Biochemical and biological analysis of Philodryas baroni (Baron’s Green Racer; Dipsadidae) venom

2013 ◽  
Vol 33 (1) ◽  
pp. 22-31 ◽  
Author(s):  
MN Sánchez ◽  
A Timoniuk ◽  
S Maruñak ◽  
P Teibler ◽  
O Acosta ◽  
...  
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mouse Skin ◽  
Protein Composition ◽  
Intradermal Injection ◽  
South American ◽  
Crude Venom ◽  
Sds Page ◽  
Leucocyte Infiltration

Philodryas baroni—an attractively colored snake—has become readily available through the exotic pet trade. Most people consider this species harmless; however, it has already caused human envenomation. As little is known about the venom from this South American opisthoglyphous “colubrid” snake, herein, we studied its protein composition by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), as well as its effects on the hemostatic system. Both reducing and nonreducing SDS-PAGE analysis demonstrated that the venom exhibits greatest complexity in the range of 50–80 kDa. The venom displayed proteolytic activity toward azocollagen, with a specific activity of 75.5 U mg−1, and rapidly hydrolyzed the Aα-chain of fibrinogen, exhibiting lower activity toward the Bβ- and γ-chains. The venom from P. baroni showed no platelet proaggregating activity per se, but it inhibited collagen- and thrombin-induced platelet aggregation. Prominent hemorrhage developed in mouse skin after intradermal injection of the crude venom, and its minimum hemorrhagic dose was 13.9 μg. When injected intramuscularly into the gastrocnemius of mice, the venom induced local effects such as hemorrhage, myonecrosis, edema, and leucocyte infiltration. Due to its venom toxicity shown herein, P. baroni should be considered dangerous to humans and any medically significant bite should be promptly reviewed by a qualified health professional.

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.

Changes in total and individual proteins during drying and ruminal fermentation of alfalfa

2005 ◽  
Vol 2005 ◽  
pp. 198-198
Author(s):  
A. A. Sadeghi ◽  
P. Shawrang ◽  
M. Moradi ◽  
A. Nikkhah
Keyword(s):  
Crude Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Plant Proteins ◽  
Ruminal Fermentation ◽  
Protein Nitrogen ◽  
Sds Page ◽  
Total Crude Protein ◽  
Hydrolysis Of

Proteolysis within plant cells occurs during wilting and drying. Changes in plant proteins during those periods usually are monitored by measurement of total crude protein and non protein nitrogen. Alternatively, changes in concentrations of individual proteins can be measured. Plants are composed of an array of different proteins. Electrophoresis can be used to separate these proteins and has been used to study effects of wilting and ensiling on proteins of some forages (Grum et al., 1991). Electrophoresis also has been used in the study of ruminal hydrolysis of oilseed meals proteins (Sadeghi et al., 2004). Most of the experiments designed to use electrophoresis to study protein metabolism in forages and ruminants have been qualitative. The main objective of this study was to determine whether sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry could be used to monitor quantitatively the changes in alfalfa protein composition during wilting, drying and ruminal exposure.

Synthesis and Ultrastructural Localization of Protein C Inhibitor in Human Platelets and Megakaryocytes

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1300-1312
Author(s):  
Maria J. Prendes ◽  
Edith Bielek ◽  
Margareta Zechmeister-Machhart ◽  
Erika Vanyek-Zavadil ◽  
Veronica A. Carroll ◽  
...  
Keyword(s):  
Protein C ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasminogen Activator Inhibitor ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Plasminogen Activator Inhibitor 1 ◽  
Sds Page ◽  
Protein C Inhibitor

The occurrence of protein C inhibitor (PCI) in human platelets and megakaryocytes was analyzed. As judged from enzyme-linked immunosorbent assays (ELISAs), PCI was present in platelets at a concentration of 160 ng/2 × 109 cells. Its specific activity was 5 times higher than that of plasma PCI. Consistently, mainly the 57-kD form (active PCI) and some high molecular weight (Mr) forms, but no bands corresponding to cleaved PCI, were detected when platelet lysates were immunoprecipitated with monoclonal anti-PCI-IgG and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The localization of PCI in platelets was studied by immunofluorescence histochemistry and immunotransmission electron microscopy: PCI was detected in  granules, in the open canalicular system, and on the plasma membrane. At these sites, colocalization with plasminogen activator inhibitor-1 was seen. Studies were performed to clarify whether platelet PCI is endogenously synthesized or taken up from plasma. Internalization of biotinylated-PCI was analyzed using platelets in suspension and gold-labeled streptavidin for visualization of incorporated biotin. Dose- and time-dependent uptake of PCI was found. PCI mRNA was detected in platelets by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, as well as in megakaryocytes by in situ hybridization of human bone marrow cryosections. We therefore conclude that platelets contain a functionally active PCI pool that is derived from both endogenous synthesis as well as internalization.

scholarly journals Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. I. Isolation and characterization of membrane and content subfractions.

1983 ◽  
Vol 96 (4) ◽  
pp. 1030-1039 ◽  
Author(s):  
W J Brown ◽  
W A Shannon ◽  
W J Snell
Keyword(s):  
Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Granule Protein ◽  
Isolation And Characterization ◽  
Cytoplasmic Face ◽  
Sds Page ◽  
Granule Membrane ◽  
Granule Membrane Proteins

The specific and azurophilic granules of rabbit polymorphonuclear heterophils (PMNs) have been isolated and fractionated into membrane and extractable subfractions. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) revealed several features of the protein composition of the two granules: (a) Whereas each type of granule had 40-60 proteins separable on one-dimensional gradient gels, few of the proteins were common to both granules. (b) The proteins of the extractable fractions (which comprised approximately 98% of the total granule protein) of each granule were distinct from the proteins of the membrane fractions (which comprised approximately 2% of the total granule protein). (c) The extractable proteins co-migrated with those collected from the medium of ionophore-treated, degranulating PMNs and therefore were defined as content proteins. These results were confirmed by radiolabeling studies. Lactoperoxidase-catalyzed iodination of intact granules did not label the content proteins but did label proteins that co-migrated with major granule membrane proteins. Moreover, disruption of the granules before iodination led to labeling of both content and membrane proteins. We conclude that the membranes of specific and azurophilic granules, which arise from different faces of the Golgi complex, are composed of unique sets of membrane proteins some of which are exposed on the cytoplasmic face of the granules.

scholarly journals Rabbit reticulocyte coated vesicles carrying the transferrin- transferrin receptor complex: I. Purification and partial characterization

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1035-1039
Author(s):  
HR Choe ◽  
ST Moseley ◽  
J Glass ◽  
MT Nunez
Keyword(s):  
Transferrin Receptor ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Receptor Complex ◽  
Assembly Factor ◽  
Excellent Source

Coated vesicles bearing the transferrin-transferrin receptor complex were isolated from rabbit reticulocytes by freeze-thaw cell lysis, followed by differential centrifugation with pelleting of vesicles at 100,000 g. Electronmicroscopy demonstrated the vesicles to have the characteristic morphology of coated vesicles, including the appearance of triskelions. The protein composition of the vesicles as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis included transferrin, transferrin receptor, and proteins of apparent mol wt of approximately 180,000, 140,000, 100,000, and 47,000 daltons. The 180,000 and 100,000 mol wt proteins were identified as clathrin and coated vesicle assembly factor proteins, respectively, by Western blot analyses. The vesicles had a Mg2+-dependent ATPase with a specific activity of approximately 8.5 nmoles ATP converted/min/mg vesicle protein. The vesicles could acidify the intravesicular space, as evidenced by the stimulation of the Mg2+-ATPase by the protonophore FCCP. Reticulocytes appear to be an excellent source of coated vesicles and as such should provide a model for studying the endocytosis of transferrin and the steps of iron uptake that proceed in these vesicles.

Synthesis and Ultrastructural Localization of Protein C Inhibitor in Human Platelets and Megakaryocytes

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1300-1312 ◽  
Author(s):  
Maria J. Prendes ◽  
Edith Bielek ◽  
Margareta Zechmeister-Machhart ◽  
Erika Vanyek-Zavadil ◽  
Veronica A. Carroll ◽  
...  
Keyword(s):  
Protein C ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasminogen Activator Inhibitor ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Plasminogen Activator Inhibitor 1 ◽  
Sds Page ◽  
Protein C Inhibitor

Abstract The occurrence of protein C inhibitor (PCI) in human platelets and megakaryocytes was analyzed. As judged from enzyme-linked immunosorbent assays (ELISAs), PCI was present in platelets at a concentration of 160 ng/2 × 109 cells. Its specific activity was 5 times higher than that of plasma PCI. Consistently, mainly the 57-kD form (active PCI) and some high molecular weight (Mr) forms, but no bands corresponding to cleaved PCI, were detected when platelet lysates were immunoprecipitated with monoclonal anti-PCI-IgG and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The localization of PCI in platelets was studied by immunofluorescence histochemistry and immunotransmission electron microscopy: PCI was detected in  granules, in the open canalicular system, and on the plasma membrane. At these sites, colocalization with plasminogen activator inhibitor-1 was seen. Studies were performed to clarify whether platelet PCI is endogenously synthesized or taken up from plasma. Internalization of biotinylated-PCI was analyzed using platelets in suspension and gold-labeled streptavidin for visualization of incorporated biotin. Dose- and time-dependent uptake of PCI was found. PCI mRNA was detected in platelets by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, as well as in megakaryocytes by in situ hybridization of human bone marrow cryosections. We therefore conclude that platelets contain a functionally active PCI pool that is derived from both endogenous synthesis as well as internalization.

Purification of a Fibrinolytic Enzyme from Bacillus Sp. Isolated from Budu

2012 ◽  
Vol 59 (1) ◽  
Author(s):  
Nurulhanis Ahmad Sanusi ◽  
Haryati Jamaluddin
Keyword(s):  
Ammonium Sulphate ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Culture Broth ◽  
Fibrinolytic Enzyme ◽  
Total Protein Content ◽  
Bacillus Sp ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page

Bacillus sp. strain B1 producing wild type fibrinolytic enzyme was isolated from Budu. The fibrinolytic enzyme was collected from the supernatant of Bacillus sp. strain B1 culture broth and purified to electrophoretic homogeneity through a combination of various purification schemes, which include ammonium sulphate precipitation, followed by anion exchange chromatography using DEAE–Sepharose Fast Flow and gel filtration chromatography on Sephadex G–75 column. During ammonium sulphate precipitation screening, it was observed that the crude enzyme from Bacillus sp. strain B1 precipitated at 40% and 50% of ammonium sulphate saturation respectively. The fibrinolytic enzyme was purified 58.5–fold with a final yield of 0.51%. The specific activity was determined to be 1.17 Units/mg using plasmin as standard and the final total protein content was 8.58 mg/ml. After the successive purification steps, the estimated molecular mass of fibrinolytic enzymes from strain B1 was estimated via sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). SDS–PAGE analysis showed a single band at 45 kDa corresponding to the purified fraction with fibrinolytic activity.

scholarly journals Purification and Characterization of the Folate Catabolic Enzyme p-Aminobenzoyl-Glutamate Hydrolase from Escherichia coli

2010 ◽  
Vol 192 (9) ◽  
pp. 2407-2413 ◽  
Author(s):  
Jacalyn M. Green ◽  
Ryan Hollandsworth ◽  
Lenore Pitstick ◽  
Eric L. Carter
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Size Exclusion ◽  
Breakdown Product ◽  
Purification And Characterization ◽  
Sds Page

ABSTRACT The abg locus of the Escherichia coli chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the p-aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by abgA and abgB. One-step purification was accomplished using a plasmid carrying abgAB with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (∼53-kDa and ∼47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1:1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a Km value for PABA-GLU of 60 ± 0.08 μM and a specific activity of 63,300 ± 600 nmol min−1 mg−1. Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the E. coli chromosome may encode a portion of a folate catabolism pathway.

scholarly journals Multimeric Association of Purified Novel Bowman-Birk Inhibitor From the Medicinal Forage Legume Mucuna pruriens (L.) DC.

2021 ◽  
Vol 12 ◽  
Author(s):  
Jafar K. Lone ◽  
Mandapanda A. Lekha ◽  
Rajiv P. Bharadwaj ◽  
Fasil Ali ◽  
M. Arumugam Pillai ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Specific Activity ◽  
Therapeutic Potential ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mucuna Pruriens ◽  
Reducing Conditions ◽  
Functional Efficiency ◽  
Sds Page ◽  
Functional Features

A Bowman-Birk protease, i.e., Mucuna pruriens trypsin inhibitor (MPTI), was purified from the seeds by 55.702-fold and revealed a single trypsin inhibitor on a zymogram with a specific activity of 202.31 TIU/mg of protein. On sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under non-reducing conditions, the protease trypsin inhibitor fraction [i.e., trypsin inhibitor non-reducing (TINR)] exhibited molecular weights of 74 and 37 kDa, and under reducing conditions [i.e., trypsin inhibitor reducing (TIR)], 37 and 18 kDa. TINR-37 revealed protease inhibitor activity on native PAGE and 37 and 18 kDa protein bands on SDS–PAGE. TINR-74 showed peaks corresponding to 18.695, 37.39, 56.085, and 74.78 kDa on ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization/quadrupole time-of-flight-mass spectrometry (ESI/QTOF-MS). Similarly, TINR-37 displayed 18.695 and 37.39 kDa peaks. Furthermore, TIR-37 and TIR-18 exhibited peaks corresponding to 37.39 and 18.695 kDa. Multiple peaks observed by the UPLC-ESI/QTOF analysis revealed the multimeric association, confirming the characteristic and functional features of Bowman-Birk inhibitors (BBIs). The multimeric association helps to achieve more stability, thus enhancing their functional efficiency. MPTI was found to be a competitive inhibitor which again suggested that it belongs to the BBI family of inhibitors, displayed an inhibitor constant of 1.3 × 10–6 M, and further demonstrates potent anti-inflammatory activity. The study provided a comprehensive basis for the identification of multimeric associates and their therapeutic potential, which could elaborate the stability and functional efficiency of the MPTI in the native state from M. pruriens.

Purification, characterization, antibacterial activity and N-terminal sequencing of buffalo-milk lysozyme

2002 ◽  
Vol 69 (3) ◽  
pp. 419-431 ◽  
Author(s):  
SUBHADRA PRIYADARSHINI ◽  
VINOD K. KANSAL
Keyword(s):  
Antibacterial Activity ◽  
Amino Acid ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Milk ◽  
Buffalo Milk ◽  
Size Exclusion ◽  
Sds Page ◽  
Wide Range

Lysozyme from buffalo milk was purified to homogeneity and its N-terminal amino acid sequence, biochemical properties and antibacterial spectrum were determined. The purification procedure, comprising ion-exchange chromatography using CM-cellulose and size-exclusion chromatography using Sephadex G-50, conferred 8622-fold purification and 39·3% recovery of lysozyme. The purified enzyme migrated as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. Immunological purity of lysozyme preparation was confirmed by immuno-electrophoresis. Molecular weight of buffalo-milk lysozyme as determined by SDS-PAGE was 16 kDa and its amino acid composition was determined by reverse phase high performance liquid chromatography (HPLC). The sequence of 23 amino acid residues at the N-terminal end showed 56·5% homology with bovine milk lysozyme and 30·4% with equine milk lysozyme. The specific activity of buffalo milk lysozyme was ten-times that of bovine milk lysozyme. Buffalo-milk lysozyme was active over a wide range of pH and its activity was strongly influenced by molarity of the medium. Antibacterial activity of buffalo-milk lysozyme was determined against 11 species of bacteria; out of seven Gram-positive bacteria tested, four were inhibited, while Gram-negative bacteria were resistant.

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