Chemical modification of bovine prothrombin fragment 1 in the presence of Tb3+ ions

The formaldehyde-morpholine method for the conversion of gamma-carboxyglutamyl (Gla) residues to gamma-methyleneglutamyl (gamma-MGlu) residues has been applied to the modification of bovine prothrombin fragment 1. In the absence of Tb3+ ions or at Tb3+ ion concentrations of 2 Km app and 25 Km app the action of 10,000-fold molar excess of formaldehyde and morpholine, pH 5.0, converts the 10 Gla residues of the protein into 10 gamma-MGlu residues. Modification of the protein using the same conditions but increasing the Tb3+ concentration to 100 Km app provided a homogeneous protein containing 3 gamma-MGlu and 7 Gla residues, bovine 3 gamma-MGlu-fragment 1. The modified protein binds the same number of Ca2+ ions (6-7) as bovine fragment 1. However, the positive cooperatively associated with Ca2+ binding is abolished and the overall affinity for Ca2+ ions is reduced. Fluorescence titrations of 3 gamma-MGlu-fragment 1 using either Ca2+ or Mg2+ ions indicate that the modified protein retains a fluorescence quenching behavior similar to that of the native protein. The modified protein does not bind to phosphatidylserine/phosphatidylcholine vesicles in the presence of Ca2+ ions. Thus the metal ion-induced fluorescence transition exhibited by the bovine protein appears to be a necessary but not sufficient condition for phospholipid binding.     

Tb3+ binding to bovine prothrombin and bovine prothrombin fragment 1

The binding of Tb3+ to bovine prothrombin and the amino-terminal 156 residues of prothrombin (F-1) was studied. On the basis of various Tb3+ emission properties, three classes of Tb3+-binding sites were described. The first class contained three high affinity sites in the F-1 region. These sites were filled noncooperatively and were saturated with Tb3+ before the other classes of sites started to fill. Ho3+ quenching of Tb3+ emission showed that these sites were in close proximity to one another (estimated distances 6-12 A). The second class of sites contained three lower affinity sites, also in the F-1 region. These sites bound Tb3+ in a stoichiometric manner and saturated prior to metal binding to the final class of sites. The number of protein ligands binding Tb3+ in the high affinity sites decreased as this second set of sites was filled. Ho3+ quenching of Tb3+ emission suggested that these sites were closely spaced and/or close to the first set of sites. The third class of sites contained 4-6 low affinity sites unique to prothrombin (not in the F-1 region). These sites were not studied extensively, but Tb3+ did not appear to bind stoichiometrically and did not saturate these sites in a manner similar to the other two classes of sites. The emission properties of Tb3+ bound to F-1 were different in KCl versus NaCl containing buffer while the emission properties of Tb3+ bound to prothrombin were not. Optimum conditions for studying lanthanide binding to F-1 (i.e. when Tb3+ bound to F-1 showed emission properties similar to Tb3+ bound to prothrombin) were when F-1 experiments were done at low F-1 concentrations in buffer containing 0.1 M KCl.     

Molecular dynamics simulation of bovine prothrombin fragment 1 in the presence of calcium ions.

Early solvation-induced structural reorganization of calcium prothrombin fragment 1 is simulated with molecular dynamics. Initial coordinates are those of the 2.2-A resolution crystal structure [Soriano-Garcia, M., Padmanabhan, K., de Vos, A. M., & Tulinsky, A. (1992) Biochemistry 31, 2554-2556]. The molecular dynamics code AMBER, appropriately modified to include long-range (less than or equal to 22.0 A) ionic forces, was employed. The solution structure appears to equilibrate within 100 ps. Although minor changes are seen in various structural domains, the early solution structure basically maintains an intricate network of nine gamma-carboxyglutamic acid (Gla) residues encapsulating seven calcium ions. However, the Gla domain moves with respect to the kringle domain. This motion is mainly due to the movement of Ser34-Leu35 that appears to be a flexible hinge between the domains. The N-terminus of Ala 1 is in a tightly bound complex with three Gla residues that remains stable in the solution structure when the long-range electrostatic cutoff is employed and the near planar alignment of the seven calcium ions is only slightly distorted. The simulation structure is discussed in terms of experiments that studied calcium ion-induced quenching of the intrinsic fluorescence, protection of the N-terminal amino group from acetylation by calcium ions, chemical modification of the N-terminus to a trinitrophenyl derivative, and the possibility of a calcium-binding site(s) in the kringle domain.     

The effects of calcium ions and pH on bovine prothrombin fragment 1. Intrinsic fluroescence studies.

The effects of pH and Ca2+ on the intrinsic fluorescence of bovine prothrombin fragment 1 were investigated to deduce the nature of protein functional groups involved in Ca2+ binding to fragment 1. From pH values of 9 to 3, increasing the H3O+ concentration results in quenching of the fluorescence of fragment 1. Reversible pH-titration curves are obtained which appear to consist of two regions. From pH 4 to pH6.5 a broad titration curve is obtained, whereas from pH6.5 to 9 a more pronounced titration behaviour is evidenced by a group or groups on fragment 1 with an apparent pKa of approx. 7.5. In contrast, the apparent association constant for Ca2+ and fragment 1 shows a sharp pH-dependence in the region between pH7 and 8 with tighter Ca2+ binding at higher pH values. A PKa of approx. 7.5 can be estimated for the group or groups on fragment 1 linked to the tight binding of Ca2+. Both H3O+ and Ca2+ result in blue-shifts in the wave-lengths of fragment-1 emission. These results are interpreted in terms of H+ - and Ca2+ - induced changes in the conformation of fragment 1 as a result of surface-charge neutralization.     

The effect of divalent metal ions on the electrophoretic mobility of bovine prothrombin and bovine prothrombin fragment 1

Examination of metal ion-dependent effects on the electrophoretic mobility of bovine prothrombin and fragment 1 provides a useful and sensitive method for investigation of conformational processes in these proteins. Utilization of this method reveals a conformational change in bovine prothrombin and fragment 1 which occurs at low metal ion concentrations. Equilibrium dialysis studies indicate that the metal ion-induced shape change occurs concomitant with binding of a single calcium ion/molecule of prothrombin or fragment 1. Mixed metal electrophoretic mobility studies with Mg2+ and Ca2+ have demonstrated the "synergistic" effect for fragment 1 observed by others. Mixed metal equilibrium dialysis has provided experimental support for this observation and allows us to conclude that two tight Ca2+ sites are not affected by low Mg2+ concentrations and that the third Ca2+ site is also a tight site for Mg2+. Thus, at low Mg2+ concentrations and upon the addition of Ca2+, there are effectively three tight sites; consequently more Ca2+ will bind to the protein at lower total Ca2+ ion concentrations.     

Homo- and heterodimer formation with prothrombin and prothrombin fragment 1 in the presence of calcium ions

The purpose of the current study is to present further evidence for prothrombin self-association as assessed by chemical crosslinking. When the self-association (evaluated by covalent crosslinking with dithiobis(succinimidylpropionate) of prothrombin or fragment 1 was evaluated at the same molar concentration of protein, similar rates of dimer formation were observed for either protein. When prothrombin and fragment 1 were incubated together with the crosslinking reagent and calcium ions, a heterodimer consisting of prothrombin and fragment 1 was observed in addition to prothrombin dimer and fragment 1 dimer. Similar experiments with prethrombin 1 showed neither significant self-association nor effect on prothrombin self-association. Comparison of the formation of prothrombin fragment 1 heterodimer formation with the effect of fragment 1 on prothrombin activation by factor Xa suggests that the anticoagulant activity of fragment 1 is not solely a result of the formation of a heterodimer between prothrombin and fragment 1.     

Decarboxylation of bovine prothrombin fragment 1 and prothrombin

Bovine prothrombin fragment 1 and prothrombin undergo decarboxylation of their gamma-carboxyglutamic acid residues when the lyophilized proteins are heated in vacuo at 110 degrees C for several hours. The fully decarboxylated fragment 1 product has lost its barium-binding ability as well as the calcium-binding function which causes fluorescence quenching in the presence of 2 mM Ca2+. There is no sign of secondary structure alteration in solution upon analysis by fluorescence emission and circular dichroic spectroscopy. A family of partially decarboxylated fragment 1 species generated by heating for shorter periods shows that the initial decrease in calcium-binding ability occurs almost twice as rapidly as the loss of gamma-carboxyglutamic acid. This is consistent with the idea that differential functions can be ascribed to the 10 gamma-carboxyglutamic acid residues in fragment 1, including both high- and low-affinity metal ion binding sites. Prothrombin itself also undergoes total decarboxylation without any apparent alteration in secondary structure. However, in this case the latent thrombin activity is progressively diminished during the heating process in terms of both clotting activity and hydrolysis of the amide substrate H-D-Phe-Pip-Arg-pNA. The present results indicate that in vitro decarboxylation of gamma-carboxyglutamic acid in dried proteins is useful for analyzing the detailed calcium-binding proteins of vitamin K dependent coagulation factors.     

Preliminary X-ray investigation of fragment 1 of bovine prothrombin

Crystals of fragment 1 of bovine prothrombin grown from phosphate at pH 7.5 are tetragonal, space group P4₁2₂2 or P4₃2₁2 with $\text{a = b = 79.5}\text{A}\text{?}\text{, c = 84.9}\text{A}\text{?}$, with probably one molecule of 22,000 molecular weight in the asymmetric unit. The presence of 17.5% carbohydrate in the fragment may account for the high liquid content (60%) of the crystals.     

Fourier transform infrared spectroscopic study of Ca2+ and membrane-induced secondary structural changes in bovine prothrombin and prothrombin fragment 1.

Fourier transform infrared (FTIR) spectroscopy was used to monitor secondary structural changes associated with binding of bovine prothrombin and prothrombin fragment 1 to acidic lipid membranes. Prothrombin and prothrombin fragment 1 were examined under four different conditions: in the presence of (a) Na2EDTA, (b) 5 mM CaCl2, and in the presence of CaCl2 plus membranes containing 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with either (c) bovine brain phosphatidyl-serine (bovPS) or (d) 1,2-dioleoyl-phosphatidylglycerol (DOPG). The widely reported Ca(2+)-induced conformational change in bovine prothrombin fragment 1 was properly detected by our procedures, although Ca(2+)-induced changes in whole prothrombin spectra were too small to be reliably interpreted. Binding of prothrombin in the presence of Ca2+ to procoagulant POPC/bovPS small unilamellar vesicles produced an increase in ordered secondary structures (2% and 3% increases in alpha-helix and beta-sheet, respectively) and a decrease of random structure (5%) as revealed by spectral analysis on both the original and Fourier-self-deconvolved data and by difference spectroscopy with the undeconvolved spectra. Binding to POPC/DOPG membranes, which are less active as procoagulant membranes, produced no detectable changes in secondary structure. In addition, no change in prothrombin fragment 1 secondary structure was detectable upon binding to either POPC/bovPS or POPC/DOPG membranes. This indicates that a membrane-induced conformational change occurs in prothrombin in the nonmembrane-binding portion of the molecule, part of which is activated to form thrombin, rather than in the membrane-binding fragment 1 region. The possible significance of this conformational change is discussed in terms of differences between the procoagulant activities of different acidic lipid membranes.     

A metal ion-binding site in the kringle region of bovine prothrombin fragment 1.

45Ca(II) binding studies (equilibrium dialysis) on the kringle domain of bovine prothrombin fragment 1 were conducted using a mixture of peptides (residues 43-156 and 46-156) resulting from limited alpha-chymotryptic hydrolysis of fragment 1. Analysis of the Scatchard plot of these data indicates a single, low affinity Ca(II)-binding site to be present. Similar results were obtained from studies on the decarboxylated fragment 1 derivative, 10-gamma-MGlu-fragment 1. Acetylation of bovine fragment 1 in the absence of Ca(II) or Mg(II) ions results in the loss of the metal ion-promoted quenching of the intrinsic Trp fluorescence of the protein and the Ca(II)-mediated binding to phosphatidylserine/phosphatidylcholine (PS/PC) vesicles. The acetylation of the NH2 alpha-group of Ala-1 has been shown (Welsch, D. J., and Nelsestuen, G. L. (1988) Biochemistry 27, 4946-4952) to abolish the PS/PC binding property of fragment 1. The present study demonstrates that acetylation of a second site possibly Ser-79 or Thr-81 using the conditions described in the preceding paper results in loss of both the fluorescence transition and the Ca(II)-mediated PS/PC binding of the resulting protein derivative. Removal of the O-acetyl group at the Ser-79/Thr-81 site is accomplished by aminolysis with 0.2 M hydroxylamine, pH 10, 50 degrees C; the fluorescence transition is partially restored. PS/PC binding is partially restored if the NH2 alpha-group of Ala-1 is trinitrophenylated but is not restored if the NH2 alpha-group of Ala-1 is acetylated. We conclude that the Ser-79/Thr-81 site may represent a portion of the metal ion-binding site within the kringle domain of fragment 1. Occupancy of this site by a Ca(II) ion appears to be important in the binding of the protein to PS/PC vesicles.     

Effect of bovine prothrombin fragment 1 on the translational diffusion of phospholipids in Langmuir-Blodgett monolayers.

Previous work has shown that bovine prothrombin fragment 1 binds to supported planar membranes composed of phosphatidylcholine and phosphatidylserine in a Ca(2+)-specific manner (Tendian et al. (1991) Biochemistry 30, 10991; Pearce et al. (1992) Biochemistry 31, 5983-5995). In the present work, fluorescence pattern photobleaching recovery has been used to examine the effect of membrane-bound fragment 1 on the translational diffusion coefficients of two fluorescent phospholipids in fluid-like phosphatidylserine/phosphatidylcholine Langmuir-Blodgett monolayers. The results show that saturating concentrations of fragment 1, in the presence of Ca2+, reduce the diffusion coefficient of nitrobenzoxadiazolyl-conjugated phosphatidylserine (NBD-PS) and nitrobenzoxadiazolyl-conjugated phosphatidylcholine (NBD-PC) by factors of approximately four and two, respectively. Ca2+ or fragment 1 alone do not have a statistically significant effect on NBD-PS or NBD-PC diffusion. In addition, a nonspecific protein (ovalbumin) does not change the diffusion coefficients of the fluorescent phospholipids either in the absence or presence of Ca2+. The fractions of the fluorescent phospholipids that are laterally mobile are approximately 0.9 for all samples. These results are interpreted with several models for possible mechanisms by which extrinsically bound proteins might retard phospholipid diffusion in membranes.     

Structure of bovine prothrombin fragment 1 refined at 2.25 A resolution.

The structure of bovine prothrombin fragment 1 has been refined at 2.25 A resolution using high resolution measurements made with the synchrotron beam at CHESS. The synchrotron data were collected photographically by oscillation methods (R-merge = 0.08). These were combined with lower order diffractometer data for refinement purposes. The structure was refined using restrained least-squares methods with the program PROLSQ to a crystallographic R-value of 0.175. The structure includes 105 water molecules with occupancies of greater than 0.6. The first 35 residues (Ala1-Leu35) of the N-terminal gamma-carboxy glutamic acid-domain (Ala1-Cys48) of fragment 1 are disordered as are two carbohydrate chains of Mr approximately 5000; the latter two combine to render 40% of the structure disordered. The folding of the kringle of fragment 1 is related to the close intramolecular contact between the inner loop disulfide groups. Half of the conserved sequence of the kringle forms an inner core surrounding these disulfide groups. The remainder of the sequence conservation is associated with the many turns of the main chain. The Pro95 residue of the kringle has a cis conformation and Tyr74 is ordered in fragment 1, although nuclear magnetic resonance studies indicate that the comparable residue of plasminogen kringle 4 has two positions. Surface accessibility calculations indicate that none of the disulfide groups of fragment 1 is accessible to solvent.     

Kinetic and equilibrium metal-ion-binding behaviour reflected in a metal-ion-dependent antigenic determinant in bovine prothrombin. Comparison with bovine prothrombin fragment 1.

Rabbit anti-(bovine prothrombin fragment 1) antibodies were fractionated by using fragment-1 affinity chromatography in the absence of metal ions, and showed an absolute requirement for the presence of metal ions in their interactions with bovine fragment 1 or prothrombin. These antibodies were employed to evaluate both the rate constants for a protein conformation change and the equilibrium metal-ion binding to isolated bovine fragment 1 and intact prothrombin. The close similarity of the rates obtained for the conformation change in fragment 1 and those observed in prothrombin indicated that the same process is involved in both proteins and that the non-fragment-1 region of the prothrombin has essentially no effect on this process in the fragment-1 region. Equilibrium metal-ion-binding studies indicate that the details of the metal-ion-binding process in fragment 1 and prothrombin are essentially the same. We conclude that the metal-ion-binding behaviour of the fragment-1 domain of intact prothrombin is identical with that of isolated fragment 1.     

Chemical modification of prothrombin fragment 1: documentation of sequential, two-stage loss of protein function

The amino groups of prothrombin fragment 1 (amino acids 1-156 of prothrombin) were derivatized by acetylation, amidination, and reductive methylation. Conditions that caused complete acetylation of protein amino groups produced a fragment 1 derivative which no longer displayed a metal ion dependent intrinsic fluorescence change and had lost its membrane binding capability as well. However, when derivatized in the presence of calcium ions, extensive acetylation yielded a product that underwent protein fluorescence quenching at metal ion concentrations similar to those observed for the native protein. This derivative bound to membranes in a calcium-dependent manner with only a small reduction in affinity. Several results showed the existence of a partially functional protein that was characterized by a high degree of calcium-dependent protein fluorescence quenching but which had a requirement for 10-fold higher calcium concentration. This derivative was produced by partial acetylation (greater than 3 equiv) of metal-free protein. This partially acetylated protein had greatly diminished membrane binding. The calcium-protected amino group, therefore, was among the most reactive acetylation sites in the metal-free protein. The second site, responsible for abolishing all metal ion induced fluorescence change, was resistant to acetylation and became derivatized at the last stages of amino group acetylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Luminescence studies on RbI:Tb3+ crystals.

Optical absorption, photoluminescence, thermoluminescence (TL) and photostimulated luminescence (PSL) studies on RbI:Tb(3+) crystals irradiated with gamma-rays is reported. Photoluminescence of these crystals exhibits characteristic Tb(3+) emissions, due to transitions from the (5)D(3) and (5)D(4) levels to various levels of the (7)F septet. On F-bleaching the gamma-irradiated crystals, Z(3) centres are observed. The TL glow curve indicates a two-step thermal annihilation process for the radiatively created defects. The presence of the characteristic emissions due to terbium ions in the photostimulation at the F-band, and TL emissions under both glow peaks, confirm the participation of Tb ions in the defect production and recombination processes. Trap parameters for the TL process are calculated and presented. The low temperature glow peak is attributable to Z(3) centres.     

The N-terminal activation fragment of bovine prothrombin. Immunological studies leading to a one step purification.

Some immunological studies on prothrombin fragment 1 from bovine prothrombin and its warfarin-induced precursor acarboxyprothrombin are reported. Based on the results, a rapid and simple immunoadsorption method for the isolation of prothrombin fragment 1 in good yield has been established. The method exploits the conformational change induced in the fragment by removal of Ca2+. The principle may be applicable to other gamma-carboxyglutamyl-containing proteins or fragments therof.     

Modifications of bovine prothrombin fragment 1 in the presence and absence of Ca(II) ions. Loss of positive cooperativity in Ca(II) ion binding for the modified proteins.

Chemical modification of bovine prothrombin fragment 1 according to the procedure of D. J. Welsch and G. L. Nelsestuen (1988) [Biochemistry 27, 4946-4952 and ealier papers] provided a series of fragment 1 derivatives in which various nitrogen-containing side chains were N-acetylated and/or N-2,4,6-trinitrophenylated. In addition the des-[Ala-1,Asn-2]- and des-[Ala-1,Asn-2,Lys-3]-fragment 1 derivatives were prepared by limited enzymatic hydrolysis of fragment 1 using cathepsin C and plasmin, respectively. Quantitative studies on the Ca(II) binding of these proteins have been accomplished using 45Ca(II) equilibrium dialysis. Binding of these fragment 1 derivatives to phosphatidylserine/phosphatidylcholine (PS/PC) vesicles (25:75) in the presence of Ca(II) ions has been studied using the light-scattering technique. Acylation of the 5 lysine residues of fragment 1 by the action of acetic anhydride (500-fold molar excess) in the presence of 75 mM Ca(II), pH 8.0, results in loss of positive cooperativity in Ca(II) binding (Scatchard plot) and an increase in the number of Ca(II) ions bound. The Ca(II)-dependent PS/PC binding of the acylated protein is reduced. Removal of 2 and 3 residues from the amino terminus likewise leads to loss of positive cooperativity in Ca(II) binding and reduced binding affinity to PS/PC vesicles. The important role of the amino-terminal 1-10 sequence is discussed. We conclude that positive cooperativity in Ca(II) binding is not a prerequisite for the Ca(II)-dependent binding of bovine prothrombin fragment 1 to PS/PC vesicles.     

Chemical studies on the isolated collagen-like and globular fragment of complement component C1q. Comparative studies on bovine and human C1q

Both the collagen-like and the globular fragments of a subcomponent C1q of the first component of bovine and human complement were highly purified by enzymic digestion followed by gel filtration. Analyses by polyacrylamide gel electrophoresis showed that the former was composed of covalently linked peptide chains with an average molecular weight of 14 000, and that the latter was composed of three non-covalently linked peptide chains each having a molecular weight of approximately 15 000. Great similarities between amino acid compositions of the globular fragments and some similarities between those of the collagen-like fragments were found. Moreover, great similarities of amino acid compositions were found among three non-covalently linked chains of each globular fragment as well as between the corresponding chains of both globular fragments. These results suggested that both the collagen-like and the globular domains on the C1q molecule remained highly conserved in its evolution.