Extraction and properties of hemagglutinin from cell wall fragments of Fusobacterium nucleatum

1982 ◽  
Vol 152 (1) ◽  
pp. 298-305
Author(s):  
P Dehazya ◽  
R S Coles
Keyword(s):  
Cell Wall ◽  
Sodium Dodecyl Sulfate ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fusobacterium Nucleatum ◽  
Blue Staining ◽  
Sephadex Column ◽  
Dodecyl Sulfate ◽  
Triton X

To study the hemagglutinin of Fusobacterium nucleatum, methods were sought to solubilize and purify this component. When cells of F. nucleatum were ruptured by passage through a French press, the fragments lost virtually all ability to agglutinate human erythrocytes. Extraction of the fragments with 2% Triton X-100 for 30 min at 22 degrees C restored hemagglutinating activity (HA). Hemagglutination by these fragments could be inhibited by arginine, as can hemagglutination by intact bacteria. Treatment of active cell wall fragments with pronase and 2% Triton X-100-EDTA at 37 degrees C or with pronase and 0.1% Triton X-100-EDTA at pH 10.0 allowed recovery of solubilized HA. The former HA was inhibited by arginine (arg+) whereas the latter was not (arg-). Fractionation of the arg+ extract by preparative isoelectric focusing showed that HA was recovered from the gel sections having a pH between 4.5 and 5.5. Hemagglutination by this preparation was still arg+. Chromatography of this hemagglutinin on DEAE-Sephadex increased the specific activity to high levels with a loss of inhibition by arginine. A fraction from the DEAE-Sephadex column containing 10,700 HA units per mg of protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Solubilization at 22 degrees C before electrophoresis revealed three Coomassie blue-staining bands which migrated with apparent molecular weights of about 21,000, 38,000 and 60,000. When the same DEAE fraction was boiled in sodium dodecyl sulfate, electrophoresis revealed only one band with an apparent molecular weight of 21,000.

Simplified sodium dodecyl sulfate-polyacrylamide gel electrophoresis of unconcentrated urine with enhanced resolution and detection sensitivity.

1987 ◽  
Vol 33 (10) ◽  
pp. 1886-1887 ◽  
Author(s):  
T Marshall ◽  
K M Williams
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Detection Sensitivity ◽  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Dodecyl Sulfate

Abstract We applied a simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis method to urine. The method, developed for serum protein analysis (Clin Chem 1984;30:475-9), has a high sample throughput and gives excellent resolution with unconcentrated urine. It clearly distinguishes and characterizes proteinuric urine (7.5 microL) by Coomassie Blue staining and gives complex silver-stained patterns with nonproteinuric urine (2 microL). The former is recommended for routine clinical screening, the latter for research purposes.

Rat lingual lipase: partial purification, hydrolytic properties, and comparison with pancreatic lipase

1984 ◽  
Vol 247 (4) ◽  
pp. G385-G393 ◽  
Author(s):  
I. M. Roberts ◽  
R. K. Montgomery ◽  
M. C. Carey
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Pancreatic Lipase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Partial Purification ◽  
Ph Optimum ◽  
Dodecyl Sulfate ◽  
Triton X

We have partially purified lingual lipase from the serous glands of rat tongue. With a combination of Triton X-100 extraction or Triton X-114 phase-separation techniques, Bio-Bead SM-2 treatment, dialysis, and gel filtration on Sephadex G-200 or Sephacryl S-300, we obtained a sparingly soluble lipid-free protein demonstrating hydrolytic activity against triglycerides and negligible phospholipase or cholesteryl esterase activities. Compared with homogenate, specific activities of the enzyme were enriched 3- to 5-fold prior to gel filtration and 10-fold after gel filtration. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration under denaturing conditions (6 M guanidine X HCl or 0.1% sodium dodecyl sulfate) revealed one major glycoprotein band with Mr approximately 50,000. Gel filtration of the active enzyme in 0.1% Triton X-100 gave an Mr approximately 270,000-300,000, suggesting extensive self-aggregation. With both tributyrin and triolein, the pH optimum of the purified enzyme was 4.0 and activity extended from pH 2.0 to 8.0. In contrast to purified human pancreatic lipase, lingual lipase hydrolyzed triglyceride emulsions and mixed micelles stabilized with both short-chain (dihexanoyl) and long-chain (egg) lecithin and were inhibited only slightly (18-25%) by micellar concentrations of two common bile salts, taurodeoxycholate and taurocholate. Our results suggest that the hydrolysis of dietary fat by lingual lipase may extend from the pharynx through the esophagus and stomach and into the upper small intestine.

Reducing the artifacts produced by impure antisera in immunoblots of low-molecular-mass proteins in urine.

1986 ◽  
Vol 32 (5) ◽  
pp. 811-815 ◽  
Author(s):  
J M Perini ◽  
B Dehon ◽  
T Marianne ◽  
A Klein ◽  
P Roussel
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rabbit Serum ◽  
Urinary Proteins ◽  
Dodecyl Sulfate ◽  
Triton X ◽  
Direct Use

Abstract Immunoblots of several urinary low-molecular-mass proteins can be very useful in investigations of pathological proteinuria. However, use of certain commercial antisera in such procedures leads to artifacts corresponding to nonspecific bands; e.g., immunoglobulins from nonimmunized rabbit serum may bind to human urinary proteins, and this binding is not inhibited by Triton X-100. We have developed a procedure to improve the specificity of detection of urinary low-Mr proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by immunoblotting with commercial antisera: we treat the protein blot with a mixture of mercaptoethanol and sodium dodecyl sulfate before incubation with the first antiserum. This allows direct use of commercial antisera without prior absorption of contaminating antibodies.

Purification of human prostatic acid phosphatase by affinity chromatography and isoelectric focusing. Part I.

1978 ◽  
Vol 24 (3) ◽  
pp. 466-470 ◽  
Author(s):  
P Vihko ◽  
M Kontturi ◽  
L K Korhonen
Keyword(s):  
Acid Phosphatase ◽  
Sodium Dodecyl Sulfate ◽  
Affinity Chromatography ◽  
Isoelectric Focusing ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Prostatic Acid Phosphatase ◽  
Polyacrylamide Gel ◽  
Dodecyl Sulfate

Abstract The main isoenzyme of human prostatic acid phosphatases was purified by affinity chromatography on L(+)-tartrate linked to agarose and by isoelectric focusing. The enzyme was a single protein when examined by polyacrylamide gel electrophoreses, either as a native protein or in the presence of sodium dodecyl sulfate. The analytical recovery of enzyme activity was 19%. The specific activity was 40 18 mumol/(min X mg) for hydrolysis of 5.5 mmol/liter p-nitrophenyl phosphate at pH 4.8 and 37 degrees C. The purification factor was as great as 1900. The molecular weight of the enzyme as measured by gel filtration was 109 000 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate 54 000, indicating that the enzyme had been isolated in the dimer form. By this method we have achieved the best purification of human prostatic acid phosphatase so far.

Purification of proteins similar to HPr and enzyme I from the oral bacterium Streptococcus salivarius. Biochemical and immunochemical properties

1983 ◽  
Vol 29 (12) ◽  
pp. 1694-1705 ◽  
Author(s):  
Christian Vadeboncoeur ◽  
Mario Proulx ◽  
Luc Trahan
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Streptococcus Salivarius ◽  
Dodecyl Sulfate ◽  
Molecular Sieving ◽  
Enzyme I

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is made of several proteins. Two of them are designated general proteins because they are required for the transport and phosphorylation of all sugars of the PTS. These two proteins are found in the soluble fraction of cellular extracts and are termed HPr and enzyme I (EI). We reported in this work the purification and the characterization of these two proteins from Streptococcus salivarius ATCC 25975. HPr was purified by DEAE-cellulose chromatography, molecular sieving on Ultrogel AcA44, and carboxymethylcellulose chromatography. Sodium dodecyl sulfate electrophoresis in the presence of urea revealed a single band with a molecular weight of 6700. The protein contained no tryptophan and had a pi of 4.8. The purification scheme of EI was as follows: DEAE-cellulose chromatography, hydroxylapatite chromatography, DEAE-Sephadex A-50 chromatography, preparative electrophoresis, and molecular sieving on Ultrogel AcA34. The five-step purification for EI produced a 199-fold purified preparation with a specific activity of 530 μmol of HPr phosphorylated per minute per milligram of protein at 37 °C. The fraction obtained after filtration on Ultrogel AcA34 gave one band (68 000) on sodium dodecyl sulfate – polyacrylamide gel electrophoresis. The molecular weight of the native enzyme determined by gel filtration at 4 °C was 135 000, suggesting that it was a dimer. Enzyme I had a pI of 4.2, a pH optimum of 6.7, a Km for HPr of about 27 μM, a Km for phosphoenolpyruvate of 0.48 mM, and kinetics that were consistent with a Ping-Pong mechanism. Evidence had been obtained which indicated that S. salivarius enzyme I was antigenically very similar to enzyme I from various strains of Streptococcus mutans, but not to the enzyme from Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, and Escherichia coli.

Solubility and adsorption behaviors of chlorophenols in the presence of surfactant

1997 ◽  
Vol 35 (7) ◽  
pp. 123-130 ◽  
Author(s):  
J. C. Liu ◽  
P. S. Chang
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Critical Micelle Concentration ◽  
Sodium Dodecyl ◽  
Adsorption Behaviors ◽  
Brij 35 ◽  
Important Index ◽  
Dodecyl Sulfate ◽  
Triton X

The solubility of chlorophenols as affected by surfactant was investigated. Three kinds of surfactant, sodium dodecyl sulfate, Triton X-100, and Brij 35, were utilized. The solubilization of chlorophenols by surfactant follows the order of 2,4,6-trichlorophenol > 2,4-dichlorophenol > 2,6-dichlorophenol > 2-chlorophenol; and the critical micelle concentration is an important index. The adsorption reactions of 2,4-dichlorophenol and 2,4,6- trichlorophenol onto hydrous montmorillonite in the presence of surfactant were examined. The presence of surfactant decreased the adsorption of chlorophenols significantly. The roles of hydrophobicity of chlorophenols in solubilization and adsorption behaviors are discussed.

Different submicellar solubilization mechanisms revealed by 1H NMR and 2D diffusion ordered spectroscopy (DOSY)

2020 ◽  
Vol 22 (19) ◽  
pp. 11075-11085
Author(s):  
Mengjian Wu ◽  
Zhaoxia Wu ◽  
Shangwu Ding ◽  
Zhong Chen ◽  
Xiaohong Cui
Keyword(s):  
Nmr Spectroscopy ◽  
Sodium Dodecyl Sulfate ◽  
1H Nmr ◽  
Sodium Dodecyl ◽  
Butyl Methacrylate ◽  
H Nmr ◽  
Molecular Scale ◽  
Two Systems ◽  
Dodecyl Sulfate ◽  
Triton X

Different submicellar solubilization mechanisms of two systems, Triton X-100/tetradecane and sodium dodecyl sulfate (SDS)/butyl methacrylate, are revealed on the molecular scale by 1H NMR spectroscopy and 2D diffusion ordered spectroscopy (DOSY).

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