Gamma radiation effects on α-lactalbumin: structural modifications

2001 ◽  
Vol 79 (2) ◽  
pp. 154-157 ◽  
Author(s):  
A Chapelier ◽  
M Desmadril ◽  
C Houée-Levin
Keyword(s):  
Thermodynamic Properties ◽  
Conformational Changes ◽  
Radiation Effects ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Aromatic Amino Acids ◽  
Size Exclusion ◽  
Structural Modifications ◽  
Exclusion Chromatography ◽  
Radiation Induced

α-Lactalbumin was irradiated in the lyophilized state in air at ambient temperature. The irradiated protein was examined by size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, circular dichroism, and microcalorimetry. Irradiation induced the loss of aromatic amino acids and of helicity so that fragmentation and aggregation products were obtained. The thermodynamic properties of the protein were also modified. The irradiated protein had lower stability, however, the temperature at which denaturation occurred process remained constant.Key words: ionizing radiation, radiation-induced conformational changes, protein structural modifications, protein thermodynamic properties.

scholarly journals The Native Monomer of Bacillus Pumilus Ribonuclease Does Not Exist Extracellularly

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Olga Ilinskaya ◽  
Vera Ulyanova ◽  
Irina Lisevich ◽  
Elena Dudkina ◽  
Nataliya Zakharchenko ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Concentration ◽  
Bacillus Pumilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Culture Fluid ◽  
Size Exclusion ◽  
High Protein Concentration ◽  
Exclusion Chromatography ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Hypochromic Effect

Supported by crystallography studies, secreted ribonuclease of Bacillus pumilus (binase) has long been considered to be monomeric in form. Recent evidence obtained using native polyacrylamide gel electrophoresis and size-exclusion chromatography suggests that binase is in fact dimeric. To eliminate ambiguity and contradictions in the data we have measured conformational changes, hypochromic effect, and hydrodynamic radius of binase. The immutability of binase secondary structure upon transition from low to high protein concentration was registered, suggesting the binase dimerization immediately after translocation through the cell membrane and leading to detection of binase dimers only in the culture fluid regardless of ribonuclease concentration. Our results made it necessary to take a fresh look at the binase stability and cytotoxicity towards virus-infected or tumor cells.

scholarly journals Impact of Botrytis cinerea Contamination on the Characteristics and Foamability of Yeast Macromolecules Released during the Alcoholic Fermentation of a Model Grape Juice

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 472
Author(s):  
Richard Marchal ◽  
Thomas Salmon ◽  
Ramon Gonzalez ◽  
Belinda Kemp ◽  
Céline Vrigneau ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Alcoholic Fermentation ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Grape Juice ◽  
Size Exclusion ◽  
Periodic Acid Schiff ◽  
Exclusion Chromatography ◽  
The Impact

Botrytis cinerea is a fungal pathogen responsible for the decrease in foamability of sparkling wines. The proteolysis of must proteins originating from botrytized grapes is well known, but far less information is available concerning the effect of grape juice contamination by Botrytis. The impact from Botrytis on the biochemical and physico-chemical characteristics of proteins released from Saccharomyces during alcoholic fermentation remains elusive. To address this lack of knowledge, a model grape juice was inoculated with three enological yeasts with or without the Botrytis culture supernatant. Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALLS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques (AgNO3 and periodic acid Schiff staining) was used in the study. When Botrytis enzymes were present, a significant degradation of the higher and medium MW molecules released by Saccharomyces was observed during alcoholic fermentation whilst the lower MW fraction increased. For the three yeast strains studied, the results clearly showed a strong decrease in the wine foamability when synthetic musts were inoculated with 5% (v/v) of Botrytis culture due to fungus proteases.

scholarly journals Characterization of a Novel β-Galactosidase fromBifidobacterium adolescentis DSM 20083 Active towards Transgalactooligosaccharides

2000 ◽  
Vol 66 (4) ◽  
pp. 1379-1384 ◽  
Author(s):  
Katrien M. J. Van Laere ◽  
Tjakko Abee ◽  
Henk A. Schols ◽  
Gerrit Beldman ◽  
Alphons G. J. Voragen
Keyword(s):  
Size Exclusion Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Extract ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Bifidobacterium Adolescentis ◽  
Exclusion Chromatography

ABSTRACT This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel β-galactosidase (β-Gal II). In cells grown on TOS, in addition to the lactose-degrading β-Gal (β-Gal I), another β-Gal (β-Gal II) was detected and it showed activity towards TOS but not towards lactose. β-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown B. adolescentis by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. β-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. β-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35°C. The enzyme showed highestV max values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. β-Gal II was active towards β-galactosyl residues that were 1→4, 1→6, 1→3, and 1↔1 linked, signifying its role in the metabolism of galactooligosaccharides by B. adolescentis.

scholarly journals Purification and partial biochemical characterization of normal human interleukin 1.

1984 ◽  
Vol 160 (3) ◽  
pp. 772-787 ◽  
Author(s):  
J A Schmidt
Keyword(s):  
High Performance ◽  
Biochemical Characterization ◽  
Interleukin 1 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Thymocyte Proliferation ◽  
Exclusion Chromatography ◽  
Normal Human

A protocol for the rapid, efficient purification of the major charged species of human interleukin 1 (IL-1) has been developed using high performance anion exchange and size exclusion chromatography. The isolated material is pure as determined by sodium dodecyl sulfate (SDS) gradient polyacrylamide gel electrophoresis (PAGE) and analytical isoelectric focusing (IEF). The molecular weight of the purified material is 15,000 and the isoelectric point (pI) is 6.8, values that are in good agreement with those previously reported for human IL-1. 10(-10) M concentrations of the purified material give half-maximal stimulation in the thymocyte proliferation assay. Amounts of IL-1 sufficient for receptor studies and detailed biochemical analysis can now be produced on a regular basis.

scholarly journals Identification, Purification, and Characterization of Iminodiacetate Oxidase from the EDTA-Degrading Bacterium BNC1

2001 ◽  
Vol 67 (2) ◽  
pp. 696-701 ◽  
Author(s):  
Yong Liu ◽  
Tai Man Louie ◽  
Jason Payne ◽  
Jan Bohuslavek ◽  
Harvey Bolton ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Size Exclusion ◽  
Prosthetic Group ◽  
Oxidase Gene ◽  
Nickel Affinity Chromatography ◽  
Degrading Bacterium ◽  
Apparent Homogeneity ◽  
Exclusion Chromatography

ABSTRACT Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O2. The temperature and pH optima for IDA oxidation were 35°C and 8, respectively. The apparent Km for IDA was 4.0 mM with a k cat of 5.3 s−1. When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed inEscherichia coli, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.

Stabilization and purification of the secondary metabolism specific enzyme, m-hydroxybenzylalcohol dehydrogenase

1986 ◽  
Vol 32 (2) ◽  
pp. 167-175 ◽  
Author(s):  
Richard E. Scott ◽  
K. S. Lam ◽  
G. M. Gaucher
Keyword(s):  
Secondary Metabolism ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Specific Enzyme ◽  
Major Band ◽  
Exclusion Chromatography ◽  
Salt Fractionation

m-Hydroxybenzylalcohol dehydrogenase (EC 1.1.1.97), a secondary metabolism associated protein from stationary phase cultures of Penicillium urticae, was stabilized in crude extracts prior to purification. Stabilization studies resulted in the formulation of an optimal cell breakage and purification buffer. This buffer increased the enzyme's in vitro half-life at 30 °C from 14 to over 800 min which greatly aided purification and enhanced yields. Purification was achieved by salt fractionation, size-exclusion chromatography, affinity chromatography, and ion-exchange chromatography. The 1200-fold purified protein gave only one major band by sodium dodecyl sulphate – polyacrylamide gel electrophoresis.

scholarly journals High-level expression and molecular characterization of a recombinant prolidase fromEscherichia coliNovaBlue

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5863 ◽  
Author(s):  
Tzu-Fan Wang ◽  
Meng-Chun Chi ◽  
Kuan-Ling Lai ◽  
Min-Guan Lin ◽  
Yi-Yu Chen ◽  
...  
Keyword(s):  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Negative Influence ◽  
Size Exclusion ◽  
State Process ◽  
Nickel Chelate ◽  
Exclusion Chromatography ◽  
Efficient Option ◽  
High Level

Long-term use of organophosphorus (OP) compounds has become an increasing global problem and a major threat to sustainability and human health. Prolidase is a proline-specific metallopeptidase that can offer an efficient option for the degradation of OP compounds. In this study, a full-length gene fromEscherichia coliNovaBlue encoding a prolidase (EcPepQ) was amplified and cloned into the commercially-available vector pQE-30 to yield pQE-EcPepQ. The overexpressed enzyme was purified from the cell-free extract of isopropyl thio-β-D-galactoside IPTG-inducedE. coliM15 (pQE-EcPepQ) cells by nickel-chelate chromatography. The molecular mass ofEcPepQ was determined to be about 57 kDa by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and the result of size-exclusion chromatography demonstrated that the enzyme was mainly present in 25 mM Tris–HCl buffer (pH 8.0) as a dimeric form. The optimal conditions forEcPepQ activity were 60 °C, pH 8.0, and 0.1 mM Mn2+ion. Kinetic analysis with Ala-Pro as the substrate showed that theKmandkcatvalues ofEcPepQ were 8.8 mM and 926.5 ± 2.0 s−1, respectively. The thermal unfolding ofEcPepQ followed a two-state process with one well-defined unfolding transition of 64.2 °C. Analysis of guanidine hydrochloride (GdnHCl)-induced denaturation by tryptophan emission fluorescence spectroscopy revealed that the enzyme had a [GdnHCl]0.5,N-Uvalue of 1.98 M. The purified enzyme also exhibited some degree of tolerance to various water/organic co-solvents. Isopropanol and tetrahydrofuran were very detrimental to the enzymatic activity ofEcPepQ; however, other more hydrophilic co-solvents, such as formamide, methanol, and ethylene glycol, were better tolerated. Eventually, the non-negative influence of some co-solvents on both catalytic activity and structural stability ofEcPepQ allows to adjust the reaction conditions more suitable forEcPepQ-catalyzed bioprocess.

scholarly journals A purified lectin with larvicidal activity from a woodland mushroom, Agaricus semotus Fr.

2021 ◽  
Vol 65 (1) ◽  
pp. 65-73
Author(s):  
Isaiah O. Adedoyin ◽  
Taiwo S. Adewole ◽  
Titilayo O. Agunbiade ◽  
Francis B. Adewoyin ◽  
Adenike Kuku
Keyword(s):  
Larvicidal Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Complete Inactivation ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Effects Of Temperature

This study investigated the larvicidal activity on Culex quinquefasciatus of lectin purified from fresh fruiting bodies of woodland mushroom, Agaricus semotus. A. semotus lectin (ASL) was purified via ion-exchange chromatography on DEAE-cellulose A-25 and size exclusion chromatography on Sephadex G-100 matrix. Molecular weight (16.6 kDa) was estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The effects of temperature, pH, metal chelation- and larvicidal activity of ASL were also investigated. The ASL indifferently agglutinated the erythrocytes of the human ABO blood system and was stable at acidic pH and below 50 °C whereas 66% of its activity was lost at 60 °C with complete inactivation at 70 °C. ASL is a metalloprotein requiring barium ion as chelation of metals by 50 mM EDTA rendered the lectin inactive, while the addition of BaCl2, among other metal salts, restored the activity. ASL showed larvicidal activity against C. quinquefasciatus larvae after 24 h with a mortality of 5 and 95% at 5 and 25 mg/mL respectively, and LC50 of 13.80 mg/mL. This study concluded that purified A. semotus lectin showed impressive larvicidal activity, which could be exploited in its development as an insecticidal agent.

Purification and some properties of β-glucosidase from the ectomycorrhizal fungus Pisolithus tinctorius strain SMF

1993 ◽  
Vol 39 (1) ◽  
pp. 125-129 ◽  
Author(s):  
Weiguo Cao ◽  
Don L. Crawford
Keyword(s):  
Molecular Weight ◽  
Ethylenediaminetetraacetic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Ectomycorrhizal Fungus ◽  
Pisolithus Tinctorius ◽  
Size Exclusion ◽  
Exclusion Chromatography ◽  
A Cell

A cell-associated β-glucosidase was purified 152-fold to homogeneity from the ectomycorrhizal fungus Pisolithus tinctorius strain SMF. The apparent molecular weight of the native protein, as determined by size exclusion chromatography, was approximately 450 000. A single band with a molecular weight of 150 000 was obtained after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Thus, the native enzyme may consist of three monomers. The pI of the enzyme was determined to be 3.8 by isoelectric focusing. The enzyme had an optimal pH of 4.0 and an optimal temperature for activity of 65 °C. It showed a high substrate specificity toward aryl-β-glucosides, such as p-nitrophenyl β-D-glucopyranoside (PNPG), and β-1,6 glucosidic linkages. Cellobiose was hydrolyzed at about two-thirds the rate of PNPG. The Km for hydrolysis of PNPG was 0.87 mM. Strong inhibitors of the enzyme were aluminum, copper, ethylenediaminetetraacetic acid (EDTA), deoxynojirimycin, gluconic acid, and SDS. Calcium, manganese, and p-hydroxymercuribenzoic acid reduced the activity to a lesser extent. Potassium, mercury, cobalt, dithiothreitol, and glucosamine had no effect on activity. Enzyme activity was slightly increased to 112% in the presence of 1% glycerol. The enzyme was more stable under acidic conditions than under alkaline conditions.Key words: Pisolithus, ectomycorrhizal, β-glucosidase, purification.

scholarly journals Isolation of HDL by sequential flotation ultracentrifugation followed by size exclusion chromatography reveals size-based enrichment of HDL-associated proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jack Jingyuan Zheng ◽  
Joanne K. Agus ◽  
Brian V. Hong ◽  
Xinyu Tang ◽  
Christopher H. Rhodes ◽  
...  
Keyword(s):  
Structural Integrity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cholesterol Acyltransferase ◽  
Density Lipoprotein ◽  
Size Exclusion ◽  
High Yield ◽  
Phospholipid Transfer ◽  
Exclusion Chromatography ◽  
Associated Proteins ◽  
Alpha 1 Antitrypsin

AbstractHigh-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.

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