HEPARIN INDEPENDENT PURIFICATION OF ANTITHROMBIN III (AT III) BY IMMUNO-AFFINITY CHROMATOGRAPHY RESULTING IN A FUNCTIONALLY INTACT MOLECULE

1987 ◽  
Author(s):  
M Jørgensen
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Binding Capacity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Step ◽  
Heparin Binding ◽  
Purification Method ◽  
Step Procedure ◽  
At Iii ◽  
Sepharose 4B

Previous methods for purification of AT III are based on its heparin-binding capacity. However, in congenital AT III deficiency abnormal inhibitor molecules with impaired binding of heparin and/or thrombin has been reported. The aim of the present study was to develop a purification method based on immuno-affinity chromatography, and thus independent of the heparin binding capacity.Rabbits were immunized with human AT III purified by a three-step procedure involving dextran sulphate precipitation, affinity chromatography on heparin-Sepharose and ion-exchange chromatography on DEAE-Sephadex A-50. Rabbit immunoglobulins against human AT III were isolated by affinity chromatography using purified human AT III coupled to CNBr-activated Sepharose 4B. Trace amounts of immunoglobulin against human albumin, IgG and IgM were removed by solid phase immunoadsorption. The highly purified immunoglobulins against human AT III were coupled to CNBr-activated Sepharose 4B. This anti-AT III-Sepharose was used for single-step purification of AT III from plasma. Elution was performed by Na-citrate buffer at pH 3.0 and the eluted fractions immediately neutralized. The recovery was more than 60%.The purified AT III appeared as a single protein band in SDS-poly-acrylamide gel electrophoresis with or without reduction. Affinity purified AT III and AT III purified by the three-step procedure were indistinguishable when analyzed by crossed immunoelectrophoresis in the absence and the presence of heparin isoelectrical focusing in polyacrylamid gel at a pH 4-6.5 gradient, and SDS-polyacrylamide gel electrophoresis. AT III antigen concentration was determined by electroimmunoassay and the reactive site concentration determined by titration with purified human thrombin using Phe-Pip-Arg-Nan (S-2238) as substrate. The ratio (active site conc.)/(antigen conc.) was identical in the two AT III preparations. It is concluded that this single-step immuno-affinity chromatography gives a high recovery from plasma of a highly purified functionally intact AT III molecule. The purification method is independent of the heparin binding capacity of AT III. This is of particular importance for the purification and characterization of abnormal AT III molecules with impaired heparin-binding site.

Purification of α2-Antiplasmin by a Single-Step Affinity Chromatographic Procedure

1979 ◽  
Author(s):  
B Wiman
Keyword(s):  
Binding Sites ◽  
Gel Electrophoresis ◽  
Major Part ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Step ◽  
High Ionic Strength ◽  
Chromatographic Purification ◽  
Purification Method ◽  
Activity Measurements ◽  
Chromatographic Procedure

A new and efficient single-step purification method for human α2-antiplasmin has been elaborated. The method is based on the interaction between α2-antiplasmin and a fragment (LBSI) constituting the three NH2-terminal triple-loop structures in plasminogen produced by elastase digestion. This fragment has been purified and coupled to Sepharose and used for affinity chromatographic purification of α2-antiplasmin using plasminogen depleted plasma as starting material. After adsorption and washing at high ionic strength the α2-antiplasmin is specifically eluted with 6-aminohexanoic acid. The inhibitor preparation obtained in this way is over 90% pure as judged from SDS polyacrylamide gel electrophoresis and activity measurements. About 40-45 mg pure α2-antiplasmin per liter plasma is obtained representing a yield of about 60%. LBS-I Sepharose has much higher capacity for α2-antiplasmin and is also much more specific than plasminogen-Sepharose. Repetitive treatment of plasma with LBS I-Sepharose failed to adsorb the last 20% of α2-antiplasmin as judged by Laurell electrophoresis. This supports the recent finding ot Clemmensen (1979) on partially purified α2-antiplasmin that a form of the inhibitor with less affinity for the lysine-binding sites in plasminogen may exist, even in unfractionated plasma. The major part of this type of α2-antiplasmin is also a functional antiplasmin since it can form a complex with plasmin.

scholarly journals Purification and properties of different forms of modeccin, the toxin of Adenia digitata. Separation of subunits with inhibitory and lectin activity

1980 ◽  
Vol 185 (1) ◽  
pp. 203-210 ◽  
Author(s):  
L Barbieri ◽  
M Zamboni ◽  
L Montanaro ◽  
S Sperti ◽  
F Stirpe
Keyword(s):  
Protein Synthesis ◽  
Affinity Chromatography ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Purification And Properties ◽  
Inhibitor Of Protein Synthesis ◽  
Sepharose 4B

1. The subunits were isolated of modeccin (subsequently referred to as modeccin 4B), the toxin purified from the roots of Adenia digitata by affinity chromatography on Sepharose 4B [Gasperi-Campani, Barbieri, Lorenzoni, Montanaro, Sperti, Bonetti & Stirpe (1978) Biochem J. 174, 491-496]. They are an A subunit (mol.wt. 26 000), which inhibits protein synthesis, and a B subunit (mol.wt. 31 000), which binds to cells. Both sununits, as well as intact modeccin, gave single bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but showed some heterogeneity on isoelectric focusing and on polyacrylamide-gel electrophoresis at pH 9.5. 2. A second form of modeccin, not retained by Sepharose 4B, was purified by affinity chromatography on acid-treated Sepharose 6B: this form is subsequently termed modeccin 6B 3. Modeccin 6B has a molecular weight indistinguishable from that of modeccin 4B, and consists of two subunits of mol.wts. 27 000 and 31 000, joined by a disulphide bond. The subunits were not isolated because of their high insolubility in the absence of sodium dodecyl sulphate. 4. As compared with modeccin 4B, modeccin 6B is slightly less toxic to animals, does not agglutinate erythrocytes, and is a more potent inhibitor of protein synthesis in a lysate of rabbit reticulocytes, giving 50% inhibition at the concentration of 0.31 microgram/ml.

scholarly journals General ligands in affinity chromatography. Cofactor–substrate elution of enzymes bound to the immobilized nucleotides adenosine 5′-monophosphate and nicotinamide–adenine dinucleotide

1972 ◽  
Vol 127 (4) ◽  
pp. 625-631 ◽  
Author(s):  
K. Mosbach ◽  
H. Guilford ◽  
R. Ohlsson ◽  
M. Scott
Keyword(s):  
Lactate Dehydrogenase ◽  
Affinity Chromatography ◽  
Cyanogen Bromide ◽  
Competitive Inhibitor ◽  
Single Step ◽  
Lactate Dehydrogenase Activity ◽  
Step Procedure ◽  
The Matrix ◽  
Subsequent Elution ◽  
Sepharose 4B

1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD+-dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation (a), NAD+–Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD+ was attached by using dicyclohexylcarbodi-imide; for preparation (b), AMP–Sepharose, N6-(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD+ yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP–Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5m. A 0.0–0.15m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP–Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5mm-NAD+); 80 (lactate+NAD+); 95 (lactate+semicarbazide+NAD+); 100 (0.5mm-NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP–Sepharose.

Purification of alpha1-fetoprotein.

1976 ◽  
Vol 22 (8) ◽  
pp. 1306-1309 ◽  
Author(s):  
S L Twomey ◽  
R V Sweet
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Pure Product ◽  
Sepharose 4B

Abstract Previously published methods for purifying alpha1-fetoprotein are inadequate because they either do not yield a completely pure product or they cause some denaturation. We present a method that does not have these serious disadvantages, and with which alpha1-fetoprotein was purified by sequential use of concanvalin A affinity-chromatography, preparative gel-electrophoresis, and immunoabsorption with anti-albumin antibody covalently coupled to Sepharose 4B. The purity of the product was monitored by discontinuous polyacrylamide-gel electrophoresis and counterimmunoelectrophoresis, both of which must be used to ascertain what proteins are present at each step of the purification.

scholarly journals Purification and characterization of rat adipose tissue lipoprotein lipase

1982 ◽  
Vol 207 (3) ◽  
pp. 485-495 ◽  
Author(s):  
S M Parkin ◽  
B K Speake ◽  
D S Robinson
Keyword(s):  
Adipose Tissue ◽  
Affinity Chromatography ◽  
Lipoprotein Lipase ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Sepharose 4B

Lipoprotein lipase (EC 3.1.1.34) extracted from adipose tissue of glucose-fed rats with 5 mM-sodium barbital, pH 7.5, containing 20% (v/v) glycerol and 0.1% (v/v) Triton X-100, was partially purified by affinity chromatography on heparin linked to Sepharose 4B. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the partially purified enzyme preparation revealed the presence of two major Coomassie-staining bands (mol.wts. 62 000 and 56 000) as well as a number of minor bands. Treatment of partially purified enzyme with [1,3-3H]di-isopropyl fluorophosphate resulted in the incorporation of radiolabel into the band of mol.wt. 56 000, but not into the band of mol.wt. 62 000. Both the amount of the 56 000-mol.wt. polypeptide and the incorporation of [1,3-3H]di-isopropyl fluorophosphate into this band were greatly reduced in the enzyme preparations isolated from adipose tissue of 48 h-starved rats. whereas the amount of the 62 000-mol.wt. polypeptide was unaffected by starvation. Purification of lipoprotein lipase from adipose tissue of glucose-fed rats was also carried out using affinity chromatography on Sepharose 4B linked to heparin with low affinity for antithrombin-III. This procedure resulted in the presence of a single band of mol.wt. 56 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These results suggest that the polypeptide of mol.wt. 56 000 corresponds to the subunit of lipoprotein lipase, whereas the 62 000-mol.wt. polypeptide probably represents antithrombin-III.

Purification of α2-Antiplasmin by a Single-Step Affinity Chromatographic Procedure

1979 ◽  
Author(s):  
B. Wiman
Keyword(s):  
Binding Sites ◽  
Gel Electrophoresis ◽  
Major Part ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Step ◽  
High Ionic Strength ◽  
Chromatographic Purification ◽  
Purification Method ◽  
Activity Measurements ◽  
Chromatographic Procedure

A new and efficient single-step purification method for human α2-antiplasmin has been elaborated. The method is based on the interaction between α2-antiplasmin and a fragment (LBSI) constituting the three NH2-terminal triple-loop structures in plasminogen produced by elastase digestion. This fragment has been purified and coupled to Sepharase and used for affinity chromatographic purification of α2-antiplasmin using plasminogen depleted plasma as starting material. After adsorption and washing at high ionic strength the α2-antiplasmin is specifically eluted with 6-aminohexanoic acid. The inhibitor preparation obtained in this way is over 90% pure as judged from SDS Polyacrylamide gel electrophoresis and activity measurements. About 40-45 mg pure α2-antiplasmin per liter plasma is obtained representing a yield of about 60%. LBS-I Sepharose has much higher capacity for α2-antiplasmin and is also much more specific than plasminogen-Sepharose. Repetitive treatment of plasma with LBS I-Sepharose failed to adsorb the last 20% of α2-antiplasmin as judged by Laurell electrophoresis. This supports the recent finding of Clemmensen (1979) on partially purified α2-antiplasmin that a form of the inhibitor with less affinity for the lysine-binding sites in plasminogen may exist, even in unfractionated plasma. The major part of this type of α2-antiplasmin is also a functional antiplasmin since it can form a complex with plasmin.

Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel

1990 ◽  
Vol 63 (03) ◽  
pp. 439-444 ◽  
Author(s):  
C Kuyas ◽  
A Haeberli ◽  
P Walder ◽  
P W Straub
Keyword(s):  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ease Of Use ◽  
Terminal Sequence ◽  
Purification Method ◽  
Human Fibrinogen ◽  
Isolation Methods ◽  
One Step ◽  
Complementary Binding

SummaryWith an immobilized synthetic pentapeptide GlyProArgProLys comprising the N-terminal sequence GlyProArg of the α-chain of fibrin, a new affinity method for the quantitative isolation of fibrinogen out of anticoagulated plasma was developed. The method proved to be superior to all known isolation methods in respect to ease of use and yield, since fibrinogen could be isolated in one step out of plasma with a recovery of more than 95% when compared to the immunologically measurable amounts of fibrinogen. Moreover the amounts of contaminating proteins such as fibronectin, factor XIII or plasminogen were negligible and the purity of the isolated fibrinogen was higher than 95% as measured by polyacrylamide gel electrophoresis. The clottability was 90% and more. Another advantage of this affinity purification method is the possibility to isolate fibrinogen quantitatively out of small plasma samples (<5 ml). Further, abnormal fibrinogen molecules, provided their complementary binding site for GlyProArg is preserved, may also be quantitatively isolated independent of any solubility differences as compared to normal fibrinogen. In addition fibrin(ogcn) fragments originating from plasmic digestion can be separated on the basis of their affinity to GlyProArg. The described affinity gel can be used more than 50 times without any loss of capacity.

scholarly journals Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

1981 ◽  
Vol 197 (3) ◽  
pp. 629-636 ◽  
Author(s):  
J L McKenzie ◽  
A K Allen ◽  
J W Fabre
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Human Brain ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Antibody Affinity ◽  
Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

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 Affinity purification and subunit structures of β-N-acetylhexosaminidases A and B from boar epididymis

1984 ◽  
Vol 219 (3) ◽  
pp. 1009-1015 ◽  
Author(s):  
H C Parkes ◽  
J L Stirling ◽  
P Calvo
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Gradient Gel Electrophoresis ◽  
Sepharose 4B ◽  
Electrophoretic Transfer ◽  
Peptide Maps

beta-N-Acetylhexosaminidase from boar epididymis was separated into two forms, A and B, on DEAE-cellulose. Both these forms were excluded from Sepharose S-200 and had apparent Mr values of 510 000 on gradient gel electrophoresis under non-denaturing conditions. Affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylam ine coupled to CNBr-activated Sepharose 4B was used to separate and purify beta-N-acetylhexosaminidases A and B that had specific activities of 115 and 380 mumol/min per mg of protein respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of denatured beta-N-acetylhexosaminidase A gave a single major component of Mr 67 000. beta-N-Acetylhexosaminidase B also had this component, and in addition had polypeptides of Mr 29 000 and 26 000. All these polypeptides were glycosylated. Antiserum to the B form precipitated form A from solution and reacted with the 67 000-Mr component or form A after electrophoretic transfer from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose sheets. The 67 000-Mr components of forms A and B yielded identical peptide maps when digested with Staphylococcus aureus V8 proteinase, and the 29 000-Mr and 26 000-Mr components in form B may be related to the 67 000-Mr polypeptide.

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