Soluble Fibrin Complexes: Separation as a Function of pH and Characterization

Summary1. The influence of the pH on the separation of high molecular weight derivatives obtained by a limited action of thrombin on fibrinogen was studied by agarose gel chromatography. When the pH of the elution buffer was 8.5, non crosslinked associations were easily separated in two peaks eluted prior to the fibrinogen peak: one contained a dimer, the other several high polymers. At pH 6.5, only the fibrinogen peak appeared: the fibrinogen molecule proteolysed by thrombin formed no stable associations at this pH and was eluted with the intact fibrinogen molecule. In the presence of factor XIII and Ca++, numerous associations were obtained which are independant of the pH.2. The polypeptide chain composition of the different species separated at pH 8.5 was studied by SDS-polyacrylamide gel electrophoresis. This technic showed Aα, Bβ and γ chains in the fibrinogen peak, whereas in the chromatographic fractions containing the dimer four bands corresponding to Aα, α, Bβ and γ chains were found. In the peak containing the high polymers, only the presence of α, Bβ and γ chains was demonstrated.3. These experimental results concerning the effect of pH on the formation of soluble complexes showed that the presence of fibrin monomers in fibrinogen solution was not sufficient to promote any associations. The formation of such derivatives is strongly dependent on the pH of the solution. This obviously can be explained by an influence of the pH either on the ionization of polymerisation sites and the intermolecular bonds between the complex units or on the unmasking of the polymerisation sites by a hypothetical pH induced conformational change of the fibrinogen molecule.

Download Full-text

Functional Relationship of Polymerization Sites of Vertebrate Fibrinogens

10.1055/s-0038-1665661 ◽

1979 ◽

Author(s):

C Cierniewski ◽

T Krajewski ◽

E Janiak

Keyword(s):

Gel Electrophoresis ◽

Functional Relationship ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Functional Similarity ◽

Fractionation Method ◽

Relationship Of ◽

Fibrin Monomers ◽

Fibrinogen Molecule ◽

Cold Ethanol Fractionation

Various studies on the interaction of immobilized mammalian fibrinogen and fibrin monomers with some fibrinogen derivatives demonstrated the presence of two sets of polymerization sites in the mammalian fibrinogen molecule. We obtained the same results while investigating the fibrinogen molecules of other classes of vertebrates /Pisces. Amphibia. Aves/. Despite significant differences among their subunit structures, all of them contain polymerization sites homologous to mammalian counterparts. Moreover, due to great functional similarity, fibrinogen or fibrin monomers of the analyzed species of Pisces. Amphibia. Aves and Mammalia interacted in a specific way with immobilized pig fibrin monomers or fibrinogen, respectively. Using these pig affinity adsorbents, fibrinogen and fibrin monomers of different vertebrates were isolated directly from plasma and analyzed by SDS polyacrylamide gel electrophoresis. Polypeptide compositions of eluted proteins were identical to those obtained for corresponding fibrinogen preparations isolated by cold-ethanol fractionation method. It appears to indicate that the nature of polymerization sites in vertebrate fibrinogens is alike.

Download Full-text

Structural Abnormality in the Gamma Polypeptide Chain of Paris I Fibrinogen

10.1055/s-0039-1689118 ◽

1975 ◽

Author(s):

Andrei Z. Budzynski ◽

Victor J. Marder ◽

Doris Ménaché ◽

Marie-Claude Guillin

Keyword(s):

Structural Defect ◽

Gel Electrophoresis ◽

Factor Xiii ◽

Polypeptide Chain ◽

Polyacrylamide Gel Electrophoresis ◽

Structural Abnormality ◽

Crosslinking Reaction ◽

Carboxy Terminal Region ◽

Carboxy Terminal ◽

Fibrin Monomers

Congenital abnormal fibrinogen Paris I has an abnormal γ chain which is probably the cause of impaired polyperization of fibrin monomers and an inability to crosslink in the presence of Factor XIII. SDS-polyacrylamide gel electrophoresis demonstrated that the Aα and Bβ chains of Paris I fibrinogen are normal. However, only 1/3 of the γ chains are normal, the remaining 2/3 migrating as a heavier species between the B β and γ chains. The conversion of Paris I fibrinogen into crosslinked fibrin in the presence of thrombin, calcium and Factor XIII is accompanied by the disappearance of the normal γ chain and the formation of γ chain dimers, but the abnormal γ Paris I chain did not disappear. There was only partial disappearance of the α chains, with approximately 2/3 not participating in the crosslinking reaction. It would appear that 2/3 of the fibrinogen molecules in the plasma of the propositus have abnormal γ Paris I chains, which are approximately 2500 daltons heavier than the normal γ chains. Plasmic digestion of Paris I fibrinogen results in the formation of an abnormal Fragment D with a heavier γ chain remnant. Degradation of Paris I fibrin yields a normal Fragment D-D and an abnormal Fragment D. The data indicate that the structural defect in γ Paris I chain occurs in the carboxy-terminal region, which can account for both the defective polymerization and imperfect crosslinking.

Download Full-text

Functional Relationship of Polymerization Sites of Vertebrate Fibrinogens

10.1055/s-0039-1684390 ◽

1979 ◽

Author(s):

C.S. Ciernlewski ◽

T. Krajewski ◽

E. Janiak

Keyword(s):

Gel Electrophoresis ◽

Functional Relationship ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Functional Similarity ◽

Fractionation Method ◽

Relationship Of ◽

Fibrin Monomers ◽

Fibrinogen Molecule ◽

Cold Ethanol Fractionation

Various studies on the interaction of immobilized mammalian fibrinogen and fibrin monomers with some fibrinogen derivatives demonstrated the presence of two sets of polymerisation sites In the mammalian fibrinogen molecule. We obtained the same results while investigating the fibrinogen molecules of other classes of vertebrates /Pisces, Amphibia, Aves/. Despite significant differences among their subunit structures, all of them contain polymerization sites homologous to mammalian counterparts. Moreover, due to great functional similarity, fibrinogen or fibrin monomers of the analyzed species of Pisces, Amphibia, Ayes and Mammalia interacted in a specific way with Immobilized pig fibrin monomers or fibrinogen, respectively. Using these pig affinity adsorbents, fibrinogen and fibrin monomers of different vertebrates were isolated directly from plasma and analyzed by SDS Polyacrylamide gel electrophoresis. Polypeptide compositions of eluted proteins were identical to those obtained for corresponding fibrinogen preparations isolated by cold-ethanol fractionation method. It appears to indicate that the nature of polymerization sites in vertebrate fibrinogens is alike.

Download Full-text

A New Fibrinogen Variant With Abnormal Gamma Chain : Fibrinogen Haifa

10.1055/s-0038-1653071 ◽

1981 ◽

Author(s):

J Soria ◽

C Soria ◽

G Palareti ◽

M Tavori ◽

M Samama ◽

...

Keyword(s):

Protective Effect ◽

Gel Electrophoresis ◽

Arterial Thrombosis ◽

Degradation Products ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Gamma Chain ◽

Peripheral Arterial ◽

Fibrin Monomers ◽

Fibrinogen Molecule

A congenital abnormal fibrinogen was detected in a 30 year old woman presenting several episodes of peripheral arterial thrombosis.The abnormality in fibrin formation is located in the fibrin monomers aggregation, since the fibrinopeptides release and the fibrin stabilization were normal.The SDS polyacrylamide gel electrophoresis of plasmic degradation products, obtained either in the presence or in the absence of calcium, revealed an absence of protective effect of calcium for plasmin degradation of Fibrinogen Haifa. Since, Ca++ protects against further plasmin degradation of the gamma chain, we suggest that the anomaly is located in this part of the fibrinogen molecule.Because the anomaly was discovered at Haifa, Israel, we suggest to call this abnormal fibrinogen : “Fibrinogen Haifa”.

Download Full-text

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657115 ◽

1982 ◽

Vol 47 (01) ◽

pp. 014-018 ◽

Cited By ~ 13

Author(s):

H Sumi ◽

N Toki ◽

S Takasugi ◽

S Maehara ◽

M Maruyama ◽

...

Keyword(s):

Trypsin Inhibitor ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Chromatography ◽

Urinary Trypsin Inhibitor ◽

Plasmin Activity ◽

Molecular Weights ◽

Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

Download Full-text

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

Canadian Journal of Biochemistry ◽

10.1139/o77-143 ◽

1977 ◽

Vol 55 (9) ◽

pp. 958-964 ◽

Cited By ~ 6

Author(s):

M. P. C. Ip ◽

R. J. Thibert ◽

D. E. Schmidt Jr.

Keyword(s):

Molecular Weight ◽

Rat Liver ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Liver Homogenate ◽

Polyacrylamide Gel ◽

Gel Chromatography ◽

Labile Hydrogen ◽

Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj1910799 ◽

1980 ◽

Vol 191 (3) ◽

pp. 799-809 ◽

Cited By ~ 41

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.

Download Full-text

Partial characterization of an abnormal lactate dehydrogenase isoenzyme, LDH-1ex, in serum from a patient with hepatocellular carcinoma.

Clinical Chemistry ◽

10.1093/clinchem/35.5.844 ◽

1989 ◽

Vol 35 (5) ◽

pp. 844-848

Author(s):

D L Kalpaxis ◽

E E Giannoulaki

Keyword(s):

Hepatocellular Carcinoma ◽

Lactate Dehydrogenase ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Chromatography ◽

Heat Stable ◽

Single Polypeptide ◽

Polypeptide Chains

Abstract Serum from a patient with hepatocellular carcinoma contained an abnormal isoenzyme of lactate dehydrogenase (LDH; EC 1.1.1.27), LDH-1ex, that on electrophoresis on 10-g/L agarose gel migrated anodally to the LDH-1 band. This isoenzyme was partly purified by ultrafiltration and preparative electrophoresis. Gel chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis studies of the resulting LDH-1ex preparation suggested that this isoenzyme is probably a tetramer made up of four single polypeptide chains (monomers), each having a molecular mass of about 32,000 Da. LDH-1ex was heat stable and reacted more readily with 2-hydroxybutyrate than did the slower migrating LDH-4 and LDH-5 isoenzymes. LDH-1ex showed no activity when lactate was omitted from the substrate solution or replaced by ethanol.

Download Full-text

An Abnormal Fibrinogen (Copenhagen) Associated with Severe Thromboembolic Disease, but with Normal Adsorption of Plasminogen

10.1055/s-0039-1684593 ◽

1979 ◽

Author(s):

Sandbjerg M. Hansen ◽

I. Clemmensen

Keyword(s):

Venous Thrombosis ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Thromboembolic Disease ◽

Polyacrylamide Gel ◽

Fibrinopeptide A ◽

Related Material ◽

Thrombotic Disease ◽

Fibrin Formation ◽

Fibrin Monomers

An autosomally inherited qualitative fibrinogen (F) defect is presented. The abnormal F was detected in the plasma of a 54 year old woman with severe arterial thrombotic disease. A decreased rate of fibrin formation of plasma, or purified F by thrombin or ancrod (Arvin (R ) ) was demonstrated. The plasma F concentration was normal, when estimated by clottability or immunologic technique. No F related material was present in the serum. The purified abnormal F was indistinguishable from normal F by Immunoelectrophoresis or SDS Polyacrylamide gel electrophoresis. The major defects detected were an abnormality of polymerization of fibrin monomers and a decreased rate of release of fibrinopeptide A. To evaluate a possible abnormality of the binding of plasminogen (P) to the abnormal fibrin, we examined the adsorption of partially degraded P (Lys-P), which has a higher affinity for fibrin than Glu-P. The adsorption was normal, but the study might be useful in the evaluation of dysfibrinogenemia associated with venous thrombosis.

Download Full-text

Cleavage of bacterial flagellin with cyanogen bromide. Chemical and physical properties of the protein fragments

Biochemical Journal ◽

10.1042/bj1130489 ◽

1969 ◽

Vol 113 (3) ◽

pp. 489-499 ◽

Cited By ~ 30

Author(s):

C. R. Parish ◽

G. L. Ada

Keyword(s):

Amino Acid ◽

Gel Electrophoresis ◽

Amino Acid Analysis ◽

Cyanogen Bromide ◽

Degradation Products ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Gel Chromatography ◽

Molecular Weights ◽

Lysine Residues

1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as ∈-N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the ‘C,D’ complex and a presumed ‘AB’ fragment. 4. The sum of the amino acid analyses of fragments A and B and the ‘C,D’ complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 ∈-N-methyl-lysine residues of the molecule were in fragment A. Reaction with [125I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the ‘C,D’ complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B–A–D–C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.

Download Full-text