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PubMed1996

Specific measurement of hypocarboxylated prothrombin in plasma or serum and application to the diagnosis of hepatocellular carcinoma.

An enzyme-linked immunosorbent assay (ELISA) was developed for measuring human hypocarboxyprothrombin, a protein induced by vitamin K antagonists (PIV KA-II). A specific monoclonal antibody (P1-2-B9) was prepared and used for coating a microELISA plate, and revelation proceeded with a rabbit polyclonal anti-human prothrombin antibody-peroxidase conjugate. This assay allowed the measurement of PIVKA-II at concentrations ranging from 2 to 200 ng/ml, with an intraassay coefficient of variation of less than 5.2% and an interassay coefficient of variation of less than 7.4%. This assay permits the direct evaluation of PIVKA-II in citrated plasma as well as in serum. The concentration of PIVKA-II is expressed in nanograms per milliliter. It offers specific measurement of PIVKA-II without any cross-reactivity from native prothrombin: the specificity for PIVKA-II respective to prothrombin is > 10(5). No reactivity was observed with other vitamin K-dependent proteins, whether fully active or decarboxylated. Complementary studies have demonstrated that the monoclonal antibody used was preferentially directed to 3 to 6 Gla hypocarboxylated prothrombin. PIVKA-II concentration was below 2.4 ng/ml in normal individuals (n = 59). In 61 patients given dicoumarol for more than 90 days (receiving a stable therapy), the measured concentrations of PIVKA-II ranged from 750 to 13,400 ng/ml. Combined with the assay of alpha-fetoprotein (AFP), measurement of PIVKA-II in patients with liver diseases introduces a complementary exploration for hepatocellular carcinoma (HCC). In a study in 59 patients with HCC, the assay sensitivity was 49.1% for PIVKA-II, 47.5% for AFP, and 71% for both markers combined.

Antibodies, Monoclonal↗
PubMed1991

Preferential ligand binding to multi-state acceptor systems: comparisons of the calcium-binding and dimerization characteristics of prothrombin and fragment 1.

Consideration is given to the interactions of a ligand with self-associating acceptor systems for which preferential binding is an ambiguous term in that ligand-mediated self-association does not necessarily imply a greater binding constant for polymeric acceptor--even in instances where binding sites are preserved in the self-association process. This dilemma is shown to arise in situations involving the binding of ligand to monomeric and polymeric forms of an acceptor that also coexist in equilibrium with inactive isomeric states. For example, the ten-fold increase in the measured dimerization constant for prothrombin Fragment 1 in the presence of a saturating concentration of Ca2+ ion may well reflect the existence of a 12% greater binding constant for the interaction of metal ion with dimeric acceptor. However, that result, as well as the detailed form of the sigmoidal binding curve, are also reasonably described by another extreme model in which the monomeric and dimeric forms of the acceptor possess equal affinities for Ca2+ ion. Likewise, the fact that the same experimental dimerization constant applies to prothrombin and its Ca(2+)-saturated complex does not preclude the possibility that the active form of dimeric zymogen exhibits a 12% greater affinity for metal ion. Numerical simulations have established that characterization of the dimerization behaviour as a function of free ligand concentration should allow greater discrimination between such models of the interplay between calcium binding and self-association of prothrombin and Fragment 1. Finally, by illustrating the likelihood that the disparity in self-association behaviour of prothrombin and Fragment 1 merely reflects minor differences in the relative magnitudes of isomerization constants and/or binding constants for monomeric and dimeric states of the two acceptors, the present investigation serves to allay concern about the validity of employing the proteolytic fragment as a model of the intact zymogen.

Calcium↗
PubMed2002

Prothrombin activation during carbon tetrachloride-induced acute liver injury in mice.

Serine protease thrombin is a central factor for blood coagulation and is an integral part of inflammatory reactions. Prothrombin activation of the liver homogenates during carbon tetrachloride-induced liver injury was investigated. At 72 h after administration of carbon tetrachloride, the prothrombin activation activity reached the maximal value, and then decreased to the basal level at 168 h. The alanine transaminase (ALT) activity, an index of tissue injury, was dramatically elevated at 36 h after treatment, and almost recovered to normal levels at 72 h. Histochemical analysis revealed that the proliferation of hepatocytes and remarkable phagocytosis by macrophages was observed at 72 h, in contrast to severe tissue damage at 36 h. Finally, we showed that prothrombinase activity, composed of factor Xa and factor Va, was involved in the injury-associated prothrombin activation. Taken together, these results strongly suggest that prothrombin activation by the prothrombinase complex occurred following tissue destruction in carbon tetrachloride-induced liver injury.

Animals↗
PubMed1997

Partially purified Echis carinatus venom cleaves active-site-mutated bovine prothrombin at two sites.

Partially purified snake venom of Echis carinatus (ECV) is a well-known enzyme used to generate meizothrombin from prothrombin. In this reaction, meizothrombin is slowly converted to two smaller fragments. It is not certain if this slow reaction is catalyzed by meizothrombin or by ECV. Using previously constructed active site mutant bovine prothrombin, we eliminated meizothrombin autolysis and examined ECV reactions. ECV did slowly generate two fragments during activation of the active site mutant prothrombin. This result indicates that ECV cleaved bovine prothrombin at two sites: at Arg323-Ile to expose the active site, at another bond at or near the known meizothrombin autolysis site. Because the former cleavage is faster than the latter one, ECV is still a reasonable tool to generate meizothrombin under proper conditions.

Animals↗
PubMed1999

The prothrombin gene G20210A mutation and the platelet glycoprotein IIIa polymorphism PlA2 in patients with central retinal vein occlusion.

The prothrombin gene G20210A mutation and the platelet glycoprotein IIIa polymorphism PlA2 have been shown to be associated with thromboembolic disease. We wondered if mutations were overrepresented in patients with central retinal vein occlusion. We studied 129 consecutive patients with a history of central retinal vein occlusion. We analysed for the prothrombin gene G20210A mutation and the platelet glycoprotein IIIa polymorphism PlA2 and compared the results to controls with no history of thrombosis. For the platelet glycoprotein IIIa polymorphism PlA2, 69% were normal, 26% were heterozygous, and 5% were homozygous. For the G20210A prothrombin mutation, 97% were normal and 3% were heterozygous. Neither the prothrombin gene G20210A mutation nor the platelet glycoprotein IIIa polymorphism PlA2 seem to be associated with central retinal vein occlusion.

Adult↗
PubMed2002

Effect of acetylcysteine on prothrombin index in paracetamol poisoning without hepatocellular injury.

Acetylcysteine treatment reduces liver damage after paracetamol overdose, but can affect the prothrombin index, which is used to assess the progress of overdose patients. We aimed to assess retrospectively the effect of intravenous acetylcysteine on the prothrombin index in patients with paracetamol poisoning without signs of hepatocellular injury. Prothrombin index had been recorded before, and serially during, acetylcysteine treatment in 87 patients. After initiation of treatment, prothrombin index decreased (mean 0.33, 95% CI 0.29-0.38) in all patients, and was strongly associated with the start of acetylcysteine infusion. In patients with uncomplicated paracetamol poisoning, a fall in this index might be misinterpreted as a sign of liver failure, leading to prolonged treatment time.

Acetaminophen↗
PubMed2002

Increase of prothrombin-mRNA after global cerebral ischemia in rats, with constant expression of protease nexin-1 and protease-activated receptors.

Prothrombin, protease-activated receptors (PARs) and the specific thrombin inhibitor protease nexin-1 (PN-1) are expressed in the brain. Recent studies have shown that the serine protease thrombin, depending on its concentration, plays an important role in neuronal degeneration or protection after cerebral ischemia. However, it is still uncertain whether a change in prothrombin or alterations in the expression of specific PAR-subtypes or PN-1 are associated with postischemic thrombin effects. Using semi-quantitative reverse transcription-polymerase chain reaction analysis, we show that prothrombin was up-regulated in the hippocampal formation 24 h after transient global ischemia in rats (two-vessel occlusion with hypotension), whereas the expression of PN-1 and the expression of PAR-subtypes 1-3 did not change significantly. Thus, control of the balance between the expression of prothrombin and PN-1 may reflect an important mechanism that underlies postischemic thrombin effects.

Amyloid beta-Protein Precursor↗
PubMed2002

gamma-Carboxyglutamic acid content of hepatocellular carcinoma-associated des-gamma-carboxy prothrombin.

Serum des-gamma-carboxy prothrombin (DCP) is a useful marker for the diagnosis of hepatocellular carcinoma (HCC), but the exact mechanism of its synthesis and its structural properties in liver diseases are unknown. DCP is measured by the monoclonal antibody MU-3. The purpose of this study was to examine the epitope of MU-3 and to characterize the differences in DCP between HCC and benign liver diseases. The epitope of MU-3 was examined by ELISA using prothrombin Gla domain polypeptides and was determined to be amino acid residues 17-27 of the prothrombin Gla domain, which has four gamma-carboxyglutamic acid residues (Gla) at positions 19, 20, 25 and 26. Peptides having a glutamic acid residue (Glu) at these positions reacted strongly to MU-3 but lost reactivity when Glu 19 or 20 was changed to Gla. In the order of gamma-carboxylation, MU-3 reacted strongly to DCP containing 0-1 Gla, weakly to 2-4 Gla and not at all to DCP containing more than five Gla. After adsorbing normal prothrombin with barium carbonate, DCP reaction to MU-3 was measured by determining the amount of DCP that was adsorbed by MU-3-coated beads. The proportion of DCP reacting to MU-3 in HCC was 41.0-76.8%, whereas in patients with benign liver diseases, only 0-42.1% reacted to MU-3. These results indicate that DCP variants preferentially synthesized in HCC have less than four Gla, which are restricted to positions 16, 25, 26 and 29, whereas DCP variants in benign liver diseases have more than five Gla.

1-Carboxyglutamic Acid↗
PubMed2002

Factor Va increases the affinity of factor Xa for prothrombin: a binding study using a novel photoactivable thiol-specific fluorescent probe.

The multiprotein complex of factor Xa, factor Va, and prothrombin efficiently generates the blood-clotting agent, thrombin. Here, the formation of the factor Xa*prothrombin complex and the effects of factor Va on this complex were examined using a photoactivable thiol-specific fluorescent probe (LWB), which was synthesized and incorporated into the active site of factor Xa. The use of fluorescent LWB illustrated that factor Xa has an increased affinity for prothrombin in the presence of factor Va. Further exposure of these components to UV light resulted in a specific photocrosslinking of LWB-factor Xa to prothrombin, suggesting a physical association between these proteins. These data demonstrate that LWB can successfully function both as a spectroscopic probe and as a photocrosslinking reagent for studying protein-protein interactions.

Cross-Linking Reagents↗
PubMed1993

Structural changes in the protease domain of prothrombin upon activation as assessed by N-bromosuccinimide modification of tryptophan residues in prethrombin-2 and thrombin.

Increases in intrinsic fluorescence (delta I), reflecting changes in tryptophan environments, occur upon bond cleavages necessary for prothrombin (II) activation to thrombin (IIa) by prothrombinase. Cleavage at Arg274-Thr275 (numbering based on bovine prothrombin sequence, with chymotrypsinogen numbering in braces) between the amino-terminal fragment 1.2 and protease (Pre2) domains of prothrombin yields delta I = 5%, and cleavage within the Pre2 domain at Arg323-Ile324 to form IIa yields delta I = 35%, while cleavage at both yields delta I = 25%. Since the change in fluorescence upon activation of prothrombin can be largely attributed to a change within the Pre2 domain, the susceptibilities of each of the 9 Trp residues of IIa and its immediate precursor Pre2 to oxidation by N-bromosuccinimide (NBS) were compared. Pre2 and IIa were titrated with increasing amounts of NBS (0.5-5 equiv of NBS/TRP), aliquots were removed and fully digested with trypsin, and tryptophan-containing peptides were separated and quantitated by RP-HPLC with fluorescence detection. Tryptic digests yielded 9 tryptophan-containing peptides, which were identified by amino acid composition. Tryptophan residues in IIa and Pre2 displayed a 10-fold range of sensitivity to modification. Tryptophans 337 and 360 (W29, W51) were modified less readily in IIa than in Pre2, while residues 373, 542, and 550 (W60D, W207, W215) were modified more readily, and other residues were equally susceptible. Residues 360 and 373 (W29, W60D) flank the active site histidine. From the crystal structure, residues 373 and 550 (W60D, W215) are implicated in substrate binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Structures of the noncovalent complexes of human and bovine prothrombin fragment 2 with human PPACK-thrombin.

Both human and bovine prothrombin fragment 2 (the second kringle) have been cocrystallized separately with human PPACK (D-Phe-Pro-Arg)-thrombin, and the structures of these noncovalent complexes have been determined and refined (R = 0.155 and 0.157, respectively) at 3.3-A resolution using X-ray crystallographic methods. The kringles interact with thrombin at a site that has previously been proposed to be the heparin binding region. The latter is a highly electropositive surface near the C-terminal helix of thrombin abundant in arginine and lysine residues. These form salt bridges with acidic side chains of kringle 2. Somewhat unexpectedly, the negative groups of the kringle correspond to an enlarged anionic center of the lysine binding site of lysine binding kringles such as plasminogens K1 and K4 and TPA K2. The anionic motif is DGDEE in prothrombin kringle 2. The corresponding cationic center of the lysine binding site region has an unfavorable Arg70Asp substitution, but Lys35 is conserved. However, the folding of fragment 2 is different from that of prothrombin kringle 1 and other kringles: the second outer loop possesses a distorted two-turn helix, and the hairpin beta-turn of the second inner loop pivots at Val64 and Asp70 by 60 degrees. Lys35 is located on a turn of the helix, which causes it to project into solvent space in the fragment 2-thrombin complex, thereby devastating any vestige of the cationic center of the lysine binding site. Since fragment 2 has not been reported to bind lysine, it most likely has a different inherent folding conformation for the second outer loop, as has also been observed to be the case with TPA K2 and the urokinase kringle. The movement of the Val64-Asp70 beta-turn is most likely a conformational change accompanying complexation, which reveals a new heretofore unsuspected flexibility in kringles. The fragment 2-thrombin complex is only the second cassette module-catalytic domain structure to be determined for a multidomain blood protein and only the third domain-domain interaction to be described among such proteins, the others being factor Xa without a Gla domain and Ca2+ prothrombin fragment 1 with a Gla domain and a kringle.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Enhanced gamma-carboxylation of recombinant factor X using a chimeric construct containing the prothrombin propeptide.

Factor Xa is the serine protease component of prothrombinase, the enzymatic complex responsible for thrombin generation. Production of recombinant factor X/Xa has proven to be difficult because of inefficient gamma-carboxylation, a critical post-translational modification. The affinities of the vitamin K-dependent propeptides for the gamma-carboxylase vary over 2 logs, with the propeptide of factor X having the highest affinity followed by the propeptides of factor VII, protein S, factor IX, protein C, and prothrombin [Stanley, T. B. (1999) J. Biol. Chem. 274, 16940-16944]. On the basis of this observation, it was hypothesized that exchanging the propeptide of factor X with one that binds the gamma-carboxylase with a reduced affinity would enhance gamma-carboxylation by allowing greater substrate turnover. A chimeric cDNA consisting of the human prothrombin signal sequence and propeptide followed by mature human factor X was generated and stably transfected into HEK 293 cells, and modified factor X was purified from conditioned medium. The results indicate that on average 85% of the total factor X produced with the prothrombin propeptide was fully gamma-carboxylated, representing a substantial improvement over a system that employs the native factor X propeptide, with which on average only 32% of the protein is fully gamma-carboxylated. These results indicate that the affinity of the gamma-carboxylase for the propeptide greatly influences the extent of gamma-carboxylation. It was also observed that regardless of which propeptide sequence is directing gamma-carboxylation (factor X or prothrombin), two pools of factor X are secreted; one is uncarboxylated and a second is fully gamma-carboxylated, supporting the notion that the gamma-carboxylase is a processive enzyme.

1-Carboxyglutamic Acid↗
PubMed1983

Characterization of the complementary deoxyribonucleic acid and gene coding for human prothrombin.

The DNA sequences of a complementary deoxyribonucleic acid (cDNA) and a portion of the gene coding for human prothrombin have been determined. The cDNA was 2005 base pairs in length and was found to code for part of a leader sequence of 36 amino acids, 579 amino acids present in the mature protein, a stop codon, a noncoding region of 97 base pairs, and a poly(A) tail of 27 base pairs. It is proposed that the leader sequence consists of a signal sequence and a pro sequence for the mature protein that circulates in plasma. The 10 glutamic acid residues that are present in the amino-terminal region of prothrombin and are converted to gamma-carboxyglutamic acid in the mature protein are coded by only the GAG codon. The cDNA for prothrombin was also employed as a probe for screening a human fetal liver genomic DNA library. One of the strongly positive phage containing a human DNA insert of 5 kilobases was mapped with restriction endonucleases and sequenced. This DNA contained approximately half of the gene for human prothrombin and included six introns and five exons coding for amino acid residues 144-448. The two largest intervening sequences in the genomic DNA contained two copies each of AluI repetitive DNA.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence of the gene for human prothrombin.

A human genomic DNA library was screened for the gene coding for human prothrombin with a cDNA coding for the human protein. Eighty-one positive lambda phage were identified, and three were chosen for further characterization. These three phage hybridized with 5' and/or 3' probes prepared from the prothrombin cDNA. The complete DNA sequence of 21 kilobases of the human prothrombin gene was determined and included a 4.9-kilobase region that was previously sequenced. The gene for human prothrombin contains 14 exons separated by 13 intervening sequences. The exons range in size from 25 to 315 base pairs, while the introns range from 84 to 9447 base pairs. Ninety percent of the gene is composed of intervening sequence. All the intron splice junctions are consistent with sequences found in other eukaryotic genes, except for the presence of GC rather than GT on the 5' end of intervening sequence L. Thirty copies of Alu repetitive DNA and two copies of partial KpnI repeats were identified in clusters within several of the intervening sequences, and these repeats represent 40% of the DNA sequence of the gene. The size, distribution, and sequence homology of the introns within the gene were then compared to those of the genes for the other vitamin K dependent proteins and several other serine proteases.

Amino Acid Sequence↗
PubMed1977

A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S.

Human prothrombin, factor IX, and factor X have been idolated in high yield and characterized as the their amino-terminal sequence, molecular weight, amino acid composition, and migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional human plasma protein, called protein S, has also been purified and its properties have been compared with those of prothrombin, factor IX, and factor X. Prothrombin (mol wt 72 000), factor IX (mol wt 57 000), and protein S (mol wt 69 000) are single-chain glycoproteins, while factor X (mol wt 59 000) is a glycoprotein composed of two polypeptide chains held together by a disulfide bond(s). The amino-terminal sequence of the light chain of human factor X is homologous with prothrombin, factor IX, and protein S. The heavy chain of human factor X is slightly larger than the heavy chain of bovine factor X and differs from bovine factor X in its amino-terminal sequence.

Amino Acid Sequence↗
PubMed1975

Structural studies on bovine prothrombin. Isolation and partial characterization of the Ca2+ binding and carbohydrate containing peptides of the N-terminus region.

Three peptides, one of which binds Ca2-+ (calcium binding fragment, CBF) but contains no carbohydrates and two of which bind no Ca2-+ but contain carbohydrates, have been isolated from the N-terminus region of bovine prothrombin. The preparation of these peptides involved (a) thrombin cleavage of prothrombin to intermediate 1 (thrombinogenic) and fragment 1 (nonthrombinogenic), (b) tryptic attack on fragment 1, and (c) separation of the CBF from the latter reaction by addition of a phosphatidylcholine-phosphatidylserine dispersion in the presence of Ca-2+. Further study on the non-calcium-binding peptides from the tryptic digest of fragment 1 revealed the presence of two low molecular weight glycopeptides, GP-1 and GP-2. A detailed examination of the chemical characteristics of CBF provided some insight into this unusual peptide. Whereas fragment 1, as well as prothrombin, exhibited two classes of Ca-2+ binding sites (one of high affinity, 3-4 mol/mol of peptide and the other of low affinity, 10-12 mol/mol of peptide), CBF bound only 3-4 mol of Ca-2+/mol of peptide. This indicated the presence of only the high affinity sites of the parent molecule. CBF contained an unusually high level of glutamic acid (approximately 30% of the total amino acids as determined in an acid hydrolysate) and had an N-terminal glycine. Most likely these glutamyl residues were present originally as the gamma-carboxyglutamyl residue as proposed by Stenflo et al. (Stenflo, J., Ferlung, P., Egan, W., and Roepstorff, P. (1974), Proc. Natl. Acad. Sci. U.S-A 71, 2730). The CBF contained no detectable carbohydrate. Its molecular weight varied inexplicably according to the procedure used and gave the following values; 8500, by gel filtration; 5200, by 6 M guanidine-HCl gel chromatography; 3490, by analytical ultracentrifugation. The glycopeptides, GP-1 and GP-2, were distinguished from each other by differences in their behavior on ion exchange chromatography and in their amino acid composition, and from CBF by their inability to bind calcium under any conditions. On the other hand, GP-1 and GP-2 had nearly identical levels of carbohydrate, 45.1 and 48.0 wt %, and possessed essentially the same percent distribution of carbohydrates: sialic acid, 16.5 plus or minus 0.5; mannose, 10.3 plus or minus 0.4; glucosamine, 11.2 plus or minus 0.1; galactose, 7.9 plus or minus 0.3. Their molecular weights were as follows: GP-1, 70000, by gel filtration; 6500, by 6 M guanidine-HCl gel chromatography; 4600, by ultracentrifugation; GP-2, 6500 by gel filtration; 6900, by 6 M guanidine-HCl gel chromatography; 1960, by analytical ultracentifugation. Though there are some obvious variations depending on method, this could be attributable to a probable error in v measurement on these carbohydrate containing peptides. The significance of these findings as they relate to prothrombin to thrombin conversion is discussed.

Amino Acid Sequence↗
PubMed1998

Autocatalytic peptide bond cleavages in prothrombin and meizothrombin.

During factor Xa-catalyzed prothrombin activation, several other reaction products accumulate as a result of proteolysis of prothrombin and its activation products by thrombin and meizothrombin. Gel electrophoretic analysis and N-terminal sequencing of reaction products showed that in the absence of Ca2+ ions thrombin cleaved the following peptide bonds: Arg51-Thr52/Arg54-Asp55 in the fragment 1 (F1) domain (k = 0.4 x 10(4) M-1 s-1), Arg155-Ser156 in prothrombin (k = 2 x 10(4) M-1 s-1), and Arg284-Thr285 in prethrombin 1 (k = 0.02 x 10(4) M-1 s-1). In the presence of 2.5 mM CaCl2, cleavage in fragment 1 (Arg51-Thr52/Arg54-Asp55) was not detectable, whereas cleavage at Arg155-Ser156 (i.e., removal of F1) was inhibited 25-fold. Cleavage at Arg284-Thr285 (formation of prethrombin 2 des-1-13) was not affected by the presence of Ca2+ ions. Meizothrombin rapidly converted itself into meizothrombin des-F1. The half-life (t1/2 = approximately 30 s) of this reaction was independent of the meizothrombin concentration (0.1-1 microM meizothrombin), which is indicative for intramolecular autocatalysis (k = 0.02 s-1 in the presence of 2.5 mM Ca2+ ions). Since the rapid removal of fragment 1 precludes investigations of the cleavage at Arg284-Thr285 in intact meizothrombin, we analyzed the cleavage of this peptide bond in R155A-meizothrombin, a recombinant product that is resistant to autocatalytic removal of the fragment 1 domain. In the absence of phospholipids, R155A-meizothrombin converted itself into thrombin des-1-13 by a combination of intramolecular (k = 0.8 x 10(-4) s-1) and intermolecular autocatalysis (k = 0.2 x 10(3) M-1 s-1). Intramolecular autocatalytic conversion of R155A-meizothrombin into thrombin was not affected by the presence of phospholipids (k = 0.8 x 10(-4) s-1), whereas intermolecular autocatalysis was accelerated 25-fold (k = 5.6 x 10(3) M-1 s-1) by phospholipid vesicles. Since factor Xa/Va-catalyzed conversion of meizothrombin into thrombin occurs with k = 5.5 x 10(8) M-1 s-1, we conclude that in reaction systems containing purified proteins autocatalysis of meizothrombin hardly contributes to thrombin formation during factor Xa-catalyzed prothrombin activation.

Animals↗
PubMed2003

Probing the kinetic landscape of transient peptide-protein interactions by use of peptide (15)n NMR relaxation dispersion spectroscopy: binding of an antithrombin peptide to human prothrombin.

Protein-ligand interactions may lead to the formation of multiple molecular complexes in dynamic exchange, affecting the kinetic and thermodynamic characteristics of the binding equilibrium. We followed the dissociation kinetics of the transient and specific complex of an antithrombotic peptide N-acetyl-Asp(55)-Phe-Glu-Glu-Ile-Pro(60)-Glu-Glu-Tyr-Leu-Gln(65) with human prothrombin by use of (15)N NMR relaxation dispersion spectroscopy of the peptide. Every one of the five (15)N-labeled adjacent residues of the peptide exhibited apparently different kinetic exchange and relaxation behaviors, which were especially evident at different concentrations of prothrombin. Binding-induced (15)N relaxation dispersion of residues Phe(56), Glu(57), Glu(58), and Ile(59) can be fitted phenomenologically to a two-site on-and-off exchange mechanism with physically feasible relaxation and kinetic parameters obtained for residues Phe(56), Glu(58), and Ile(59), independent of the prothrombin concentration. The apparent kinetic parameters of Glu(57) show some dependence on the concentration of prothrombin and the extracted transverse relaxation rate for Glu(57) in the bound state was severalfold higher than that expected for a protein-peptide complex with a size of approximately 72 kDa. In addition, the equilibrium population of the bound peptide obtained for Glu(57) was inconsistent with those for Phe(56), Glu(58), and Ile(59) and with the prothrombin/peptide ratios used in the experiments. These discrepancies can be explained by the presence of two conformations for the peptide-protein complex exchanging at a rate of approximately 100 s(-)(1). In all, our study shows that fast dissociation of protein-peptide complexes can be studied quantitatively using peptide (15)N NMR relaxation dispersion measurements without a precise knowledge of the peptide and protein concentrations. In addition, protein titration was found to improve the accuracy of quantitative analysis and may make it possible to determine the rate of conformational changes within the protein-peptide complex.

Antithrombins↗