scholarly journals Anticoagulant function of a 24-Kd fragment isolated from human fibrinogen A alpha chains

Blood ◽  
1993 ◽  
Vol 81 (12) ◽  
pp. 3277-3284
Author(s):  
HK Lau
Keyword(s):  
Amino Acid ◽  
Maximum Level ◽  
Polymerization Reaction ◽  
Amino Acid Residues ◽  
Clotting Time ◽  
Human Fibrinogen ◽  
Apparent Homogeneity ◽  
Lag Period ◽  
Anticoagulant Function ◽  
Fibrin Monomers

A fibrinogen fragment obtained by limited-plasmin proteolysis has been isolated and purified to apparent homogeneity by gel filtrations. This fragment, denoted as 24-Kd fragment, has an apparent M(r) approximately 24,000 and contains an N-terminal sequence of met-glu-leu-glu-arg-pro- gly-gly-asn-glu-ile. The fragment contains a large number of acidic amino acid residues, and its amino acid composition is similar to several fibrinogen A alpha chains degradation fragments isolated previously. It corresponds to a peptide of the fibrinogen A alpha chains, the N-terminal of which starts at alpha Met-240. This peptide delays thrombin plasma clotting time. It does not bind calcium ions and does not inhibit thrombin's amidolytic activity. It binds to immobilized fibrin but not fibrinogen. It also inhibits the polymerization of desAA and desAABB fibrin monomers by simultaneously decreasing the maximum rate and the maximum level of the polymerization reaction. However, the initial lag period of this reaction is not affected by the fragment.

scholarly journals Anticoagulant function of a 24-Kd fragment isolated from human fibrinogen A alpha chains

Blood ◽  
1993 ◽  
Vol 81 (12) ◽  
pp. 3277-3284 ◽  
Author(s):  
HK Lau
Keyword(s):  
Amino Acid ◽  
Maximum Level ◽  
Polymerization Reaction ◽  
Amino Acid Residues ◽  
Clotting Time ◽  
Human Fibrinogen ◽  
Apparent Homogeneity ◽  
Lag Period ◽  
Anticoagulant Function ◽  
Fibrin Monomers

Abstract A fibrinogen fragment obtained by limited-plasmin proteolysis has been isolated and purified to apparent homogeneity by gel filtrations. This fragment, denoted as 24-Kd fragment, has an apparent M(r) approximately 24,000 and contains an N-terminal sequence of met-glu-leu-glu-arg-pro- gly-gly-asn-glu-ile. The fragment contains a large number of acidic amino acid residues, and its amino acid composition is similar to several fibrinogen A alpha chains degradation fragments isolated previously. It corresponds to a peptide of the fibrinogen A alpha chains, the N-terminal of which starts at alpha Met-240. This peptide delays thrombin plasma clotting time. It does not bind calcium ions and does not inhibit thrombin's amidolytic activity. It binds to immobilized fibrin but not fibrinogen. It also inhibits the polymerization of desAA and desAABB fibrin monomers by simultaneously decreasing the maximum rate and the maximum level of the polymerization reaction. However, the initial lag period of this reaction is not affected by the fragment.

scholarly journals Amino acid composition of human fibrinogen and anticoagulant derivatives

1967 ◽  
Vol 105 (1) ◽  
pp. 393-400 ◽  
Author(s):  
E. Triantaphyllopoulos ◽  
D. C. Triantaphyllopoulos
Keyword(s):  
Amino Acid ◽  
Partial Specific Volume ◽  
Amino Acid Residues ◽  
Human Fibrinogen ◽  
Molecular Weights ◽  
Small Peak ◽  
Ammonium Sulphate Fractionation ◽  
Large Peak ◽  
Ionizable Groups ◽  
Protein Moiety

1. The amino acid compositions of human fibrinogen and three intermediate anticoagulant derivatives were determined by column chromatography. The derivatives were isolated by ammonium sulphate fractionation and column electrophoresis from solutions of fibrinogen undergoing spontaneous breakdown. One derivative, isolated as the large electrophoretic peak at the end of the clottable period (100% CP) of the parent fibrinogen solution, was labelled LP100 and others obtained at twice this period (200% CP) were designated as LP200 and SP200 (LP, large peak; SP, small peak). 2. Maximal ‘molecular’ weights of approx. 294000 for LP100, 137000 for LP200 and 37000 for SP200 were calculated for the protein moieties. At least 265 amino acid residues must have been lost from each fibrinogen molecule during the formation of LP100, and 1362 during the formation of the other two derivatives. 3. Only one derivative (LP200) had a partial specific volume ([unk] 0·725ml./g.) different from that of fibrinogen ([unk] 0·721ml./g.). 4. No significant differences in refractive index at 589mμ were detected. 5. Calculation of the total number of ionizable groups/105g. of each protein moiety showed a preponderance of the following numbers of negative charges: 22 in fibrinogen; 24 in LP100; 26 in LP200; 49 in SP200. The isoionic points were estimated to be approx.+0·03pH unit (for fibrinogen), −0·06pH unit for (LP100) and +0·28pH unit (for LP200) from the pK of imidazole, and 0·78pH unit above the average pK of aspartyl and glutamyl ions (for SP200). These figures agree closely with experimentally determined values of the isoelectric point of fibrinogen and its derivatives.

scholarly journals On the Primary Structure of Human Fibrinogen

1977 ◽  
Author(s):  
A. Henschen ◽  
F. Lottspeich ◽  
E. Töpfer-Petersen ◽  
R. Warbinek
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Primary Structure ◽  
Cyanogen Bromide ◽  
Attachment Site ◽  
Complete Sequence ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Human Fibrinogen ◽  
Β Chain

The aim of the present investigation is to elucidate the primary structure of human fibrinogen. Through the work of several laboratories including our own large parts of the structure are now known. Here will be reported the complete amino acid sequence of the γ-chain (409 residues). Furthermore, the carbohydrate linkage site in the β-chain and plasmin cleavage sites in the β- and γ-chains have been identified.The peptide chains were isolated by CM-cellulose chromatography following mercaptolysis and alkylation. The γ-chain was cleaved in a way to produce large fragments suitable for automatic sequencing, e. g. with cyanogen bromide or trypsin after citraconylation. The sequences of the isolated fragments allowed reconstruction of the complete sequence of the γ-chain.The carbohydrate linkage site in the β-chain could be isolated by affinity chromatography on concanavalin A-agarose following cleavage of the chain by trypsin or cyanogen bromide. The sequence of 21 amino acid residues around the carbohydrate attachment site was determined.The plasmin cleavage site giving rise to N-terminal glycine in the γ-chain D-fragment was identified. The amino acid sequence linking plasmic fragments E and D in the β-chain was determined.

scholarly journals Some Characteristics of Fibrin Monomer Preparations Made from Dissolved Fibrin Clots or from Fibrinogen and Immobilized Thrombin in Urea

1977 ◽  
Author(s):  
F. Brosstad ◽  
H. C. Godal ◽  
P. Kierulf
Keyword(s):  
Amino Acid ◽  
Liquid Nitrogen ◽  
Gel Chromatography ◽  
Complete Conversion ◽  
Terminal Amino Acid ◽  
Human Fibrinogen ◽  
Fibrin Monomer ◽  
Terminal Amino ◽  
Sds Electrophoresis ◽  
Fibrin Monomers

Fibrin monomer was prepared from purified human fibrinogen by the action of insolubilized thrombin in the presence of 3.3 M urea. As judged by N-terminal amino acid analyses, complete conversion of fibrinogen to fibrin was achieved. The preparation proved homogeneous by gel chromatography and SDS electrophoresis and had retained clotting abilities identical to those of fibrin monomers arising from fibrinogen exposed to large amounts of thrombin. No changes were observed after freezing in liquid nitrogen or after storage at +4°C for 4 weeks. As to homogeneity and ability to polymerize the preparation proved superior to those fibrin monomer preparations obtained by dissolution of polymerized fibrin in various solvents.

Radioiodinated Human Fibrinogen. In Vitro Effects of Increasing Iodination

1975 ◽  
Author(s):  
B. E. Ly ◽  
P. Kierulf
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Clotting Time ◽  
Terminal Amino Acid ◽  
Human Fibrinogen ◽  
Terminal Amino ◽  
In Vitro Effects ◽  
Polypeptide Chains

Fibrinogen preparations with increasing contents of iodine, ranging from 0.2 to 20 atoms of iodine per molecule fibrinogen, were obtained with the ICl method. Aggregation and shortening of the thrombin clotting time occurred when the content of iodine exceeded 3 atoms per molecule.Upon the action of thrombin, the increase in N-terminal glycine, reflecting fibrin formation, was almost identical in native and iodinated fibrinogen. At visible gelation, however, decreased amounts of N-terminal glycine were found in heavily iodinated fibrinogen, thus indicating enhanced fibrin polymerization. N-terminal analysis of heavily iodinated fibrinogen demonstrated a deficiency in N-terminal tyrosine concomitantly with the apparance of a new N-terminal amino acid, identified as mono-iodo-tyrosine.Polyacrylamide gel electrophoresis at pH 8.9 revealed an increase in mobility following extensive iodination, but no shift in the isoelectric point was observed upon isofocusing.Neither clottability nor the behaviour of fibrinogen and its subunit polypeptide chains on SDS-gel electrophoresis was affected by iodination.

scholarly journals Cloning, Expression, Purification, and Characterization of Glutaredoxin from Antarctic Sea-Ice BacteriumPseudoalteromonassp. AN178

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Quanfu Wang ◽  
Yanhua Hou ◽  
Yonglei Shi ◽  
Xiao Han ◽  
Qian Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sea Ice ◽  
Amino Acid Residues ◽  
E Coli ◽  
Antarctic Sea Ice ◽  
Apparent Homogeneity ◽  
Cold Active ◽  
Catalytic Motif ◽  
Protein Disulfide

Glutaredoxins (Grxs) are small ubiquitous redox enzymes that catalyze glutathione-dependent reactions to reduce protein disulfide. In this study, a full-length Grx gene (PsGrx) with 270 nucleotides was isolated from Antarctic sea-ice bacteriumPseudoalteromonassp. AN178. It encoded deduced 89 amino acid residues with the molecular weight 9.8 kDa. Sequence analysis of the amino acid sequence revealed the catalytic motif CPYC. RecombinantPsGrx (rPsGrx) stably expressed inE. coliBL21 was purified to apparent homogeneity by Ni-affinity chromatography. rPsGrx exhibited optimal activity at 30°C and pH 8.0 and showed 25.5% of the activity at 0°C. It retained 65.0% of activity after incubation at 40°C for 20 min and still exhibited 37.0% activity in 1.0 M NaCl. These results indicated that rPsGrx was a typical cold active protein with low thermostability.

Evidence for the Existence of Foetal Fibrinogen

1969 ◽  
Vol 22 (01) ◽  
pp. 101-109 ◽  
Author(s):  
I Witt ◽  
H Müller ◽  
W Künzer
Keyword(s):  
Molecular Structure ◽  
Amino Acid ◽  
Umbilical Cord Blood ◽  
Primary Structure ◽  
Gel Electrophoresis ◽  
Deae Cellulose ◽  
Clotting Time ◽  
Chemical Modifications ◽  
Human Fibrinogen ◽  
Ph Dependency

SummaryHuman fibrinogen was isolated from the blood of adults and from umbilical cord blood. The fibrinogen preparations obtained were clottable with thrombin to between 97 and 98% and homogeneous in acrylamide gel electrophoresis. Comparative analyses of these highly purified foetal and adult fibrinogen showed that they have the same quantitative amino acid composition and the same mobility in acrylamide gel electrophoresis at pH 9.2.Chromatography on DEAE-cellulose made it likely that foetal and adult fibrinogen have a slightly different electrical charge. The pH-dependency of the thrombin clotting time is strikingly different. The fingerprints of the tryptic peptides showed at least 3 peptides with different mobility in chromatography.From these data it is assumed that foetal and adult fibrinogen have a different molecular structure. Hitherto it cannot be decided if the two proteins have a different primary structure, which is most probable, or if they are chemical modifications.

scholarly journals Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing

1983 ◽  
Vol 209 (2) ◽  
pp. 291-297 ◽  
Author(s):  
C S Jones ◽  
P Shankaran ◽  
D J Davidson ◽  
A Poulos ◽  
J W Callahan
Keyword(s):  
Amino Acid ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Isoelectric Focusing ◽  
Protein Profile ◽  
Minor Component ◽  
Intact Protein ◽  
Amino Acid Residues ◽  
Apparent Homogeneity ◽  
A Minor

Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100.

scholarly journals Studies on mammalian intestinal peroxidase

1982 ◽  
Vol 205 (2) ◽  
pp. 271-279 ◽  
Author(s):  
S Kimura ◽  
P H Jellinck
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Spectral Properties ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rat Small Intestine ◽  
Amino Acid Residues ◽  
Apparent Homogeneity ◽  
Order Of Magnitude ◽  
Benzhydroxamic Acid ◽  
Intestinal Enzyme

A peroxidase, purified from rat small intestine to apparent homogeneity as judged by polyacrylamide-gel electrophoresis, exhibited an absorbance ratio (A412/A280) of 0.783. Its Mr (44000 +/- 1000) and spectral properties were similar to those of the pig intestinal enzyme. The velocity constant for the reaction between rat intestinal peroxidase and hydrogen peroxide was found to be 1.8×10(7) M-1 . s-1. Benzhydroxamic acid inhibited the peroxidative oxidation of guaiacol by intestinal peroxidase from both species but the concentration required to cause half-inhibition of the enzyme from the rat was higher by one order of magnitude than for the pig enzyme. The amino acid composition of highly-purified pig intestinal peroxidase showed a relative abundance of basic amino acids (lysine and arginine) and was similar to that of lactoperoxidase, but not that of myeloperoxidase. The initial ten amino acid residues of this enzyme (the first reported partial sequence for a mammalian peroxidase) were also determined.

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