scholarly journals Heparin-sepharose affinity chromatography for purification of bull seminal-plasma hyaluronidase

1979 ◽  
Vol 183 (3) ◽  
pp. 531-537 ◽  
Author(s):  
P N Srivastava ◽  
A A Farooqui
Keyword(s):  
Hyaluronic Acid ◽  
Affinity Chromatography ◽  
Seminal Plasma ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Single Band ◽  
National Formulary ◽  
Turnover Number

Bull seminal-plasma hyaluronidase was purified 180-fold by chromatography on concanvalin A-Sepharose, heparin Sepharose, Sephadex G-200 and Sephacryl S-200. With hyaluronic acid as the substrate, the specific activity and turnover number of purified hyaluronidase were 3.63 mumol/min per mg (104000 National Formulary units/mg of protein) and 214 min-1 (mol of product formed/mol of enzyme per min) respectively. Polyacrylamide-gel electrophoresis indicated that the purified enzyme migrated as a single band on 7.5 and 10% (w/v) gels at pH 4.3 and 5.3. Bull seminal-plasma hyaluronidase was markedly inhibited by hydroxylamine, phenylhydrazine and semicarbazide. Purified hyaluronidase (1.25 munits; 1 unit = 1 mumol of N-acetylglucosamine liberated/min at 37 degrees C) dispersed the cumulus clot of rabbit ova in 1 h at 22 degrees C.

Isolation of carboxypeptidase N by affinity chromatography on column of CNBr-activated Sepharose with immobilized antibody

1979 ◽  
Vol 44 (2) ◽  
pp. 626-630 ◽  
Author(s):  
Eva Simonianová ◽  
Marie Petáková
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rabbit Antiserum ◽  
Serum Analysis ◽  
Polyacrylamide Gel ◽  
Rat Serum ◽  
Carboxypeptidase N

The isolation of rat serum carboxypeptidase N (EC 3.4.2.2) by affinity chromatography on a column of CNBr-activated Sepharose with immobilized antibody is described. The ligands used were either rabbit antiserum to rat carboxypeptidase N or the IgG fraction prepared from this serum. The coupling of the isolated antibodies to CNBr-activated Sepharose increased the capacity of the column approximately three times. The specific activity of the enzyme prepared by this method was 109-times higher than the activity of the serum. Analysis of the final product by polyacrylamide gel electrophoresis showed carboxypeptidase N and traces of albumin.

Purification of Porcine and Human Tissue Activator

1979 ◽  
Author(s):  
P Wallen ◽  
M Rånby ◽  
N Bergsdorf ◽  
P Kok
Keyword(s):  
Affinity Chromatography ◽  
Human Tissue ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Potassium Thiocyanate ◽  
Polyacrylamide Gel ◽  
Main Band ◽  
Reference Preparation

Tissue activator from pig heart: A highly purified activator preparation from pig heart has been prepared, essentially using two affinity adsorbtion steps. 1) Affinity adsorbtion to fibrin and elution with potassium thiocyanate. 2) Affinity chromatography on Sepharose-arginine. The final product, which is obtained by gel filtration on Sephacryl S-200 contains according to SDS-polyacrylamide gel electrophoresis one main band with Mw 64000. Reduced samples still appear as one component but with Mw 31000. The specific activity is about 500000 IU/mg (WHO Reference Preparation for Urokinase) and the yield 15-25 %. Tissue activator from human uterus: A highly purified preparation of human tissue activator has been prepared from uterus by an immunosorbent technique using antibodies produced in goats against the porcine tissue activator and coupled to Sepharose. A crude preparation from 1 kg uterus tissue and containing about 100000 IU tissue activator was adsorbed on 30 g of the immunosorbent. The activity was eluted with a KSCN-gradient. Further purification was obtained by affinity chromatography on Sepharose-arginine. The yield in the active fraction was 30-35 % and the specific activity 200000 to 300000 IU/mg. SDS-polyacrylamide gel electrophoresis showed one main band and 1-2 additional trace components.

scholarly journals Purified human liver acid β-d-galactosidases possessing activity towards G(M1)-ganglioside and lactosylceramide

1977 ◽  
Vol 165 (3) ◽  
pp. 591-594 ◽  
Author(s):  
A L Miller ◽  
R G Frost ◽  
J S O'Brien
Keyword(s):  
Affinity Chromatography ◽  
Enzyme Activities ◽  
Gel Electrophoresis ◽  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Single Band

Our studies with purified human liver acid beta-D-galactosidases (EC 3.2.1.23) indicate that 4-methylumbelliferyl beta-D-galactosidase and G(M1)-ganglioside beta-D-galactosidase activities are identical with lactosylceramidase II activity. Evidence for this includes co-purification of all enzyme activities by affinity chromatography to yield a single band on polyacrylamide-gel electrophoresis and coincident elution from Sepharose 6B of all three enzyme activities.

Purification Of Factors IX And X From Clinical Concentrate

1981 ◽  
Author(s):  
G C Russell ◽  
G Kemble ◽  
E G D Tuddenham
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Single Band ◽  
Factor Ix ◽  
Factor X ◽  
Clinical Factor

Human factors IX and X have been purified to homogeneity from clinical factor IX concentrate that had been rejected for therapeutic use due to particulate contamination. (It was necessary to start with this material since in the UK, plasma is not commercially available). The procedure involved barium citrate adsorption followed by ammonium sulphate elution, DEAE- cellulose chromatography, gel filtration on Sephacryl S-200 and affinity chromatography on heparin sepharose gel. The preparation of factor IX at this stage showed a single band on SDS-polyacrylamide gel electrophoresis, of molecular weight 58,000. No change in molecular weight was observed in the presence of 2-mercaptoethanol. A further affinity chromatography column - poly (homoarginine) Sepharose or dextran sulphate sepharose - was necessary to obtain homogeneous factor X. The preparation obtained showed a single band on SDS-polyacrylamide gel electrophoresis of molecular weight 67,000. In the presence of 2-mercaptoethanol, two bands were obtained of molecular weights 49000 and 17000 representing the heavy and light chains respectively of factor X. The purified coagulation proteins contained no activated species detectable by nonactivated partial thromboplastin time or by chromogenic substrate (S2222) assay. Prothrombin protein Sand protein C are by-products of this purification procedure.

scholarly journals Studies on human pregnancy-associated plasma protein A. Purification by affinity chromatography and structural comparisons with α2-macroglobulin

1980 ◽  
Vol 191 (3) ◽  
pp. 799-809 ◽  
Author(s):  
R G Sutcliffe ◽  
B M Kukulska-Langlands ◽  
J R Coggins ◽  
J B Hunter ◽  
C H Gore
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Plasma Protein ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Carbohydrate Content ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Tryptic Fragment

Pregnancy-associated plasma protein-A (PAPP-A) has been purified by a combination of methods including antibody-affinity chromatography. The resultant protein, obtained in 16% yield from maternal serum, appeared as a single major component on non-denaturing polyacrylamide and SDS/polyacrylamide gel electrophoresis. The protein showed a single component when analysed by isoelectric focusing under denaturing conditions in the presence and absence of reduction and had a pI of 4.34 and 4.42 respectively. These pI values were indistinguishable from those of alpha 2-macroglobulin (alpha 2M). The molecular weight of the PAPP-A polypeptide as shown by SDS/polyacrylamide-gel electrophoresis was 187000, with a minor component of mol.wt. 82500 that was attributed to proteolysis. Since native PAPP-A had a molecular weight on gel chromatography very similar to that of alpha 2M (620000–820000), it was concluded that PAPP-A was a homotetramer. In the absence of reduction, a high-molecular-weight (420000) protomer of PAPP-A was found. It was deduced that PAPP-A, like alpha 2M, is a dinner, whose protomers are composed of disulphide-linked polypeptide chains. It was found that the molecular weight of the PAPP-A polypeptide exceeded that of alpha 2M by 3.3%, but that the total carbohydrate content of PAPP-A exceeded that of alpha 2M by 10% and that its neutral carbohydrate content exceeded that of alpha 2M by between 7.4 and 9.0%. The significance of the estimated molecular weights of alpha 2M (181000) and its major tryptic fragments is discussed in the light of published values. A tryptic fragment alpha 2M (82500 mol.wt.) was apparently the same size as the major tryptic fragment of PAPP-A.

scholarly journals Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

1983 ◽  
Vol 213 (1) ◽  
pp. 225-234 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

Two-dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins

1998 ◽  
Vol 19 (5) ◽  
pp. 797-801 ◽  
Author(s):  
Michele Mortarino ◽  
Gabriella Tedeschi ◽  
Armando Negri ◽  
Fabrizio Ceciliani ◽  
Luciano Gottardi ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Gel Electrophoresis ◽  
Plasma Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Two Dimensional ◽  
Seminal Plasma Proteins

scholarly journals Purification, characterization and radioimmunoassay of adenosine deaminase from human leukaemic granulocytes

1981 ◽  
Vol 195 (2) ◽  
pp. 389-397 ◽  
Author(s):  
D A Wiginton ◽  
M S Coleman ◽  
J J Hutton
Keyword(s):  
Molecular Weight ◽  
Adenosine Deaminase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Periodic Acid Schiff ◽  
Apparent Homogeneity

Adenosine deaminase was purified 3038-fold to apparent homogeneity from human leukaemic granulocytes by adenosine affinity chromatography. The purified enzyme has a specific activity of 486 mumol/min per mg of protein at 35 degrees C. It exhibits a single band when subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, non-denaturing polyacrylamide-gel electrophoresis and isoelectric focusing. The pI is 4.4. The enzyme is a monomeric protein of molecular weight 44000. Both electrophoretic behaviour and molecular weight differ from those of the low-molecular-weight adenosine deaminase purified from human erythrocytes. Its amino acid composition is reported. Tests with periodic acid-Schiff reagent for associated carbohydrate are negative. Of the large group of physiological compounds tested as potential effectors, none has a significant effect. The enzyme is specific for adenosine and deoxyadenosine, with Km values of 48 microM and 34 microM respectively. There are no significant differences in enzyme function on the two substrates. erythro-9-(2-Hydroxy non-3-yl) adenine is a competitive inhibitor, with Ki 15 nM. Deoxycoformycin inhibits deamination of both adenosine and deoxyadenosine, with an apparent Ki of 60-90 pM. A specific antibody was developed against the purified enzyme, and a sensitive radioimmunoassay for adenosine deaminase protein is described.

scholarly journals Phosphoenolpyruvate carboxylase from the crassulacean plant Bryophyllum fedtschenkoi Hamet et Perrier. Purification, molecular and kinetic properties

1978 ◽  
Vol 175 (2) ◽  
pp. 391-406 ◽  
Author(s):  
R Jones ◽  
M B Wilkins ◽  
J R Coggins ◽  
C A Fewson ◽  
A D B Malcolm
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Maximum Velocity ◽  
Single Band ◽  
Kinetic Properties ◽  
Subunit Structure

Phosphoenolpyruvate carboxylase from the Crassulacean plant Bryophyllum fedtschenkoi has been purified to homogenetity by DEAE-cellulose treatment, (NH4)2SO4 fractionation,, and chromatography on DEAE-cellulose and hydroxyapatite. Poly(ethylene glycol) is required in the extraction medium to obtain maximum enzyme activity. The purified enzyme has a specific activity of about 26 units/mg of protein at 25 degrees C. It gives a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a mol.wt. of 105,000, and gives a single band on non-denaturing gel electrophoresis at pH8.4. Cross-linking studies at pH8.0 indicate that the subunit structure is tetrameric but that the dimer may also be an important unit of polymerization. Gel filtration results at pH6.7 confirm that the native enzyme is tetrameric with a concentration-dependent dissociation to a dimer. The kinetic behaviour is characterized by (i) relatively small variations in maximum velocity between pH5.5 and 9.0 with a double optimum, (ii) a reversible temperature-dependent inactivation between 30 and 45 degrees C, (iii) inhibition by malate, which is pH-sensitive, and (iv) almost Michaelis-Menten behaviour with phosphoenolpyruvate as the varied ligand but sigmoidal behaviour under suitable conditions with malate as the varied ligand. The findings are related to other studies to the possible role phosphoenolpyruvate carboxylase in controlling a circadian rhythm of CO2 fixation.

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