scholarly journals PARTIAL PURIFICATION AND CHARACTERIZATION OF BACTERIOCIN-LIKE PEPTIDE PRODUCED BY STAPHYLOCOCCUS XYLOSUS

2017 ◽  
Vol 3 ◽  
pp. 213
Author(s):  
Veslava Matikeviciene ◽  
Saulius Grikiškis ◽  
Erika Lubyte ◽  
Gervydas Dienys
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Assay Method ◽  
Partial Purification ◽  
Small Peptides ◽  
Antibiotic Resistant ◽  
Staphylococcus Xylosus ◽  
Lowry Method ◽  
Food Applications

The extensive use of antibiotics leads to an increasing number of antibiotic-resistant pathogenic microorganisms. The development of new antimicrobials is needed for clinical, veterinary, and food applications. Bacteriocins are small peptides with antimicrobial activity ribosomally synthesized by bacteria and could be applied as an alternative to classical antibiotics. In this study, the bacteriocin-like (BLIS) peptide, produced by Staphylococcus xylosus was partially purified and main characteristics (pH, thermal stability, resistance to some protease enzymes and molecular weight) were evaluated. Ammonium sulfate precipitation, acetone extraction and ion-exchange chromatography methods were applied for purification of bacteriocin. The activity of bacteriocin was detected using a well diffusion assay method and the amount of protein concentration was estimated by Lowry method. Molecular weight (~ 6 kDa) of purified bacteriocin was determined by sodium dodecyl sulphate polyacrilamide gel electrophoresis (SDS PAGE) method. The highest purification yield (80 %) was obtained using ion-exange chromatography and SP-sepharose as sorbent. The purified bacteriocin remained stable at pH values between 2.0 and 12.0 for 4 h. No decrease in antibacterial activity was estimated after 30 min at 121º C temperature. The purified bacteriocin was resistant to papain, pepsin and trypsin action. The BLIS inhibits a growth of Listeria monocytogenes (93 ± 3.0 %), Bacillus subtilis (85 ± 4.0 %), Pediococcus pentosaceus (79 ± 4.0 %), Staphylococcus aureus (51 ± 5.0 %) and Propionibacterium acnes (70 ± 5 %) up to 24 hours. Such bacteriocin preparation could be applied as antimicrobial agent in medical and food industry.

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

1990 ◽  
Vol 10 (2) ◽  
pp. 131-139
Author(s):  
Oyewole Adeyemo ◽  
E. O. Okegbile ◽  
O. O. Olorunsogo
Keyword(s):  
Molecular Weight ◽  
Membrane Protein ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Boar Sperm ◽  
Sperm Membrane ◽  
Triton X

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.

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 Separation and partial characterization of guinea-pig caseins

1978 ◽  
Vol 173 (2) ◽  
pp. 633-641 ◽  
Author(s):  
R K Craig ◽  
D McIlreavy ◽  
R L Hall
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Guinea Pig ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.

scholarly journals Purification and subunit structure of legumin of Vicia faba L. (broad bean)

1974 ◽  
Vol 141 (2) ◽  
pp. 413-418 ◽  
Author(s):  
David J. Wright ◽  
Donald Boulter
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Broad Bean ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Subunit Structure ◽  
Isoelectric Precipitation ◽  
Molecular Weights

Zonal isoelectric precipitation was shown to be an effective method for the preparation of legumin which was homogeneous as judged by ultracentrifugation and polyacrylamide-gel electrophoresis. The subunit structure of legumin was investigated by preparative sodium dodecyl sulphate–polyacrylamide-gel electrophoresis and ion-exchange chromatography in urea. Five distinct subunits, of which two were acidic (α) and had a molecular weight of 37000, and three were basic (β) with molecular weights of 20100, 20900 and 23800, were identified. The α and β subunits were present in equimolar amounts in the legumin molecule and, in view of this and molecular-weight considerations, an α6β6 subunit model was proposed for legumin.

Purification and partial characterization of a plasma inhibitor of tonin

1981 ◽  
Vol 59 (4) ◽  
pp. 256-261 ◽  
Author(s):  
J. Tremblay ◽  
G. Thibault ◽  
J. Gutkowska ◽  
R. Boucher ◽  
J. Genest
Keyword(s):  
Molecular Weight ◽  
Plasma Proteins ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Velocity ◽  
Exchange Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
Angiotensin I ◽  
Stokes Radius

A plasma inhibitor of tonin activity in the rat, was purified by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Its purity was investigated by analytical electrophoresis on polyacrylamide gel and by ultracentrifugation sedimentation velocity. The molecular weight (360 000) of the purified inhibitor was determined by sodium dodecyl sulfate electrophoresis and its isoelectric point (4.5) by gel isoelectrofocusing. The Stokes radius (640 nm) was evaluated by gel filtration studies and a frictional ratio (f/f0) of 1.95 was calculated from the molecular weight and Stokes radius.Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by the purified inhibitor was noncompetitive and does not exceed 70%. Electrophoresis showed the same mobility for [125I]tonin bound to plasma proteins and for [125I]tonin bound to the purified inhibitor. The inhibitor may be a protein resembling half of the dimeric protease inhibitor rat α1-macroglobulin or human α2-macroglobulin

scholarly journals Partial purification of topoisomerase IB from serum of diabetic patients and study it's kinetic properties and molecular weight

2019 ◽  
Vol 23 (10) ◽  
pp. 46
Author(s):  
Saif M. Hasan ◽  
Firas T. Maher ◽  
Nagham Q. Kadhim
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Maximum Velocity ◽  
Partial Purification ◽  
Diabetic Patients ◽  
Kinetic Properties ◽  
Topoisomerase Ib ◽  
Electrophoresis Method

This study was done to partially purification of  topoisomerase IB from serum of diabetic patients using Gel filtration technique, by using Sephadex G 100 gel. A single peak in fraction four has been obtained, and the degree of purification (17.1) fold, enzyme yield (108.2%) and specific activity (0.189ng/mg). Kinetics studies for the partial purified enzyme were carried out which showed optimal concentration of  substrate which was (0.1ng/ml), Michael's - Menten constant (Km=0.033ng) and maximum velocity (Vmax=0.90 ng/ml), while optimum Temperature was (37C°) and optimum pH was (7.5). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method, in presence of polyacrylamide  gel and sodium dodecyl sulphate (SDS-PAGE) which showed that the approximated molecular weight was (66KD).   http://dx.doi.org/10.25130/tjps.23.2018.168 

scholarly journals Purification and characterization of subcomponent C1q of the first component of mouse complement

1981 ◽  
Vol 193 (2) ◽  
pp. 621-629 ◽  
Author(s):  
K Yonemasu ◽  
T Sasaki
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Molecular Weight Range ◽  
Covalently Linked

1. Mouse C1q, a subcomponent of the first component of complement, has been purified in a highly haemolytically active form by a combination of precipitation with EGTA, ion-exchange chromatography and gel filtration. Yields ranged from 3 to 5 mg/200 ml of serum, and the activity of final preparations was in the range of 2 × 10(13)-4 × 10(13) C1q effective molecules/mg. 2. The molecular weight of mouse C1q was 439 500 +/- 1586, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 3. Mouse C1q was shown to be composed of non-covalently linked subunits, all being in the molecular-weight range 45 000-46 000, and three covalently linked chains each having a molecular weight of approx. 23 000 as determined on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate by using non-covalently and covalently linked subunits of human C1q as markers with known molecular weights calculated theoretically previously [Porter & Reid (1978) Nature (London) 275, 699-704]. 4. Mouse C1q contained hydroxyproline, hydroxylysine, a high percentage of glycine and approx. 9% carbohydrate. The absorption coefficient and nitrogen content of C1q were also determined.

Purification and partial characterization of a rat kidney aldehyde dehydrogenase that oxidizes retinal to retinoic acid

1993 ◽  
Vol 71 (1-2) ◽  
pp. 85-89 ◽  
Author(s):  
Jean Labrecque ◽  
Pangala V. Bhat ◽  
André Lacroix
Keyword(s):  
Molecular Weight ◽  
Retinoic Acid ◽  
Vitamin A ◽  
Aldehyde Dehydrogenase ◽  
Rat Kidney ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Exchange Chromatography ◽  
Size Exclusion

A NAD-dependent aldehyde dehydrogenase (EC 1.2.1.3) which catalyzes the oxidation of retinal to retinoic acid was purified to homogeneity from rat kidney by using Affi-Gel blue affinity chromatography and chromatofocusing, followed by Mono-Q anion-exchange chromatography. The apparent molecular weight of the native enzyme determined by size-exclusion fast protein liquid chromatography was 140 000. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis gave a subunit molecular weight of 53 000. The isoelectric point as measured by chromatofocusing was 8.5. The enzyme also catalyzed the oxidation of acetaldehyde, but showed much lower Km value for the retinal substrate. We suggest that aldehyde dehydrogenase found in the kidney may be a specific retinal dehydrogenase, involved in vitamin A metabolism.Key words: aldehyde dehydrogenase, vitamin A, retinal, retinoic acid, kidney.

scholarly journals Study of some hormones and Partial purification of prolidase from serum of women with polycystic ovary syndrome

2019 ◽  
Vol 24 (7) ◽  
pp. 59
Author(s):  
Reemy M. Mohamed Saleh ◽  
Firas T . Maher ◽  
Nagham Q. Kadhim
Keyword(s):  
Molecular Weight ◽  
Polycystic Ovary Syndrome ◽  
Specific Activity ◽  
Polycystic Ovary ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Ovary Syndrome ◽  
Electrophoresis Method ◽  
High Level

This study was done by partially purification of prolidase from serum of patients with polycystic ovary syndrome by Gel filtration technique, and using sephadex G100 gel as a stationary phase. The degree of purification (15.1) fold, enzyme yield (95.5%) and specific activity (0.00176 IU/I), were carried out .Kinetics studies for the partial purified enzyme technique showed optimal concentration of substrate which was 5 mmol/l Km = 0.66ng and Vmax =0.80 mM, while optimum Temperature was (35C°) and optimum pH was (8). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method , in presence of polyacrylamide gel and sodium dodecyl sulphate (SDS_PAGE) which showed that the approximates molecular weight was (54KD),we found high level of prolactin in the patient with polycystic ovary syndrome which was(24.03) when in the control was( 10.09),the value of TSH in the patient was( 17.08) which is high value and in the control was( 1.49), the value of T4 in the patient was (100.2) and in control was (118.4),the value of T3 in the patient was (0.3)and in control was (1.3).Testosterone in patient was (0.391) and in control was (0.206).    http://dx.doi.org/10.25130/tjps.24.2019.130

scholarly journals The isolation and partial characterization of low-molecular-weight phosphorylated component of the non-histone proteins of mouse nuclei

1978 ◽  
Vol 175 (1) ◽  
pp. 35-46 ◽  
Author(s):  
A J MacGillivray ◽  
C Johnston ◽  
R MacFarlane ◽  
D Rickwood
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Low Molecular Weight ◽  
Gel Chromatography ◽  
Liver Nuclei ◽  
Acidic Proteins

After labelling of mouse liver nuclei with [gamma-32P]ATP in vitro, 10-20% of the radioactivity incorporated into the saline-soluble nuclear and HAP2 chromatin fractions was located in a low-molecular-weight component (component 10) with pI near 4.5 in urea. By using combinations of ion-exchange chromatography, preparative thin-layer isoelectric focusing and gel filtration, this component was isolated from both nuclear fractions. Recovery from the saline-soluble fraction was poor under conditions that allow endogenous phosphatases to be active. Component 10 was shown to be a phosphoprotein on the basis of enzyme-digestion experiments and the detection of phosphoserine and phosphothreonine. The 32P radioactivity did not appear to be associated with phosphorylated basic amino acids. Its molecular weight was determined by gel chromatography and electrophoresis in sodium dodecyl sulphate/polyacrylamide gels as approx. 10000, and tryptic digestion of the reduced carboxymethylated protein in urea yielded two 32P-labelled peptides. It has not been possible as yet to assign a function to component 10, though its similarity to other low-molecular-weight acidic proteins is discussed.

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