Human fibrinopeptides isolation, characterization and structure

Blombäck, Birger; Blombäck, Margareta; Edman, Pehr; Hessel, Birgit

doi:10.1016/0304-4165(66)90437-5

Cited by 429 publications

(143 citation statements)

References 57 publications

Supporting

Mentioning

133

Contrasting

Unclassified

Order By: Relevance

“…FPB accounted for approximately one-half of the total mass. The remaining peptides consisted of the three FPA fragments: FPA -70%, FPA-P -20%, desAla FPA -10% (21). Previous studies with fibrinogen have demonstrated that phosphorylation of the Aa chain serine, a post-translational modification occurring prior to secretion from the liver (22)(23)(24), and removal of the amino-terminal alanine from the Aa chain are regulated with respect to subject parameters, such as age, trauma, or illness (24)(25)(26)(27), and are in place on fibrinogen prior to thrombin action (22)(23)(24).…”

Section: Fpa Peptides In Serummentioning

confidence: 99%

“…Importantly, this size-discriminated protein fraction is depleted of fibrinogen, and therefore cannot clot upon a thrombin addition, thus facilitating specific examination of any proteolytic activity of a thrombin beyond its well-characterized and supposedly ''highly specific'' role in clot formation (2,5,6,21,28). Figure 2 clearly demonstrates that a thrombin activity results in significant plasma protein digestion, in fact on the same scale as trypsin; the latter being traditionally used for intentionally caused and extensive protein degradation typical of ''bottom-up'' proteomics experiments (29).…”

Section: Variability Of Serum Protein and Peptide Contentmentioning

confidence: 99%

See 1 more Smart Citation

Thrombin induces broad spectrum proteolysis in human serum samples

O'Mullan¹,

Craft²,

Yi³

et al. 2009

Clinical Chemistry and Laboratory Medicine

View full text Add to dashboard Cite

Background: During clotting, a thrombin cleaves fibrinogen releasing fibrinopeptide A (FPA). FPA is easily identified in serum using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Using MALDI-TOF MS, we observed multiple, progressively shorter fragments of serum FPA. Following ambient incubation of serum, variations in the content of FPA fragments occur over time. Denaturation of a thrombin by heating the serum sample appears to minimize this variation. These observations suggest that intrinsic proteolytic and peptidolytic activity is elevated in serum and perhaps originates from the coagulation cascade enzymes themselves, especially a thrombin. Methods: Extrinsic addition of a thrombin to a subset (3-30 kDa) of plasma proteins was carried out to induce proteolysis and to examine the resultant peptides to reveal a thrombin susceptible parent proteins. One of these identified proteins, hemopexin, was directly digested by a thrombin and the peptides examined to confirm the observations from the initial plasma protein digestion. Results: Extrinsic addition of a thrombin to a subset (3-30 kDa) of plasma proteins results in wide-spread digestion of proteins unrelated to coagulation, revealing a substrate range encompassing more than fibrinogen. Direct digestion of one of these proteins, hemopexin, by a thrombin confirms these observations. Conclusions: The resulting peptides indicate broad tolerance beyond the consensus R-G cleavage site of fibrinogen; in fact, there appears to be no bias for the amino acid following the R/K residue. These data support our hypothesis that the enzymatic activities inherent to coagulation, or at least to thrombin, contribute to destabilization of the protein and peptide content of serum. Clin Chem Lab Med 2009;47:685-93.

show abstract

Section: Fpa Peptides In Serummentioning

confidence: 99%

Section: Variability Of Serum Protein and Peptide Contentmentioning

confidence: 99%

Thrombin induces broad spectrum proteolysis in human serum samples

O'Mullan¹,

Craft²,

Yi³

et al. 2009

Clinical Chemistry and Laboratory Medicine

View full text Add to dashboard Cite

show abstract

“…Edman degradations of the A peptide were performed as described by Blomblck et al [20]. Isoleucine and leucine were identified after hydrolysis of the PTH derivatives with 0.1 M NaOH [21].…”

Section: Determination Of N-terminal Amino Acidsmentioning

confidence: 99%

Thioredoxin 2: Cleavage with Cyanogen Bromide

Holmgren

Reichard

1967

European Journal of Biochemistry

141

View full text Add to dashboard Cite

A simple and highly reproducible method for the preparation of thioredoxin from Escherichia coli B was worked out.On treatment with cyanogen bromide thioredoxin was cleaved into two peptides which were separated by chromatography on Sephadex 6-50. The smaller peptide (peptide B) contained 37 amino acids and represented the N-terminal fragment of thioredoxin, while the larger peptide (peptide A) contained the remaining 71 or 72 C-terminal amino acids. The cystine and tryptophan residues of thioredoxin were found in peptide B.Both peptides were characterized with respect to amino acid composition and were studied by spectrofluorimetry. The increase in quantum yield of the tryptophan emission resulting from the reduction of the disulfide bridge of peptide B was considerably smaller than the corresponding increase resulting from the reduction of the disulfide bridge of thioredoxin [l].Peptidc B was not reduced by TPNH and thioredoxin reductase and showed no activity in the reduction of cytidine diphosphate wit,h ribonucleoside diphosphate reductase from Escherichia coli.Thioredoxin was discovered during an investigation on the enzymatic reduction of ribonucleotides in Escherichia coli [Z]. It is a heat-stable, small acidic protein with a molecular weight of about 12,000. Thioredoxin was found to consist of a single polypeptide chain with about 106 amino acids. The molecule contains one residue each of the amino acids met,hioninc and cystine [ Z ] .Thioredoxin can occur in the oxidized or the reduced state. The reduced form (= thioredoxin -(SH),) contains two cystein residues and is formed from the oxidized form (= thioredoxin -S2)

show abstract

“…The finding that the peptides 21-29 and 22-29, obtained from cyanogen bromide cleavage of the Sprotein, were isolated in a ratio of 1:1 (38) or 1:1.5 (58) were taken to indicate that the initial attack of subtilisin BPN' occured at the Ser2~-Ser22 bond. The further degradation of the isolated S-peptide (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) by subtilisin BPN' (149) involved hydrolysis of the Ala,9-Alaz0 bond, indicating that the enzyme possesses carboxypeptidase actitivy. However, it should be pointed out that the free amino acids liberated in the experiments mentioned above have not been isolated, and that contamination of the subtilisin BPN' preparations by carboxypeptidase has not been ruled out.…”

Section: Natural Peptides and Proteinsmentioning

confidence: 99%

“…The only bonds cleaved are four Arg-Gly bonds located in the a(A) and fl(B) chains of the hexamer [a(A) 13(B) ~']2 (14,17). The hydrolysis of the two a(A) Arg-Gly bonds leads to blood clotting while the rate of hydrolysis of the two fl(B) Arg-Gly bonds first becomes maximal when the formation of fibrin is complete (18).…”

Section: Thromhinmentioning

confidence: 99%

Chemical modifications of the subtilisins with special reference to the binding of large substrates. A review

Svendsen

1976

Carlsberg Res. Commun.

View full text Add to dashboard Cite

Subtilisin type Carlsberg and subtilisin BPN' are two well characterized extracellular proteases from Bacillus subtilis and Bacillus amyloquefaciens, respectively. The present review summarizes the various chemical modification studies which have been performed with these enzymes. Of special interest has been those modifications which lead to changes in the enzymatic properties with regard to the hydrolysis of polypeptide substrates but not small synthetic ester substrates. In this way it is possible to obtain information about how large an area of the surface of the enzyme is involved in the binding of natural substrates (e.g. clupein, gelatine, casein). Nitration, iodination, glutarylation or succinylation of the subtilisins leads to increases in the hydrolyses of clupein and gelatine, while modification of carboxyl groups leads to a decrease. In all these cases the hydrolysis of small ester substrates is almost unaffected. A section of the review deals with the comparison between the results obtained by chemical modification studies and the two other methods available for the study of "secondary binding sites": X-ray diffraction analyses and kinetic studies with synthetic polypeptides. The productive binding mode for polypeptides proposed by KRAUT and coworkers is in agreement with the results obtained by nitration of the subtilisins. However, results from our laboratory and the literature point towards more than one mode of productive binding. These results are discussed. Finally a comparison is made between the secondary binding sites in subtilisin and other proteolytic enzymes, especially the serine proteases. The importance of secondary interactions between enzyme and substrate is discussed.

show abstract

Human fibrinopeptides isolation, characterization and structure

Cited by 429 publications

References 57 publications

Thrombin induces broad spectrum proteolysis in human serum samples

Thrombin induces broad spectrum proteolysis in human serum samples

Thioredoxin 2: Cleavage with Cyanogen Bromide

Chemical modifications of the subtilisins with special reference to the binding of large substrates. A review

Contact Info

Product

Resources

About