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PubMed2000

Management of patients with suspected deep vein thrombosis in the emergency department: combining use of a clinical diagnosis model with D-dimer testing.

The management of patients presenting to hospital Emergency Departments with suspected deep vein thrombosis is problematic since urgent diagnostic imaging is at times unavailable. We evaluated the accuracy of a rapidly available D-dimer test and the potential of combining D-dimer testing with an explicit clinical model to improve the management of patients with suspected deep vein thrombosis. Two hundred and fourteen patients with suspected deep vein thrombosis presenting to the Emergency Departments of two tertiary care institutions were enrolled in this prospective cohort study. Patients were evaluated by an Emergency Physician who determined the pre-test probability for deep vein thrombosis to be either low, moderate, or high using an explicit clinical model. Patients were managed according to their pre-test probability category by specific algorithms that in all cases included venous ultrasound imaging within 24 h and a 90-day follow-up for the development of thromboembolic complications. Patients also underwent fingerstick SimpliRED(R) whole blood agglutination D-dimer testing; however, D-dimer results did not influence subsequent patient management. D-dimer had a sensitivity of 82.5% and a specificity of 84.9% for the diagnosis of deep vein thrombosis. The observed negative predictive value of D-dimer was 96.9% (95% CI, 93.0% to 99.1%) overall, and 100% (95% CI, 96.3% to 100%) in low probability patients, 94.1% (95% CI, 83.8% to 98.8%) in moderate probability patients, and 86.7% (95% CI, 59.4% to 98.3%) in high probability patients. SimpliRED(R) D-dimer has a high negative predictive value and may be useful in excluding the diagnosis in patients at low pre-test probability for deep vein thrombosis.

Agglutination Tests↗
PubMed1999

Combination of a clinical risk assessment score and rapid whole blood D-dimer testing in the diagnosis of deep vein thrombosis in symptomatic patients.

PURPOSE: The clinical diagnosis of deep vein thrombosis (DVT) is unreliable in more than 50% of cases, and, in recent years, plasma D-dimer assays have been used to predict the presence of DVT with high sensitivity and negative predictive values. This study investigated the use of a rapid whole blood test that could be performed and interpreted by the bedside in addition to a clinically derived risk assessment score (RAS) to determine whether a practical and cost-effective clinical model could be developed for the diagnosis and exclusion of DVT in symptomatic patients. METHODS: Two hundred consecutive patients who were referred to the vascular laboratory with clinically suspected DVT underwent the following procedures: (1) clinical assessment and stratification into low, moderate, or high risk for DVT on the basis of an RAS related to history, symptoms, and physical examination findings; (2) rapid (5-minute) whole blood D-dimer testing with a semi-qualitative agglutination technique on a fingerprick blood sample; and (3) color flow duplex ultrasound scanning with standard criteria for the diagnosis of proximal and calf DVT. RESULTS: Forty-six patients (23%) had acute DVT on duplex ultrasound scanning. Of these cases, 28 (61%) had proximal DVT and 18 (39%) were confined to the calf. A total of 88 patients were classified as low risk, 67 were classified as moderate, and 45 were classified as high risk on the basis of the RAS method. Of the patients who were classified at low risk, 4.5% (4/88) had DVT, as compared with 17.9% (12/67) in the moderate clinical risk group and 66.7% (30/45) in the high risk group. Isolated calf DVT was found in 30% (9/30), 50% (6/12), and 75% (3/4) of the high, moderate, and low risk groups, respectively. The sensitivity (8.7%, 26.1%, 65.2%), specificity (45.5%, 64.3%, 90.3%), positive predictive value (4.5%, 17.9%, 66.7%), and overall accuracy rate (37%, 55.5%, 84.5%) of the low, moderate, and high risk groups, respectively, in the diagnosis of DVT increased significantly with increasing risk score stratification. The sensitivity, specificity, positive and negative predictive value, and overall accuracy of the rapid D-dimer test for all DVT were 91.3%, 81.9%, 60%, 96.9%, and 84%, respectively, with a 100% sensitivity and negative predictive value for proximal DVT. A combination of the RAS and D-dimer identified a low risk group with a negative D-dimer as having less than a 1% likelihood of DVT and a high risk group with positive D-dimer as having a likelihood of more than 90%. CONCLUSION: A combination of clinical assessment and rapid D-dimer testing provides an effective means of excluding proximal DVT in symptomatic patients. The application of a clinical diagnostic model on the basis of these parameters has the potential for saving a large proportion of unnecessary duplex scans with the associated time and costs.

Female↗
PubMed2005

[Usefulness of fast ELISA determination of D-dimer levels for diagnosing pulmonary embolism in an emergency room].

OBJECTIVE: To determine the sensitivity and negative predictive value of D-dimer levels measured by fast enzyme-linked immunoabsorbent assay (ELISA) in pulmonary embolism. PATIENTS AND METHODS: Prospective study of consecutive patients with suspicion of pulmonary embolism attended in the Emergency Room of the Hospital de La Princesa in Madrid, Spain. Thromboembolism was diagnosed with an algorithm established in the hospital, and D-dimer levels were determined by fast ELISA (VIDAS D-dimer Assay) in each patient suspected of pulmonary embolism. Patients with negative findings from a test not considered a reference method for thromboembolism were followed for 3 months. RESULTS: Of 132 patients with clinical suspicion, 28 (21.2%) were positive and 104 (78.7%) were negative for embolism. D-dimer levels were below 0.5 microg/mL in 31 patients, 30 of whom did not have pulmonary thromboembolism whereas 1 did. D-dimer levels were abowe 0..5 microg/mL in 101 patients; thromboembolism did not occur in 74 of these but was reported in the remaining 27. For a value of 1 microg/mL, 66 patients had values below the cut off, 3 of whom presented pulmonary embolism. The remaining 66 patients had D-dimer levels above or equal to 1 microg/mL; 25 of them had a positive diagnosis for embolism and 41 had a negative diagnosis. Sensitivity and negative predictive values were 96.4% (95% confidence interval [CI], 79.8%-99.9%) and 96.8% (95% CI, 81.5%-98.8%), respectively, at a cut off of 0.5 microg/mL; and 89.2% (95% CI, 70.6%-97.2%) and 95.45% (95% CI, 86.4%-98.8%), respectively, at a cut off of 1 microg/mL. CONCLUSIONS: In an emergency room, thromboembolism can be excluded if plasma levels of D-dimer measured by fast ELISA are below 0.5 microg/mL because of the high negative predictive value at this cut off.

Aged↗
PubMed1995

A short autocomplementary sequence in the 5' leader region is responsible for dimerization of MoMuLV genomic RNA.

Previous work has shown that a region of Moloney murine leukemia virus (MoMuLV) RNA located between nucleotides 280 and 330 in the PSI region (nt 215-565) is implicated in the dimerization process. We show with a deletion from nucleotides 290-299 in PSI RNA transcripts and through an antisense oligonucleotide complementary to nucleotides 275-291 that the 283-298 region is involved in RNA dimer formation in vitro. In an attempt to further characterize the mechanism of dimer formation, a series of short RNA transcripts was synthesized which overlapps the PSI region of MoMuLV RNA. The dimerization of these RNAs is temperature dependent. The predicted secondary structure of the 278-303 region, as a function of temperature, reveals that this sequence is able to adopt two conformations: (1) the U288 AGCUA293 sequence in a loop; (2) part of the same nucleotides implicated in a stem. These results, together with thermodynamic analysis, strongly suggest that (1) the loop conformation of the UAGCUA sequence modulates the relative amount of RNA dimer and (2) a 16 bp long Watson-Crick base pairing is involved in RNA dimer formation. We propose that loop-loop recognition via the U288 AGCUA293 sequence leads to a stable structure induced by a stem-loop opening. Furthermore, our results do not support purine quartet formation as necessary for the dimerization of the 5' leader MoMuLV RNA.

Base Composition↗
PubMed1993

Assembly of the Arc repressor-operator complex: cooperative interactions between DNA-bound dimers.

Arc repressor, a member of the beta-ribbon family of DNA binding proteins, binds to its 21-base-pair operator as a tetramer. Here, the Arc dimer is shown to bind specifically to DNA fragments containing operator half-sites, and the equilibrium and kinetic constants for these reactions are determined. DNA-bound dimers are also shown to be transient intermediates in association experiments, indicating that assembly of the Arc tetramer-operator complex occurs by sequential addition of dimers to operator half-sites. When the left or right operator half-site is occupied by an Arc dimer, cooperative interactions increase the affinity of the second dimer by approximately 5900-fold [delta delta G = -5.1 (+/- 0.5) kcal/mol]. This increase in affinity is largely caused by an increase in the half-life of the complex; "non-cooperatively" bound dimers dissociate with a half-life of a few seconds while "cooperatively" bound dimers have half-lives of more than 1 h.

Base Sequence↗
PubMed1992

Nonoxidative cadmium-dependent dimerization of Cd7-metallothionein from rabbit liver.

The effect of free Cd(II) ions on monomeric Cd7-metallothionein-2 (MT) from rabbit liver has been studied. Slow, concentration-dependent dimerization of this protein was observed by gel filtration chromatographic studies. The dimeric MT form, isolated by gel filtration, contains approximately two additional and more weakly bound Cd(II) ions per monomer. The incubation of MT dimers with complexing agents EDTA and 2-mercaptoethanol leads to the dissociation of dimers to monomers. The results of circular dichroism (CD) and electronic absorption studies indicate that the slow dimerization process is preceded by an initial rapid Cd-induced rearrangement of the monomeric Cd7-MT structure. The 113Cd NMR spectrum of the MT dimer revealed only four 113Cd resonances at chemical shift positions similar to those observed for the Cd4 cluster of the well-characterized monomeric 113Cd7-MT. This result suggests that on dimer formation major structural changes occur in the original three-metal cluster domain of Cd7-MT.

Animals↗
PubMed1992

Molten globule monomer to condensed dimer: role of disulfide bonds in platelet factor-4 folding and subunit association.

Platelet factor 4 (PF4) exhibits high affinity for heparin and exists as a tetramer in solution under physiologic conditions. Reduction of the two disulfide bridges in PF4 increases the protein's dissociation constant for heparin approximately 20-fold and shifts the highest apparent aggregation state from tetramer to dimer as evidenced by gel filtration, chemical cross-linking, and 1H-NMR studies. 1H-NMR spectra of reduced PF4 monomers generally show narrower, less dispersed, upfield-shifted NH and alpha H resonances, suggesting the presence of an unfolded monomer state. Reduced PF4 monomer folding, however, is evidenced by the presence of about 12 relatively long-lived backbone NHs and by CD spectra that indicate conservation of overall secondary structure. These data suggest the presence of a molten globule-type state. Urea denaturation shifts this apparent molten globule to a fully unfolded state characterized by more random coil-like resonance shifts. The reduced PF4 dimer state yields NMR and CD data consistent with preservation of tertiary structural folds found for the native species. In this regard, the reduced PF4 folding transition is thermodynamically linked with dimer formation which stabilizes tertiary structure. Monomer-dimer association equilibria for reduced PF4 essentially follow the same pH and salt titration trends as reported previously for native PF4 dimers [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469-9478], indicating that that dimer interface is generally conserved in the absence of disulfide constraints. Reduced PF4 tetramers are not apparent under any conditions investigated, suggesting that disulfides are necessary for efficient antiparallel beta-sheet alignment between dimer pairs.

Chromatography, Gel↗
PubMed1993

Structure/function studies of HIV-1(1) reverse transcriptase: dimerization-defective mutant L289K.

Virion-derived HIV-1 reverse transcriptase (RT) has subunits of molecular mass 66 and 51 kDa (p66 and p51, respectively) in an approximately 1:1 ratio. Since enzyme activity appears to depend on dimerization of these subunits, identification of critical regions of primary sequence required for proper dimerization could lead to potential targets for antiviral therapy. A central region of primary sequence contains a leucine hepta-repeat motif from leucine 282 to leucine 310 that has been suggested to be involved in dimerization [Baillon, J. G., Nashed, N. T., Kumar, A., Wilson, S. H., & Jerina, D. M. (1991) New Biol. 3, 1015-1019]. A region including this hepta-repeat was recently shown to be involved in protein-protein interactions required for dimerization [Becerra, S. P., Kumar, A., Lewis, M. S., Widen, S. G., Abbotts, J., Karawya, E. M., Hughes, S. H., Shiloach, J., & Wilson, S. H. (1991) Biochemistry 30, 11708-11719]. To investigate the role of this repeat motif in dimerization, we performed site-directed mutagenesis of these leucine residues from position 282 to position 310. Mutations were introduced into p66 and p51 RT coding sequences, and the individually purified RT subunit polypeptides were compared with wild-type polypeptides for dimerization. Physical characterization of the purified mutant peptides was conducted by circular dichroism analysis. Binding between p66 and p51 was studied by gel filtration, ultracentrifugation, and CD analysis. L289K-p66 was unable to dimerize with itself and wild-type or L289K-p51. The leucine repeat motif in the p66 subunit appears to be critical in formation of the heterodimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Circular dichroism study on the conformational stability of the dimerization domain of transcription factor LFB1.

LFB1, a dimeric DNA binding protein, is a major determinant of hepatocyte-specific transcription. The thermal and chemical equilibrium unfolding of a 32-residue alpha-helical peptide comprising its dimerization domain (B1-Dim) was monitored by circular dichroism spectroscopy. The conformational stability of this peptide is shown to be concentration dependent, and the unfolding reaction is described as a two-state transition between folded dimers and unfolded monomers. The thermodynamic parameters associated with the unfolding reaction were determined under the two-state assumption by the van't Hoff procedure. The enthalpy of unfolding increases linearly with temperature, and the corresponding value of delta Cp, the difference in heat capacity between the unfolded and the folded forms of the peptide, is estimated to be ca. 0.7 kcal mol-1 K-1. The dimeric folded structure of the peptide is stabilized, at 25 degrees C, by a delta G of about 11.5 kcal mol-1, which is equivalent to a dimerization constant greater than 10(8) mol-1. These results indicate that the dimerization domain of LFB1 can fold and dimerize independently of the rest of the protein, with a thermodynamic stability comparable to that of a small globular protein.

Amino Acid Sequence↗
PubMed1991

Low-affinity platelet factor 4 1H NMR derived aggregate equilibria indicate a physiologic preference for monomers over dimers and tetramers.

Low-affinity platelet factor 4 (LA-PF4), unlike another related, sequentially homologous (about 50%) platelet-specific protein, platelet factor 4 (PF4), is an active mitogenic and chemotactic agent. PF4 exhibits a high binding affinity for heparin, while LA-PF4 does not. Both PF4 and LA-PF4 can exist in dimer and tetramer aggregate states. Equilibrium constants for PF4 aggregation have recently been estimated from fractional populations derived from proton nuclear magnetic resonance (NMR) integrals assigned to resonances in monomer, dimer, and tetramer states [Mayo & Chen (1989) Biochemistry 28, 9469]. On a 500-MHz NMR time scale, relatively slow exchange among LA-PF4 aggregate species has also allowed Tyr 15 ring proton resonances to be assigned for monomer, dimer, and tetramer states in LA-PF4. As a function of pH and ionic strength, equilibrium association constants for LA-PF4 dimer (KD) and tetramer (KT) formation have been estimated from Tyr 15 ring proton resonance integrals. At low ionic strength, KD reaches a minimum value of 12 M-1 at pH 3 where KT is at its maximum value of 1.6 x 10(5) M-1. At pH 4.1, KD and KT have the same value, 1.1 x 10(3) M-1, which is the minimum value for KT. KD plateaus off to its maximum value of 2.2 x 10(4) M-1 by pH 5.5. These values are significantly lower than those for PF4. Analysis of the pH dependence of KD and KT suggests that electrostatic interactions probably among Glu/Asp and Lys/Arg side chains form the predominant force in the monomer-monomer binding process, i.e., KD, while like-charge repulsion due to proximal, intersubunit Glu/Asp residues decreases KT as the pH is raised. At pH 7 and low ionic strength, the dimer state is highly favored over the tetramer state. Elevating the solvent ionic strength at pH 7 destabilizes the dimer state. Under these more physiologic conditions, i.e., pH 7 and 0.1-0.2 M NaCl, LA-PF4 monomers are highly favored over dimers and tetramers. For PF4 under similar solvent conditions, tetramers predominate. Differences in biological activities between these homologous platelet-specific proteins may be the result, at least in part, of differing aggregation properties. The biologically active state for PF4 is tetramer, while for LA-PF4 it is monomer. Quaternary structure may, therefore, account for strong heparin binding in PF4, most likely by presenting a more favorable structural matrix for effective glycosaminoglycan interactions.

Amino Acid Sequence↗
PubMed1983

Dimeric half-molecules of human fibrinogen are joined through disulfide bonds in an antiparallel orientation.

Human fibrinogen is a dimer composed of two identical halves. Each dimeric half contains three peptide chains (alpha, beta, and gamma) linked by disulfide bonds. The two half-molecules are joined by three disulfide bonds, one between the two alpha-chains (residue alpha-28) and two between the two gamma-chains (residues gamma-8 and gamma-9). In the absence of any difinitive experimental evidence, it has been presumed that the joined halves were aligned in a parallel orientation similar to the situation found in immunoglobulins. We have now determined that the two gamma-chains--hence, the dimeric halves--are connected in an antiparallel manner. A tryptic peptide containing gamma-chain residues 6-14 was isolated as a disulfide-linked dimer from CNBr-treated fragment E. Synthetic peptides corresponding to this sequence were prepared, from which parallel and antiparallel dimers were constructed. During the syntheses, cysteine thiol groups were protected as p-methoxybenzyl and acetamidomethyl sulfides; the peptides were dimerized by selective deprotection and disulfide bond formation. First, the p-methoxybenzyl groups were removed by liquid hydrogen fluoride and the newly exposed thiols oxidized in the presence of potassium ferricyanide. Then the monocystine compound was converted to the double-cystine product by iodolytic cleavage of the acetamidomethyl group with concomitant disulfide bond formation. This selectivity was used to prepare peptide dimers which modeled both parallel and antiparallel arrangements. The antiparallel-oriented synthetic peptide was indistinguishable from the native tryptic peptide as judged by elution from reverse-phase high-performance liquid chromatography and circular dichroism spectroscopy. The parallel-oriented synthetic peptide differed from the native material by both criteria.

Amino Acid Sequence↗
PubMed1987

Dissociation of the octameric bifunctional enzyme formiminotransferase-cyclodeaminase in urea. Isolation of two monofunctional dimers.

Partial denaturation of the circular octameric bifunctional enzyme formiminotransferase-cyclodeaminase in increasing urea concentrations leads to sequential dissociation via dimers to inactive monomers. In potassium phosphate buffer, dissociation to dimers in 3 M urea coincides with loss of both activities and a major decrease in intensity of intrinsic tryptophan fluorescence. In the presence of folic acid, these dimers retain the deaminase activity, but with folylpolyglutamates, both activities are protected and the native octameric structure is retained. The protection profiles with polyglutamates are cooperative with a Hill coefficient greater than 2, suggesting that binding of more than one folylpolyglutamate per octamer is required to stabilize the native structure. In triethanolamine hydrochloride buffer, transferase-active dimers that retain the intrinsic tryptophan fluorescence can be obtained in 1 M urea and stabilized at higher urea concentration by the addition of glutamate. Deaminase-active dimers are obtained by the protection of folate in 3 M urea. Proteolysis of the two kinds of dimers by chymotrypsin leads to very different fragmentation patterns, indicating that they are structurally different. We propose that the two dimers retain different subunit-subunit interfaces, one of which is required for each activity. This suggests that the native octameric structure is required for expression of both activities and therefore for "channeling" of intermediates.

Ammonia-Lyases↗
PubMed1989

Isolation and preliminary characterization of wild-type OmpC porin dimers from Escherichia coli K-12.

Escherichia coli K-12 strain PLB3255 contains a mutation in the ompF gene that results in a 15 amino acid deletion in the porin protein. The mutation (dex) appears to increase the OmpF channel size, allowing the PLB3255 cells to grow on maltodextrins in the absence of a functional maltoporin. Porin isolated from strain PLB3255, which contains a wild-type ompC gene, was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and shown to contain 50-60% trimer aggregates and 35-40% of a 50-kDa "dimer". When the porin isolate was heated to 100 degrees C and separated on SDS gels containing 8 M urea, both the trimer and the "dimer" were recovered in a single band at 36 kDa that corresponded in mobility to wild-type OmpC porin. An analysis of the temperature stability of the isolate showed that the OmpC "dimer" was less stable and denatured at 66 degrees C compared to 81 degrees C for the trimer. To separate these aggregates, the unheated porin was suspended in 30% SDS, applied to a Sephadex G-200 gel filtration column, and eluted with 0.5% sodium deoxycholate. Two peaks were recovered containing separated trimers and "dimers". Circular dichroism spectra of isolated dimers and trimers indicated similar amounts of beta-structure. The isolated dimers and trimers were reconstituted into artificial membranes. Electrical conductance across planar bilayer lipid membranes and liposome swelling assays showed that the two isolates had similar channel-forming activity. Four other ompF deletion mutants of the same phenotype were also shown to produce 50-kDa OmpC porin "dimers".(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Outer Membrane Proteins↗
PubMed1990

Purification of PDGF-AB and PDGF-BB from human platelet extracts and identification of all three PDGF dimers in human platelets.

We have developed a panel of monoclonal antibodies to platelet-derived growth factor (PDGF) which have variable specificities for the three dimeric forms of the molecule (AA, AB, and BB). We have used these antibodies to detect and immunoaffinity purify the individual dimers from human platelet rich plasma. Extracts of outdated platelet preparations were initially chromatographed over CM-Sepharose and then passed over the Sepharose-coupled monoclonal antibodies in series in selectively isolate the three dimeric forms of PDGF. The PDGF eluted from the affinity columns was subsequently further purified by reversed-phase HPLC. From 300 units of outdated platelet preparations, we purified 58 micrograms of PDGF-BB and 140 micrograms of PDGF-AB. Using the monoclonal antibodies to develop PDGF dimer-specific ELISAs, it was observed that all three PDGF dimer forms are present in fresh human platelet extracts and that the ratios of the three dimer forms vary depending upon the extraction conditions used. The identification of all three PDGF dimer forms in human platelets point to the need to view PDGF isolated from human platelets by conventional techniques as a mixture of all three forms and not solely as PDGF-AB.

Amino Acid Sequence↗
PubMed1990

Quaternary structure of the Mr 46,000 mannose 6-phosphate specific receptor: effect of ligand, pH, and receptor concentration on the equilibrium between dimeric and tetrameric receptor forms.

The Mr 46,000 mannose 6-phosphate specific receptor exists in solution as a mixture of noncovalently associated dimeric and tetrameric forms. The two quaternary forms were separated by sucrose density centrifugation, and their composition was assessed by cross-linking with bifunctional reagents followed by SDS-polyacrylamide gel electrophoresis. The dependence of equilibrium between the dimeric and tetrameric forms on pH, receptor concentration, and presence of mannose 6-phosphate was studied. The formation of tetrameric forms is favored by pH values around 7, high receptor concentration, and presence of mannose 6-phosphate ligand. Tetrameric forms bind stronger at pH 7 to phosphomannan-Sepharose 4B than dimeric forms. Both quaternary forms dissociate at the same pH from a mannose 6-phosphate affinity matrix. When starting with dimeric or tetrameric forms, the equilibrium between dimeric and tetrameric forms is reached at pH 7.5 and 4 degrees C after 6-8 days. The presence of 5 mM mannose 6-phosphate shifts the equilibrium toward tetrameric forms. At pH 4.5 and 4 degrees C, the association of dimeric to tetrameric forms is negligible, while tetrameric forms dissociate to dimeric forms within 12 h. The results demonstrate that oligomerization is an intrinsic property of MPR-46 that is affected by ligand binding, pH, and receptor concentration.

Carrier Proteins↗
PubMed1980

Sites of D-domain interaction in fibrin-derived D dimer.

We have examined the plasmic digestion products of fibrin formed in the presence of dansylcadaverine, the fluorescent D dimer, to determine whether they are held together not only by the cross-link region on the gamma chain but also by other interactions on the D domain. Antibodies to the D dimer reacted 8X more strongly with sites on the D dimer (purified or in the presence of E) than with sites on fibrinogen or plasmin-digested fibrinogen. The reactivity of this surface site was lost when the gamma chain was cleaved by plasmin after the molecule had been destabilized by the removal of calcium ions, thus breaking the covalent linkage of the homodimer. The noncovalent D dimer retained its dimeric structure by the criteria of molar volume, measured by fluorescence polarization, and molecular sieving. The noncovalently attached, cross-link-containing peptide bound tightly to the parent molecules at higher temperatures but rotated more freely below 15 degrees C, and could be lost from the parent molecules without destroying the dimeric structure. We therefore propose that the forces maintaining the dimeric structure of the noncovalently joined molecule are not solely located at the gamma-chain cross-link region. These other sites on the D domain are therefore candidates for the initial fibrinogen polymerization site and may also have a role in fibrinogen half-molecule assembly.

Animals↗
PubMed1996

Characterization of human cytomegalovirus protease dimerization by analytical centrifugation.

Human cytomegalovirus, a member of the herpesvirus family, encodes a maturational protease required for processing of the assembly protein and virus replication. The protease is synthesized as a precursor protein that undergoes autoproteolytic cleavage to yield a mature, 28-kDa enzyme. It has recently been demonstrated that mature human cytomegalovirus protease is capable of dimerization and that the dimer is the active species [Darke, P. L., Cole, J. L., Waxman, L., Hall, D. L., Sardana, M. K., & Kuo, L. C. (1996) J. Biol. Chem. 271, 7445-7449; Margosiak, S. A., Vanderpool, D. L., Sisson, W., Pinko, C., & Kan, C.-C. (1996) Biochemistry 35, 5300-5307]. Here, analytical equilibrium and velocity sedimentation measurements were used to define the thermodynamics of protease dimerization. Protease dimerization is well described by a homogeneous, reversible mass-action equilibrium. The apparent molecular weight of the protease decreases at higher protein concentrations, and good global fits to sedimentation equilibrium data require a positive value of the second virial coefficient, indicating that the protein exhibits thermodynamic nonideality. The magnitude of the nonideality is higher than expected on the basis of excluded volume and electrostatic effects and is not very sensitive to salt concentration, as would be expected for electrostatic effects. The dimer dissociation constants are in agreement with the values we previously determined by activity measurements and hydrodynamic techniques. Dimerization is enhanced by addition of glycerol or NaCl. The temperature dependence of the dimerization constant indicates that both delta H degree and delta S degree are negative, which is commonly observed in protein self-association reactions.

Centrifugation↗
PubMed2000

Copper(II) benzoate nitroxide dimers and chains: structure and magnetic studies.

Copper(II) benzoate dimers and linear chains have been synthesized and exhibit very different magnetic behaviors. The benzoate dimers, 1a, show typical dimeric singlet-triplet transitions and strong antiferromagnetic coupling (JST = -206 K (-143 cm-1); H = -2JSTSa.Sb). The bromobenzoate dimer can be converted into linear chains of hydrogen-bonded monomers, showing 1-D ferromagnetic coupling (6a, theta = +9 K). Copper(II) sites can also be bridged by nitroxide-substituted benzoates, 1b and 1c, that is, 2-(4'-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (NNBA, 3a), with JST = -216 K (-150 cm-1), with comparable interactions between the nitroxide and triplet Cu(II) spins, theta T = -157 K. A 1-D chain similar to the bromobenzoate monomers can also be produced with NNBA, also exhibiting ferromagnetic coupling (6b, theta = +0.67 K), albeit much weaker. Other nitroxides have been introduced into the Cu(II) dimer system by capping copper(II) acetate with the polydentate 2-(4'-pyridyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PYNN, 3b), which exhibits almost no coupling to the copper centers when both ends of the dimer are capped (1d, theta = -5.8 K). In contrast, strong coupling is observed when only one PYNN is used (2, theta = -300 K), which is the result of direct coordination of the nitroxide to the copper centers, producing a chain of the dimer units.

Journal Article↗