Selective purification of intestinal maltase complex by affinity chromatography employing an uncompetitive inhibitor as the ligand

The microstructure of polyacrylonitrile (PAN) beads for affinity chromatography bioseparations was studied by TEM of stained ultramicrotomed thin-sections. Microstructural aspects such as overall pore size distribution, the distribution of pores within the beads, and surface coverage of functionalized beads affect performance properties. Stereological methods are used to quantify the internal structure of these chromatographic supports. Details of the process for making the PAN beads are given elsewhere. TEM specimens were obtained by vacuum impregnation with a low-viscosity epoxy and sectioning with a diamond knife. The beads can be observed unstained. However, different surface functionalities can be made evident by selective staining. Amide surface coverage was studied by staining in vapor of a 0.5.% RuO4 aqueous solution for 1 h. RuO4 does not stain PAN but stains, amongst many others, polymers containing an amide moiety.

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Affinity Chromatography

10.1016/s0301-4770(08)x6029-4 ◽

1978 ◽

Keyword(s):

Affinity Chromatography

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Plasminogen-Plasmin System IX. Specific Binding of Tranexamic Acid to Plasmin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647851 ◽

1975 ◽

Vol 33 (03) ◽

pp. 573-585 ◽

Cited By ~ 20

Author(s):

Masahiro Iwamoto

Keyword(s):

Tranexamic Acid ◽

Affinity Chromatography ◽

Dissociation Constant ◽

Factor Xiii ◽

Specific Binding ◽

The Other ◽

Inhibitory Mechanism ◽

Binding Studies ◽

Similar Affinity ◽

Fibrin Structure

SummaryInteractions between tranexamic acid and protein were studied in respect of the antifibrinolytic actions of tranexamic acid. Tranexamic acid did neither show any interaction with fibrinogen or fibrin, nor was incorporated into cross-linked fibrin structure by the action of factor XIII. On the other hand, tranexamic acid bound to human plasmin with a dissociation constant of 3.5 × 10−5 M, which was very close to the inhibition constant (3.6 × 10−5 M) for this compound in inhibiting plasmin-induced fibrinolysis. The binding site of tranexamic acid on plasmin was not the catalytic site of plasmin, because TLCK-blocked plasmin also showed a similar affinity to tranexamic acid (the dissociation constant, 2.9–4.8 × 10−5 M).In the binding studies with the highly purified plasminogen and TLCK-plasmin preparations which were obtained by affinity chromatography on lysine-substituted Sepharose, the molar binding ratio was shown to be 1.5–1.6 moles tranexamic acid per one mole protein.On the basis of these and other findings, a model for the inhibitory mechanism of tranexamic acid is presented.

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The Immunological Characterization of Human Antibodies to Factor VIII Isolated by Immuno-Affinity Chromatography

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650129 ◽

1981 ◽

Vol 45 (01) ◽

pp. 060-064 ◽

Cited By ~ 10

Author(s):

M L Kavanagh ◽

C N Wood ◽

J F Davidson

Keyword(s):

Factor Viii ◽

Affinity Chromatography ◽

Gel Filtration ◽

Immunological Characterization ◽

Human Antibodies ◽

Heavy Chains ◽

Light Chain Type ◽

Antibody Preparations ◽

Chain Type

SummaryNine human antibodies to factor VIII were isolated from haemophilic plasmas by affinity chromatography and gel filtration and six were subsequently subjected to immunological characterization. Three partially purified preparations were similarly characterized. Eight of the antibodies were characterized as being exclusively IgG and one preparation was found to contain IgM. Seven of the antibodies contained only a single light chain type, four being of type lambda and three of type kappa. Two antibody preparations contained both kappa and lambda light chains. In four of the preparations, only a single heavy chain sub-class could be demonstrated, three of IgG3 and one of IgG4. Of the remainder, three were a mixture of IgG3 and IgG4 sub-classes and one contained both IgG2 and IgG4. IgG sub-classification could not be achieved with the IgM-containing preparation. These results demonstrate a restricted heterogeneity of light and heavy chains in human antibodies to factor VIII.

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The Different Forms of Antithrombin III in Serum

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651855 ◽

1977 ◽

Vol 38 (02) ◽

pp. 0494-0503 ◽

Cited By ~ 18

Author(s):

D. S Pepper ◽

D Banhegyi ◽

J. D Cash

Keyword(s):

Molecular Weight ◽

Affinity Chromatography ◽

Human Serum ◽

Degradation Product ◽

Antithrombin Iii ◽

Gel Filtration ◽

Free Form ◽

Polymeric Form ◽

At Iii ◽

Human Thrombin

SummaryAntithrombin III (AT III) complexes were isolated from human serum by affinity chromatography and gel filtration. In the first step of the preparation, using heparin-agarose chromatography, we observed that the complexed form of AT III bound less strongly to the gel than the free form and that about half of the AT III was free. With further purification a 2.5 × 105 molecular weight complex was isolated. Using 125I labelled human thrombin, this complex was radioactive indicating the presence of thrombin. Only in a synthetic thrombin-AT III system was a 9 × 104 molecular weight complex detected, but not in serum. These facts suggest that in serum AT III complexes may exist in a polymeric form. Also, an AT III antigen derived from the original AT III molecule, but not complexed, was isolated which may be a degradation product.Abbreviations used: AT-III, antithrombin III. Hepes, N-2-Hydroxyethylpiperazine-N-2-Ethanesulphonic acid.

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Kinetic Study of the Effect of Heparin on the Amidase Activity of Trypsin, Plasmin and Urokinase

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657362 ◽

1983 ◽

Vol 49 (03) ◽

pp. 199-203 ◽

Cited By ~ 10

Author(s):

V M Yomtova ◽

N A Stambolieva ◽

B M Blagoev

Keyword(s):

Kinetic Study ◽

Active Center ◽

Simultaneous Presence ◽

Amidase Activity ◽

Competitive Inhibitors ◽

Uncompetitive Inhibitor

SummaryIt was found that the effect of heparin on the amidase activity of urokinase (E C 3.4.21.31), plasmin (E C 3.4.21.7) and trypsin (E C 3.4.21.4) depended on the substrate used. No effect of heparin on the amidase activity of urokinase and trypsin was observed when Pyro Glu-Gly-Arg-p-nitroanilide (S-2444) and α-N-acetyl-L-lysine-p-nitroanilide (ALNA) were used as substrates. Heparin acted as a uncompetitive inhibitor of trypsin (Ki = 1.2×10-6 M), plasmin (Ki = 4.9×10-6 M) and urokinase (Ki = l.0×10-7 M) when Bz-Phe-Val-Arg-p-nitroanilide (S-2160), H-D-Val-Leu-Lys-p-nitroanilide (S-2251) and plasminogen, respectively, were used as substrates. These results, as well as the data obtained by studying the effect of the simultaneous presence of heparin and competitive inhibitors suggest that although heparin is not bound at the active center of these enzymes, it may influence the effectivity of catalysis.

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Quantitative Assessment of Soluble Fibrin in Plasma by Affinity Chromatography – A Comparative Study with desAA-Fibrin, desAABB-Fibrin and Fibrinogen

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657891 ◽

1985 ◽

Vol 54 (02) ◽

pp. 533-538 ◽

Cited By ~ 4

Author(s):

Wilfried Thiel ◽

Ulrich Delvos ◽

Gert Müller-Berghaus

Keyword(s):

Affinity Chromatography ◽

Calcium Ions ◽

Quantitative Assessment ◽

Fluid Phase ◽

Soluble Fibrin ◽

Binding Characteristics ◽

Different Temperatures ◽

Immobilized Proteins ◽

Sepharose 4B

SummaryA quantitative determination of soluble fibrin in plasma was carried out by affinity chromatography. For this purpose, desAA-fibrin and fibrinogen immobilized on Sepharose 4B were used at the stationary side whereas batroxobin-induced 125I-desAA-fibrin or thrombin-induced 125I-desAABB-fibrin mixed with plasma containing 131I-fibrinogen represented the fluid phase. The binding characteristics of these mixtures to the immobilized proteins were compared at 20° C and 37° C. Complete binding of both types of fibrin to the immobilized desAA-fibrin was always seen at 20° C as well as at 37° C. However, binding of soluble fibrin was accompanied by substantial binding of fibrinogen that was more pronounced at 20° C. Striking differences depending on the temperature at which the affinity chromatography was carried out, were documented for the fibrinogen-fibrin interaction. At 20° C more than 90% of the applied desAA-fibrin was bound to the immobilized fibrinogen whereas at 37° C only a mean of 17% were retained at the fibrinogen-Sepharose column. An opposite finding with regard to the tested temperature was made with the desAABB-fibrin. Nearly complete binding to insolubilized fibrinogen was found at 37° C (95%) but only 58% of the desAABB-fibrin were bound at 20° C. The binding patterns did not change when the experiments were performed in the presence of calcium ions. The opposite behaviour of the two types of soluble fibrin to immobilized fibrinogen at the different temperatures, together with the substantial binding of fibrinogen in the presence of soluble fibrin to insolubilized fibrin in every setting tested, devaluates affinity chromatography as a tool in the quantitative assessment of soluble fibrin in patients’ plasma.

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Factors Influencing the Separation of Glu-Plasminogen Affinity Forms I and II by Affinity Chromatography

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661211 ◽

1984 ◽

Vol 52 (03) ◽

pp. 347-349 ◽

Cited By ~ 6

Author(s):

Daan W Traas ◽

Bep Hoegee-de Nobel ◽

Willem Nieuwenhuizen

Keyword(s):

Affinity Chromatography ◽

Dissociation Constants ◽

Ionic Composition ◽

Chloride Ions ◽

Factors Influencing ◽

Two Factors ◽

Human Plasminogen

SummaryNative human plasminogen, the proenzyme of plasmin (E. C. 3.4.21.7) occurs in blood in two well defined forms, affinity forms I and II. In this paper, the feasibility of separating these forms of human native plasminogen by affinity chromatography, is shown to be dependent on two factors: 1) the ionic composition of the buffer containing the displacing agent: buffers of varying contents of sodium, Tris, phosphate and chloride ions were compared, and 2) the type of adsorbent. Two adsorbents were compared: Sepharose-lysine and Sepharose-bisoxirane-lysine. Only in the phosphate containing buffers, irrespective of the type of adsorbent, the affinity forms can be separated. The influence of the adsorbent can be accounted for by a large difference in dissociation constants of the complex between plasminogen and the immobilized lysine.

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