Biomedical subjects

C M Jackson

Publications and source records attributed to C M Jackson.

At least 55 records · Page 3Linked to original sources

PubMed1995

The use of Japanese and bobwhite quail as indicator species in avian toxicity test. ECPA/GIFAP Environmental Round Table.

A comparative analysis was made between the toxicity of plant protection products to two species of quail used in regulatory testing. It was shown that in general differences in the sensitivity between bobwhite quail and Japanese quail were small, and not statistically significant. It was therefore concluded that both species should be seen as equally suitable models for avian safety assessment. The mutual acceptance of studies carried out on either of these species for US and EC registration procedures, would lead to a reduction in vertebrate sacrifice and resource spending.

Animals↗

PubMed1994

Asthma control.

Explore the source record for details and available documents.

Albuterol↗

PubMed1994

Factor X-activating glycoprotein of Russell's viper venom. Polypeptide composition and characterization of the carbohydrate moieties.

There is contradictory information regarding the molecular weight and polypeptide chain composition of RVV-X, a glycoprotein in Russell's viper venom that is capable of activating factor X to Xa. We show that RVV-X is a 92,880-Da glycoprotein. It consists of three disulfide-linked polypeptide chains, one heavy chain (alpha-chain, M(r) 57,600) and two light chains (beta- and gamma-chains, M(r) 19,400 and 16,400, respectively). The two light chains seen on SDS-polyacrylamide gel electrophoresis under reducing conditions are two distinct components of the molecule, rather than a heterogeneous mixture of a single chain as previously reported (Takeya, H., Nishida, S., Miyata, T., Kawada, S., Saisaka, Y., Morita, T., and Iwanaga, S. (1992) J. Biol. Chem. 267, 14109-14117). The following evidence supports this conclusion. (i) The two light chains of RVV-X are present in equal proportion. (ii) The estimated molecular weight of an alpha 1 beta 1 gamma 1-structure closely matches the molecular weight determined by matrix-assisted laser desorption mass spectrometry. (iii) The amino acid compositions and NH2-terminal sequences of the beta- and gamma-chains are different. (iv) Although both the beta- and gamma-chains contain one N-linked oligosaccharide chain each, they are glycosylated differentially. RVV-X contains six N-linked oligosaccharides, four in the alpha-chain and one in each of the beta- and gamma-chains. The carbohydrate structures are different from those known for other snake venom glycoproteins, and they resemble closely those in various mammalian glycoproteins. The majority of the oligosaccharides are complex bi-, tri-, and tetraantennary structures, with a small proportion of multiantennary and high mannose-type structures. Two notable structural features of RVV-X oligosaccharides are as follows. (i) Sialic acid residues are linked to beta-galactosyl residues solely by alpha 2,3-linkages, and (ii) bisecting N-acetylglucosamine residues are present in the majority of the oligosaccharides.

Amino Acid Sequence↗

PubMed1994

A comparison of the systemic bioactivity of inhaled budesonide and fluticasone propionate in normal subjects.

1. The aim of this study was to compare the systemic bioactivity of low and high doses of inhaled budesonide and fluticasone propionate given by respective dry powder inhaler devices. 2. A randomised, single blind cross-over design was used in nine healthy subjects who were given 800 micrograms day-1 of budesonide Turbohaler (B800) for 1 week, followed by 1 week of 1600 micrograms day-1 (B1600), or fluticasone Diskhaler 750 micrograms day-1 (F750) for 1 week followed by 1 week of 1500 micrograms day-1 (F1500). There was a 1 week washout between treatments with fluticasone or budesonide. A twice daily dosing regime was used and mouth-rinsing was employed to reduce gut bioavailability as well as to obviate local adverse effects. 3. Parameters of hypothalmic-pituitary adrenal (HPA) axis activity and bone metabolism were measured at baseline (B0/F0), at the end of each week of treatment and after the 1 week washout (F0 or B0). 4. Both fluticasone and budesonide significantly (P < 0.05) attenuated the post tetracosactrin serum cortisol at low and high doses whilst early morning cortisol was unchanged. No dose-response effect was observed with either drug, and there was no significant difference between treatment with fluticasone or budesonide. 5. Neither budesonide nor fluticasone produced significant suppression of plasma osteocalcin, although the higher doses of both drugs significantly reduced fasting urinary calcium levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗

PubMed1993

Thrombin exosite for fibrinogen recognition is partially accessible in prothrombin.

The procoagulant alpha-thrombin is produced by the proteolytic cleavages of a minimum of two peptide bonds Arg274-Thr275 and Arg323-Ile324 in prothrombin. The Arg323-Ile324 cleavage is required for the expression of the active site of thrombin (Morita, T., Iwanaga, S. Suzuki, T. (1976) J. Biochem. (Tokyo) 79, 1089-1108; Hibbard, L. S., Nesheim, M. E., and Mann, K. G. (1982) Biochemistry 21, 2285-2292). It is not yet clear to what extent the proteolytic events are responsible for exposing protein recognition exosites on thrombin. We employed high resolution NMR spectroscopy to examine interactions of prothrombin and thrombin with synthetic hirudin peptides targeted toward the fibrinogen recognition exosite of thrombin. The hirudin tail synthetic analogues (acetyl-Asp55-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln65/G ly65-OH) exhibited similar NMR relaxation enhancements (line broadening patterns and transferred nuclear Overhauser effects) with human prothrombin as with human alpha-thrombin, indicating that both proteins bind the peptide in a similar manner. The protein-induced relaxation enhancements are specific to the interaction of the hirudin peptides with the fibrinogen recognition exosite of thrombin since no significant effects were observed with either human serum albumin or with human gamma-thrombin, which has an impaired recognition exosite. The binding affinities were determined from NMR relaxation time measurements, which gave approximate Kd values of 500 microM and < 100 microM for prothrombin and alpha-thrombin, respectively. Since the hirudin tail fragment binds specifically to the fibrinogen recognition exosite in alpha-thrombin, this exosite appears to be partially accessible in prothrombin in a proenzyme form.

Amino Acid Sequence↗

PubMed1993

Bronchodilator reversibility to low and high doses of terbutaline and ipratropium bromide in patients with chronic obstructive pulmonary disease.

BACKGROUND: There is uncertainty regarding the use of monotherapy or combination therapy with beta 2 agonists and anticholinergic drugs in patients with chronic obstructive pulmonary disease (COPD). The measurement of forced expiratory volume in one second (FEV1) or relaxed vital capacity (RVC) in the assessment of reversibility in these patients has also caused considerable debate. METHODS: Twenty seven patients with COPD were evaluated on two occasions. Patients received the following treatments in sequence: (sequence 1) low dose terbutaline 500 micrograms, high dose terbutaline 5000 micrograms, low dose ipratropium 40 micrograms, high dose ipratropium 200 micrograms; (sequence 2) low dose ipratropium 40 micrograms, high dose ipratropium 200 micrograms, low dose terbutaline 500 micrograms, high dose terbutaline 5000 micrograms. RVC, FEV1 and FVC were measured at baseline and 30 minutes after successive treatments. RESULTS: Values for FEV1 at baseline on the first and second study days were not significantly different: 0.90 (0.87-0.93) 1 v 0.90 (0.87-0.93) 1. Likewise, baseline values for RVC and FVC were not different. The number of patients showing a greater than 330 ml overall improvement in RVC was 20 of 27 for sequence 1 and 22 of 27 for sequence 2; similar trends were observed for FEV1 and FVC. For all three parameters there was a significant difference between mean responses to low and high doses of terbutaline when the latter was given as the first drug in sequence 1. When ipratropium was given first in sequence 2 there was, however, no significant improvement with high dose terbutaline over and above the response to low dose terbutaline. The latter effect was more noticeable with RVC than with either FEV1 or FVC. The total bronchodilator response at the end of each sequence was similar whether ipratropium was given first or second. CONCLUSIONS: The measurement of RVC, FEV1, and FVC were equally effective at picking up those patients who had a significant overall bronchodilator response to combined therapy with inhaled beta 2 agonist and anticholinergic medication. There was no significant benefit of adding a higher dose of terbutaline when ipratropium bromide had been given previously, particularly when using RVC as the parameter of response.

Administration, Inhalation↗

PubMed1993

An audit of hospital discharge letters in patients admitted with acute asthma. Tayside Asthma Audit Group.

The management of patients following discharge with acute asthma is dependent on effective communication with general practitioners. A retrospective audit was therefore performed on copies kept of 81 typed discharge letters with a diagnosis of acute asthma between March and October 1991. A subset of 42 interim (handwritten) letters were also audited where case notes could be retrieved. Details on clinic follow up were also obtained from the case notes (n = 42). In the typed letter documentation of severity and treatment of the acute attack was accurate in most cases. Information concerning a precipitating factor was provided in 54% of cases and a smoking history in 57%. Deficiencies were found in specifying inhaler delivery devices (40% recorded), and whether inhaler technique had been formally assessed whilst in hospital (17%). Drug prescribing on discharge was as follows: oral steroid (69%), inhaled steroids (77%), inhaled B2-agonists (92%), theophylline slow release (38%), salbutamol controlled release (20%), and antibiotics (30%). The implementation of a self-management plan and domiciliary peak flow was mentioned in 66% of the letters. The interim letter was generally poor in particular for mention if discharge peak flow (2%), clinic follow-up (64%) and prednisolone regime (61%). Mean +/- s.d. time for clinic follow-up (n = 42) was 4.7 +/- 1.7 weeks (range 1-13 weeks) with 24% non-attendance. Thus, improvements in discharge letters are clearly required for optimum continuity of care in the community.

Acute Disease↗

PubMed1992

Study of high-affinity interactions by quantitative affinity chromatography. Analytical expressions in terms of total ligand concentration.

Analytical expressions are derived for the description of ligand-facilitated and ligand-retarded desorption of partitioning solute in terms of total ligand concentration in quantitative affinity chromatography. Their application is then illustrated by consideration of results from recycling partition equilibrium studies of the heparin-facilitated desorption of thrombin from heparin-Sepharose, and of the competition between methyl-alpha-D-mannoside and p-nitrophenyl-alpha-D- mannoside for concanavalin A immobilized on CPG-170. Finally, published frontal affinity chromatographic data for the NADH-dependent elution of rabbit muscle lactate dehydrogenase from oxamate-Sepharose are reanalysed using these equations to demonstrate the characterization of a system reflecting the binding of a solute-ligand complex to an affinity matrix. This investigation extends the scope of quantitative affinity chromatography to include not only the study of solute-ligand interactions governed by larger binding constants but also the characterization of interactions in which the partitioning solute and ligand are both macromolecular, and eliminates the need for prior dialysis to establish the free ligand concentration required for application of earlier analytical expressions.

Animals↗

PubMed1992

An audit of acute asthma admissions to a respiratory unit.

A criterion based audit was performed on 90 admissions with acute asthma to a specialist respiratory unit using the guidelines set by the British Thoracic Society. Three main areas were audited including: documentation of severity markers in the case notes on admission, acute treatment given on admission, and further investigations performed. Case notes were found to be deficient in documentation of objective severity markers such as peak expiratory flow rate (52% recorded) and blood gases (72% recorded), as well as subjective markers such as speech (27%), air entry (58%) and exhaustion (4%). Of the total 90 admissions, 78% were identified as having at least three out of five objective markers for acute severe asthma. Most of these severe cases (93%) were given corticosteroids but none were prescribed greater than 35% concentration of inspired oxygen. The majority of admissions had a chest radiograph (87%), and 56% had measurement of serum potassium. These results show that even in a specialised respiratory unit, guidelines such as those produced by the British Thoracic Society are not in general being adhered to. It also indicates that assessment of severity is inadequate, particularly for peak flow measurement, and that management is deficient in established treatments such as the use of high flow oxygen. As a result of the audit, a severity marker stamp for the case notes along with guidelines for treatment have been implemented in order to improve the quality of care delivered to patients admitted with acute asthma.

Acute Disease↗

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↗

PubMed1991

Effects of thermodynamic nonideality in kinetic studies: evidence of an expanded intermediate complex in the inactivation of thrombin by antithrombin III.

The technique of competitive chromogenic substrate hydrolysis is used to examine the inhibitory effects of sucrose and glycerol on the inactivation of thrombin by antithrombin III. This inhibition is attributed to the existence of a slight increase in volume/asymmetry associated with formation of the thrombin-antithrombin complex that subsequently undergoes covalent modification in an irreversible inactivation step. Partial reversal of the equilibrium step is thus considered to result from the effects of molecular crowding in the highly concentrated environment that is generated by the inclusion of these small insert solutes.

Antithrombin III↗

PubMed1991

Use of quantitative affinity chromatography for characterizing high-affinity interactions: binding of heparin to antithrombin III.

The versatility of quantitative affinity chromatography (QAC) for evaluating the binding of macromolecular ligands to macromolecular acceptors has been increased substantially as a result of the derivation of the equations which describe the partitioning of acceptor between matrix-bound and soluble forms in terms of total, rather than free, ligand concentrations. In addition to simplifying the performance of the binding experiments, this development makes possible the application of the technique to systems characterized by affinities higher than those previously amenable to investigation by QAC. Addition of an on-line data acquisition system to monitor the concentration of partitioning solute in the liquid phase as a function of time has permitted the adoption of an empirical approach for determining the liquid-phase concentration of acceptor in the system at partition equilibrium, a development which decreases significantly the time required to obtain a complete binding curve by QAC. The application of these new QAC developments is illustrated by the determination of binding constants for the interactions of high-affinity heparin (Mr 20,300) with antithrombin III at three temperatures. Association constants of 8.0 +/- 2.2 x 10(7), 3.4 +/- 0.3 x 10(7), and 1.0 +/- 0.2 x 10(7) M-1 were observed at 15, 25, and 35 degrees C, respectively. The standard enthalpy change of -4.2 +/- 0.6 kcal/mol that is calculated from these data is in good agreement with a reported value obtained from fluorescence quenching measurements.

Antithrombin III↗

PubMed1990

Kinetic intermediates in prothrombin activation. Bovine prethrombin 1 conversion to thrombin by factor X.

Two pathways are possible during the proteolytic formation of alpha-thrombin (alpha-IIa) from prothrombin (II) or prethrombin 1 (P1). One of the pathways, with prethrombin 2 or prethrombin 2 associated with fragment 2 (P2F2) as intermediates, has long been known to exist when activation is catalyzed by Factor Xa (Xa) alone. The second pathway, with meizothrombin or meizothrombin (des fragment 1) (MzIIa(-F1)) as intermediate, has been shown to exist when Factor Va and phospholipids are present with Xa. Until now, MzIIa(-F1) has not been detected in reactions catalyzed by Xa alone. In this study, we demonstrate that P1 activation by Xa alone occurs via both pathways, and we provide rate constants and kinetic equations for calculating the relative contributions of each of the pathways to the formation of alpha-IIa by Xa. Investigation of the initial rates of proteolytic cleavage of P2F2 and P1 by Xa alone indicated first-order dependence on substrate concentration with no evidence of saturation of Xa with either substrate at concentrations as high as 200 microM. Apparent second-order rate constants (kc/Km) of 113 +/- 9 M-1 s-1 for the formation of thrombin from P2F2 and 1,410 +/- 19 M-1 s-1 for the disappearance of P1 were determined at pH 7.5, 25 degrees C, 10 mM CaCl2, 0.15 M ionic strength. A two-step sequential first-order pathway employing these rate constants for thrombin activity production from P1 via P2F2 could not, however, account for the quantity of thrombin that was produced during the early stages of P1 activation. Addition of a parallel first-order reaction to produce thrombin activity from P1 independently of P2F2, tentatively identified as the formation of MzIIa(-F1), yielded progress curves in quantitative agreement with the experimental data. kc/Km for the parallel reaction was estimated to be 98 +/- 10 M-1 s-1. Independent determination of the second-order rate constant for the cleavage of isolated MzIIa (-F1), 15,000 +/- 420 M-1 s-1, indicated that MzIIa(-F1) could meet the kinetic requirements for an intermediate in the parallel activation pathway. The transient formation of MzIIa (-F1), as well as the generation of alpha-IIa, was directly demonstrated during activation of P1 by active site-affinity labeling of the reaction products with a biotin derivative of D-Phe-Pro-Arg chloromethyl ketone and visualization by semiquantitative Western blotting.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Heparin promotes the binding of thrombin to fibrin polymer. Quantitative characterization of a thrombin-fibrin polymer-heparin ternary complex.

The binding of human alpha-thrombin (IIa) to fibrin polymer (FnIIp) was studied in the presence and absence of a high affinity 20,300 Mr heparin (H) at pH 7.4, I 0.15, and 23 degrees C. In the absence of heparin, thrombin interacts with a high affinity class of binding sites on fibrin polymer with a dissociation constant of 301 +/- 36 nM in a manner which is independent of the enzyme active site. Studies of thrombin binding as a function of heparin and fibrin polymer concentrations imply that a ternary thrombin-fibrin polymer-heparin complex (IIa.FnIIp.H) is formed. Assembly of the ternary complex occurs randomly through the interactions of all three possible intermediate binary complexes; IIa.H, IIa.FnIIp, and FnIIp.H. Using an independently determined value of 280 +/- 35 nM for the FnIIp.H dissociation constant, global fits of the binding data yield a dissociation constant of 15 +/- 6 nM for the IIa.H interaction and 47 +/- 9 nM for the IIa.H intermediate binary complex interaction with FnIIp. These studies indicate that heparin enhances the binding of thrombin to fibrin polymer 6.4-fold with an overall dissociation constant for ternary complex formation of 705 nM2. The effect of heparin molecular weight on ternary complex formation has also been investigated. Heparins of molecular weights 11,200-20,300 behave similarly with respect to their influence on ternary complex formation, whereas heparins of lower molecular weight are less effective in promoting thrombin binding to fibrin polymer. This effect of heparin is also independent of whether it has high or low affinity for antithrombin III. The demonstration of the formation of a ternary IIa.FnIIp.H complex complements kinetic evidence indicating the formation of an analogous ternary complex with fibrin II monomer (Hogg, P. J., and Jackson, C. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3619-3623). The possible implications of these findings for the in vivo distribution and actions of thrombin and the clinical efficacy of heparin are also discussed.

Antithrombin III↗

PubMed1990

Formation of a ternary complex between thrombin, fibrin monomer, and heparin influences the action of thrombin on its substrates.

The consequences of the combined effects of fibrin II monomer (FnIIm) and heparin (H) on the hydrolysis of peptidyl p-nitroanilide substrates by thrombin (IIa), the cleavage of prothrombin by thrombin and the thrombin-catalyzed release of fibrinopeptides from fibrinogen have been studied at pH 7.4 and I 0.15. The effects of fibrin II monomer and heparin on chromogenic substrate hydrolysis can be described by a hyperbolic mixed inhibition model in which substrate can interact with four possible enzyme species (IIa, IIa.H, IIa.FnIIm, and IIa.FnIIm.H) that arise as a result of random formation of a ternary complex among thrombin, fibrin II monomer, and heparin (Hogg, P. J. and Jackson, C. M. (1990) J. Biol. Chem. 265, 241-247). The formation of the ternary IIa.FnIIm.H complex results in an increase in the Km values of 7.03 +/- 1.17-fold (1.37-9.65 microM) and 1.94 +/- 0.60-fold (38.1-73.9 microM) for H-D-Ile-Pro-Arg-pNA and Cbz-Gly-Pro-Arg-pNA hydrolysis, respectively, and a decrease in the kc values of 0.45 +/- 0.08-fold (49.5-22.3 s-1) and 0.52 +/- 0.05-fold (93.1-48.4 s-1). Fibrin II monomer and heparin in combination also decrease the efficiency (kc/Km) with which thrombin cleaves prothrombin to produce Fragment 1 and Prethrombin 1 by 2.3-fold from 607 +/- 30 to 264 +/- 13 M-1 s-1. In contrast to the effects of fibrin II monomer and heparin on thrombin hydrolysis of chromogenic substrates, its proteolysis of prothrombin and its inactivation by antithrombin III (Hogg, P. J., and Jackson, C. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3619-3623), these components have no discernible influence on the ability of thrombin to cleave fibrinogen. These observations indicate that the substrate specificity of thrombin is altered when it is bound in a complex with fibrin II monomer and heparin and suggest that the catalytic efficiency of thrombin for its physiological substrates will be affected differentially by these interactions. Such ternary complex formation involving thrombin, fibrin II monomer, and heparin may provide a mechanism for selectively regulating thrombin action.

Amino Acid Sequence↗

PubMed1990

Mechanism of heparin action.

Heparin catalysis of clotting proteinase inactivation occurs most efficiently through the reaction of the proteinase with the antithrombin-heparin complex. The efficiency of a heparin molecule in this reaction depends on the presence of a specific pentasaccharide sequence in it, and its molecular weight. The mechanism by which such high affinity heparin acts when antithrombin III is the inhibitor is promotion of the formation of an intermediate proteinase-heparin-antithrombin complex. Heparin promotion of thrombin inactivation by heparin cofactor II may occur by a similar mechanism. The requirement for a specific oligosaccharide sequence within the heparin molecule does not, however, exist for heparin cofactor II. Binding of heparin to both thrombin and antithrombin III interferes with thrombin inactivation. This binding is very dependent on the ionic strength of the reaction mixture and may explain some of the discordant results and interpretations from early studies on the mechanism of heparin action. Low ionic strength in in vitro reactions also results in cleavage of antithrombin III by thrombin in the presence of heparin and effectively converts antithrombin III from an inhibitor to a substrate.

Antithrombin III↗

PubMed1989

Fibrin monomer protects thrombin from inactivation by heparin-antithrombin III: implications for heparin efficacy.

Fibrin II monomer has a dramatic inhibitory effect on the rate of heparin-catalyzed inactivation of human alpha-thrombin by antithrombin III. At 6 microM fibrin II monomer, equivalent to the concentration of fibrinogen in plasma, the second-order rate constant was reduced by a factor of 308--from 2.05 x 10(8) M-1.s-1 to 6.65 x 10(5) M-1.s-1. Fibrin II monomer minimally affected the uncatalyzed rate of thrombin inactivation showing a reduction in the second-order rate constant by a factor of only 1.6. Fibrinogen and the product of plasmin degradation of fibrinogen, fragment E, at 6 microM concentrations also decreased the second-order rate constant for heparin-catalyzed thrombin inactivation, but by factors of only 2.7 and 1.9, respectively. On the basis of these observations it is proposed that protection of thrombin from inactivation by heparin-antithrombin III by fibrin II monomer can explain the limited efficacy of heparin in preventing coronary reocclusion in patients treated with tissue plasminogen activator and other fibrinolytic agents.

Antithrombin III↗

PubMed1987

Evidence for self-association of prothrombin fragment 1 in the absence of calcium ions. Implications for the interpretation of cooperativity of calcium binding.

Sedimentation equilibrium studies have demonstrated that prothrombin fragment 1 from either human or bovine plasma reversibly dimerizes in the absence of Ca2+ with an equilibrium constant of 1,000 M-1. In the presence of 10 mM Ca2+ this association constant increased to 10,000 M-1. A model for preferential binding of Ca2+ to the pre-existing dimer has been found capable of accounting quantitatively for the cooperative Ca2+ binding to this prothrombin fragment, and for the dependence of its sedimentation coefficient on protein concentration in the presence and absence of metal ion. Sedimentation equilibrium studies of intact bovine and human prothrombins have confirmed previous reports that these prothrombins dimerize. For both prothrombins the association constant is 10,000 M-1, both in the absence and presence of Ca2+.

Animals↗