◽  
Author(s):  
Ho-Young Shin ◽  
Sun-Young Park ◽  
Jong Hwan Sung ◽  
Dong-Hyun Kim
Keyword(s):  
Ammonium Sulfate ◽  
Column Chromatography ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Bifidobacterium Breve ◽  
Ammonium Sulfate Fractionation ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Ginsenoside Rb2

ABSTRACT Two arabinosidases, α-l-arabinopyranosidase (no EC number) and α-l-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizing Bifidobacterium breve K-110, which was isolated from human intestinal microflora. α-l-Arabinopyranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, QAE-cellulose, and Sephacryl S-300 HR column chromatography, with a final specific activity of 8.81 μmol/min/mg.α -l-Arabinofuranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, Q-Sepharose, and Sephacryl S-300 column chromatography, with a final specific activity of 6.46 μmol/min/mg. The molecular mass ofα -l-arabinopyranosidase was found to be 310 kDa by gel filtration, consisting of four identical subunits (77 kDa each, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), and that ofα -l-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. α-l-Arabinopyranosidase and α-l-arabinofuranosidase showed optimal activity at pH 5.5 to 6.0 and 40°C and pH 4.5 and 45°C, respectively. Both purified enzymes were potently inhibited by Cu2+ and p-chlormercuryphenylsulfonic acid.α -l-Arabinopyranosidase acted to the greatest extent on p-nitrophenyl-α-l-arabinopyranoside, followed by ginsenoside Rb2. α-l-Arabinofuranosidase acted to the greatest extent on p-nitrophenyl-α-l-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted on p-nitrophenyl-β-galactopyranoside or p-nitrophenyl-β-d-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified α-l-arabinosidases. This is the first reported purification ofα -l-arabinopyranosidase from an anaerobic Bifidobacterium sp.

scholarly journals PURIFICATION AND CHARACTERIZATION OF SALIVARY AMYLASE INHIBITOR EXTRACTED FROM BARLEY

2016 ◽  
Vol 47 (4) ◽  
Author(s):  
Abood & Hakeem
Keyword(s):  
Ammonium Sulfate ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Alpha Amylase ◽  
Ionic Exchange ◽  
Inhibition Activity ◽  
Amylase Inhibitors

Amylase inhibitors were purified by many sequential steps included concentration by gradual addition of ammonium sulfate at  saturation ratios. ranged from 0 to 90% . The best ratio of saturation was found to be 70% as the specific activity and inhibition activity toward Human alpha-amylase(HAS)  were the highest ( 8 U/mg and 6 U/ml respectively as compared to those of the rest ratios, the ratio of saturation with ammonium sulfate 60 % and then 50%, (5.8 ,5.5  )U/ml and( 7.7 ،7 )U/mg respectively for inhibition activity and specific activity and for  40% ,30%20%  saturation  the inhibition activity and specific activity were(5 ،4.8 ،4 ) u/ml (6.6 ،6 ،5.8) u/mg respectively .The precepitation step was followed by ionic exchange chromatography technique by DEAE-cellulose column( 3×11 )cm and the results showed that there was one peak with inhibition activity toward (HAS). Further  purification steps were conducted using gel filtration on Sephacryl S-200 column    (1.5  ×  60)cm; the purification folds was5.59 times with outcome of 46.5%.The results of alpha-amylase inhibitors characterization showed that the molecular weight was about 23.44 and 22.9  kDa  as determined by electrophoresis and gel filteration respectively.                                         

scholarly journals Purification and Characterization of Melanogenic Enzyme Tyrosinase from Button Mushroom

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Kamal Uddin Zaidi ◽  
Ayesha S. Ali ◽  
Sharique A. Ali
Keyword(s):  
Agaricus Bisporus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Edible Mushroom ◽  
Total Activity ◽  
Protein Band ◽  
Exchange Chromatography ◽  
Ammonium Sulphate Precipitation ◽  
Key Enzyme

Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme, tyrosinase, catalyzes the first and only rate-limiting steps in melanogenesis. Since the discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it’s taking place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently cited in the literature in relation to their nutritional value. In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total activity in the crude extract and final specific activity of 52.19 U/mg. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM. This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.

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