scholarly journals A Simple Method for On-Gel Detection of Myrosinase Activity

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2204 ◽  
Author(s):  
Sándor Gonda ◽  
Zsolt Szűcs ◽  
Tamás Plaszkó ◽  
Zoltán Cziáky ◽  
Attila Kiss-Szikszai ◽  
...  
Keyword(s):  
Functional Foods ◽  
Allyl Isothiocyanate ◽  
Cruciferous Vegetables ◽  
Methyl Red ◽  
Native Page ◽  
Simple Method ◽  
Sds Page ◽  
Reversible Method ◽  
Myrosinase Activity

Myrosinase is an enzyme present in many functional foods and spices, particularly in Cruciferous vegetables. It hydrolyses glucosinolates which thereafter rearrange into bioactive volatile constituents (isothiocyanates, nitriles). We aimed to develop a simple reversible method for on-gel detection of myrosinase. Reagent composition and application parameters for native PAGE and SDS-PAGE gels were optimized. The proposed method was successfully applied to detect myrosinase (or sulfatase) on-gel: the detection solution contains methyl red which gives intensive red bands where the HSO4− is enzymatically released from the glucosinolates. Subsequently, myrosinase was successfully distinguished from sulfatase by incubating gel bands in a derivatization solution and examination by LC-ESI-MS: myrosinase produced allyl isothiocyanate (detected in conjugate form) while desulfo-sinigrin was released by sulfatase, as expected. After separation of 80 µg protein of crude extracts of Cruciferous vegetables, intensive color develops within 10 min. On-gel detection was found to be linear between 0.031–0.25 U (pure Sinapis alba myrosinase, R2 = 0.997). The method was successfully applied to detection of myrosinase isoenzymes from horseradish, Cruciferous vegetables and endophytic fungi of horseradish as well. The method was shown to be very simple, rapid and efficient. It enables detection and partial characterization of glucosinolate decomposing enzymes without protein purification.

Purification and Characterization of a Yam Glycoprotein Isolated by Alkaline Extraction from Yam Tuber

2012 ◽  
Vol 560-561 ◽  
pp. 368-373
Author(s):  
Li Ming Zhang ◽  
Yan Qiao Wang ◽  
Li Chao Zhang ◽  
Shu Li Man ◽  
Bei Mei Zuo
Keyword(s):  
Functional Foods ◽  
Gel Filtration ◽  
Processing Method ◽  
Purification And Characterization ◽  
Automatic Amino Acid Analyzer ◽  
Sds Page ◽  
Amino Acid Analyzer ◽  
Layer Chromatography ◽  
Protein Moiety

Yam glycoprotein (YGP) is an important source of bioactives for functional foods. To investigate the effects of alkaline extracting method on the features of YGP, A glycoprotein was isolated from yam tubers by using alkaline processing method, and purified by ion-exchange and gel filtration column chromatography. During the SDS-PAGE eletrophoresis, the result shows a band with approximately 30 kDa molecular weight. The YGP consists of protein moiety (62.34%) and carbohydrate moiety (37.51%), respectively. By the automatic amino acid analyzer detecting, it indicated that the YGP consists of 17 kinds of amino acids and contains a high percentage of glutamic acid and aspartic acid. The results of thin-layer chromatography show that oligosaccharides of the YGP contain D-glucose, D-galactose and mannose.

scholarly journals Purification and characterization of N-glycanase, a concanavalin A binding protein from jackbean (Canavalia ensiformis)

1998 ◽  
Vol 330 (1) ◽  
pp. 13-20 ◽  
Author(s):  
S. Philip SHELDON ◽  
N. Jeffrey KEEN ◽  
J. Dianna BOWLES
Keyword(s):  
Concanavalin A ◽  
Principal Components ◽  
Enzymic Activity ◽  
Purification And Characterization ◽  
Con A ◽  
Glycoprotein Precursor ◽  
Ph Optimum ◽  
Native Page ◽  
Sds Page

Removal of the N-glycan from the concanavalin A (Con A) glycoprotein precursor is a key step in its conversion into an active lectin. N-Glycanase (EC 3.5.1.52), the enzyme from jackbean catalysing this process, has been purified to homogeneity as judged by native PAGE. One of the purification steps is binding of the enzymic activity to Con A-Sepharose and its elution by methyl α-mannoside. On SDS/PAGE the principal components were found to be 78 kDa, 74 kDa, 54 kDa, 32 kDa and 30 kDa polypeptides. These did not react with Con A on an affinity blot. Cleveland mapping indicated that some of these polypeptides had related primary structures. The enzyme has a broad pH optimum in the region of 5.0.

scholarly journals Immunological Characterization of Acid Phosphatase in an Endophytic Fungus

2007 ◽  
Vol 7 ◽  
pp. 77 ◽  
Author(s):  
Rajani Malla ◽  
Md Zeyaullah ◽  
Utprekshya Pokharel ◽  
Ajit Varma
Keyword(s):  
Acid Phosphatase ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Phosphate Metabolism ◽  
Immunoblotting Analysis ◽  
Native Page ◽  
Selective Precipitation ◽  
Denatured Protein ◽  
Sds Page

Piriformospora indica produced only one form of intracellular acid phosphatase irrespective of the phosphate concentration and was purified. The enzyme was possibly a constitutive enzyme showing molecular mass of 66kDa as separated by SDS PAGE. Antibodies raised against cytosolic acid phosphatase of P. indica using gel band in native PAGE after selective precipitation of ammonium sulfate followed by gel filtration and ion exchange chromatography, gave productive antibody and immunoblotting analysis. Its reaction with native protein as well as denatured protein was significant. The antibody immunoprecipitated a single band of approximately 66kDa protein in SDS gel. The antibody localized the enzyme on the polyphosphate granules, cell-wall, vacuoles and cytoplasm of the mycelium indicating the possible sites of phosphate metabolism. <i> Nepal Journal of Science and Technology</i> Vol. 7, 2006

scholarly journals Isolation and partial characterization of a D-galactose-binding lectin from the latex of Synadenium carinatum

2005 ◽  
Vol 48 (5) ◽  
pp. 705-716 ◽  
Author(s):  
Maria Aparecida Souza ◽  
Francielle Amâncio-Pereira ◽  
Cristina Ribeiro Barros Cardoso ◽  
Adriano Gomes da Silva ◽  
Edmar Gomes Silva ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Apparent Molecular Weight ◽  
Size Exclusion ◽  
Terminal Sequence ◽  
Native Page ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Polypeptide Chains ◽  
Binding Lectin

A lectin from the latex of Synadenium carinatum was purified by affinity chromatography on immobilized-D-galactose-agarose and shown to be a potent agglutinin of human erythrocytes. The haemagglutination of human red cells was inhibited by 3.0 mM N-acetyl-D-galactopyranoside, 6.3 mM methyl-beta-D-galactopyranoside, 50 mM methyl-alpha-D-galactopyranoside and 50 mM D-fucose but not by L-fucose, demonstrating an anomeric and a conformational specificity. According to SDS-PAGE analysis, the lectin appeared to be a glycoprotein composed of two polypeptide chains of ca. 28 and 30 kDa, but size exclusion chromatography (Sephadex G-100) and native PAGE revealed a protein of apparent molecular weight 120 - 130 kDa made up of 28 and 30 kDa subunits. The lectin was stable in the range pH 6 - 9, and 4 - 56ºC. The N-terminal sequence of the 30 kDa subunit contained the conserved consensus sequence GPN observed in other D-galactose-binding lectins found in latex of members of the Euphorbiaceae.

scholarly journals Renaturation of telomere-binding proteins after the fractionation by SDS-polyacrylamide gel electrophoresis

2008 ◽  
Vol 53 (No. 7) ◽  
pp. 317-320
Author(s):  
G. Rotková
Keyword(s):  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Simple Method ◽  
Telomeric Sequences ◽  
Sds Page ◽  
Identification And Characterization

A simple method for identification and characterization of telomere-binding proteins is described in this article. After Sodium Dodecyl Sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE), proteins are eluted, renatured and used for retardation analysis with labelled oligonucleotides corresponding to human and plant of telomeric sequences. We show here that this method is efficient to recover sequence-specific DNA-binding abilities of putative telomere-binding proteins.

scholarly journals Biochemical Isolation and Characterization of Hyaluronidase Enzyme from Venom of Egyptian Honey Bee Apis Mellifera Lamarckii

2020 ◽  
Vol 64 (1) ◽  
pp. 153-164
Author(s):  
Mohammed M. Abdel-Monsef ◽  
Hind A. Zidan ◽  
Doaa A. Darwish ◽  
Hassan M. Masoud ◽  
Mohamed S. Helmy ◽  
...  
Keyword(s):  
Specific Activity ◽  
Deae Cellulose ◽  
Bee Venom ◽  
Native Form ◽  
Native Page ◽  
Isolation And Characterization ◽  
Sds Page ◽  
Wide Range ◽  
First Time

AbstractThe hyaluronidase enzyme has been used in many such fields of medicine as ophthalmology, orthopaedia, internal medicine, gynecology, surgery, oncology and dermatology. In this study, the hyaluronidase enzyme was purified and characterized for the first time from Egyptian bee venom homogeneously using DEAE-cellulose and Sephacryl S-300 columns. Bee venom hyaluronidase specific activity was 411.7 units/mg protein with 49.9% yield and 3.23-fold purification. The molecular weight of the purified bee venom hyaluronidase native form was 37 kDa. The purified enzyme was found homogeneous on native PAGE and SDS-PAGE, with two congruent subunits of 18.4 kDa and isoelectric point (pI) of 8.6–8.8. The enzyme was found to be stable over a wide range of temperature (20–60°C) and pH (4.5–6.5), and its optimum activity at 37°C, pH 5.4 and 0.15 M NaCl. Km for bee venom hyaluronidase was 0.029 mg/ml hyaluronic acid and its activity was elevated in presence of MgCl2 and ZnCl2 and lowered in presence of FeCl2. Heparin inhibited the hyaluronidase enzyme noncompetitively with a Ki value of 2.9 units heparin and one binding site on the enzyme molecule.

Characterization of Venoms of Deinagkistrodon acutus and Bungarus multicinctus Using Proteomics and Peptidomics

2020 ◽  
Vol 17 (3) ◽  
pp. 241-254
Author(s):  
Yaqiong Zhang ◽  
Zhiping Jia ◽  
Yunyang Liu ◽  
Xinwen Zhou ◽  
Yi Kong
Keyword(s):  
Snake Venom ◽  
Protein Families ◽  
Traditional Medicines ◽  
Phospholipases A2 ◽  
Inhibitory Peptides ◽  
Sds Page ◽  
Deinagkistrodon Acutus ◽  
Venom Proteins ◽  
Proteins And Peptides

Background: Deinagkistrodon acutus (D. acutus) and Bungarus multicinctus (B. multicinctus) as traditional medicines have been used for hundreds of years in China. The venoms of these two species have strong toxicity on the victims. Objective: The objective of this study is to reveal the profile of venom proteins and peptides of D. acutus and B. multicinctus. Method: Ultrafiltration, SDS-PAGE coupled with in-gel tryptic digestion and Liquid Chromatography- Electrospray Ionization-Tandem Mass Spectrometry (LC-ESI-MS/MS) were used to characterize proteins and peptides of venoms of D. acutus and B. multicinctus. Results: In the D. acutus venom, 67 proteins (16 protein families) were identified, and snake venom metalloproteinases (SVMPs, 38.0%) and snake venom C-type lectins (snaclecs, 36.7%) were dominated proteins. In the B. multicinctus venom, 47 proteins (15 protein families) were identified, and three-finger toxins (3FTxs, 36.3%) and Kunitz-type Serine Protease Inhibitors (KSPIs, 32.8%) were major components. In addition, both venoms contained small amounts of other proteins, such as Snake Venom Serine Proteinases (SVSPs), Phospholipases A2 (PLA2s), Cysteine-Rich Secreted Proteins (CRISPs), 5'nucleotidases (5'NUCs), Phospholipases B (PLBs), Phosphodiesterases (PDEs), Phospholipase A2 Inhibitors (PLIs), Dipeptidyl Peptidases IV (DPP IVs), L-amino Acid Oxidases (LAAOs) and Angiotensin-Converting Enzymes (ACEs). Each venom also had its unique proteins, Nerve Growth Factors (NGFs) and Hyaluronidases (HYs) in D. acutus, and Cobra Venom Factors (CVFs) in B. multicinctus. In the peptidomics, 1543 and 250 peptides were identified in the venoms of D. acutus and B. multicinctus, respectively. Some peptides showed high similarity with neuropeptides, ACE inhibitory peptides, Bradykinin- Potentiating Peptides (BPPs), LAAOs and movement related peptides. Conclusion: Characterization of venom proteins and peptides of D. acutus and B. multicinctus will be helpful for the treatment of envenomation and drug discovery.

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