Chemical Modification of Bovine Prothrombin Fragment 1 in the ...

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Sep 26, 1985 - General Hospital, Cleveland, Ohio 44109. The formaldehyde-morpholine .... available from Waverly Press. within approximately 4 h at 37 "C.
THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc

Vol. 261, No. 23, Issue of August 15, pp. 10598-10605,1386 Printed in U.S A .

Chemical Modificationof Bovine Prothrombin Fragment1 in the Presence of Tb3+Ions* (Received for publication, September 26, 1985)

Steven F.Wright$, Pola BerkowitzS, David W. Deerfield, II$, Patricia A. Byrd$, Dean L. Olson$, Richard S. Larsonz, Gregory C. Hinn$, Karl A. Koehlers, Lee G. PedersenS, and RichardG. Hiskey$ From the $Department of Chemistry, The Universityof North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514 and the §Departments of Biochemistry and Surgery, Case Western Reserve University School of Medicine, Cleveland Metropolitan General Hospital, Cleveland, Ohio 44109

The formaldehyde-morpholine method for the conversion of y-carboxyglutamyl (Gla) residues to y-methyleneglutamyl (y-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 K Z p and 25 K Z p 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 y-MGlu residues. Modification of the protein using the same conditions but increasing the Tb3+concentration to 100 K Z p provided a homogeneous protein containing 3 yMGlu and 7 Gla residues, bovine 3 y-MGlu-fragment 1. The modified protein binds the same number of Ca2+ ions (6-7) as bovine fragment 1. However, the positive cooperativity associated with Ca2+binding is abolished and the overall affinityfor Ca2+ions is reduced. Fluorescence titrations of 3 y-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.

ion specificity for protein conformational processes, typified by fluorescence quenching and phospholipid binding processes. Whilethis may be aconsequence of the fact that phospholipidbinding is themostsensitivecriterion of a correct phospholipid binding conformation of the protein, such considerations have led to models involving two classes of metal ion-binding sites on the protein. One class exhibits low cation selectivity and is intimately involved with the protein conformational response to metal ion binding. The second class of metal ion-binding sitesshows high selectivity forCa2+ ions and is involved in the phospholipid-binding process. Reporting differences in the metal ion concentration dependence of the fragment l fluorescence quenching process and self-associative processes involving fragment I, Prendergast et al. (3) and Bloom and Mann (4) similarly have suggesteda model involving two classes of metal ion-binding sites on prothrombin fragment 1. The first two metal ionbinding sites are believed to be nonselective for metal ions but capable of binding Ca2+ ions with high affinity (halfmaximal effect at 0.2 mM) and with positive cooperativity. It is suggested the two high affinity sites will accommodate a variety of metal ions.Filling the high affinitysiteswith various metal ions leads to a conformational change in the protein which is expressed by quenching of intrinsic Trp fluorescence. The second class of sites hasa lower affinity for Ca2+ ions (half-maximaleffect a t 1 mM) but binds Ca2+ ions relatively specifically. Phospholipid binding is believed to be In an effort to deduce the structural and the functional roles played by metal ions in the interaction of prothrombin a consequence of Ca2+ion binding to thelower affinity sites. Stronger evidenceforclasses of metalion-bindingsites with phospholipid surfaces, a number of laboratories have derives from work examining metal ion competition. Several attempted to correlate prothrombin and prothrombin fragment 1 spectroscopic data obtained in the presence of metal studies will be noted here. Prendergast and Mann(5)observed ions with functional properties such asphospholipid binding synergistic effects of Mg2+ ions on the Ca2+ ion dependence of prothrombinactivation by an in uitro prothrombinase or thrombin generation. While such attempts presume that system. These authors conclude that these data are consistent the interaction of prothrombin with phospholipidsurfaces with the notion that M$+ occupies one class of binding sites reflects in uiuocoagulation processes it is generally accepted that at leasttwo classes of metal ion-binding sites exist in theon prothrombin fragment 1, thereby exposing a second class of Mg2+ of sites. Wei et al. (6) have observed that the presence amino-terminal Gla-containing region of prothrombin. It is ions significantly affects the rateof dissociation of prothromfurther believed that metal ion binding to these sites has distinct structural and functional consequences for the pro- bin from the surface of phospholipid vesicles as a result of Ca2+ ion chelation. The metal ion NMR studies of Marsh et thrombin molecule. The evidence for such conclusions can be generally sum- al. (7) examined CaZ+/M$+ competition.Thisstudyalso marized. Nelsestuen et al. (1,2) have noted thediffering metal indicated theexistence of at least two types of sites at which either CaZ+or M$+ can bind. There also exist Mg2+-binding * This investigation was supported by Grants HL-20161 (R. G. H.), sitesto which Ca2+has difficultaccess or whichpossess HL-27995 (L. G . P.), and HL-32159 (K. A. K.) from the National striking differential affinity for Ca2+or M$+ ions. Deerfield Institutes of Health, United States Public Health Service. The costs et al. (8)have recently studied the effects of Ca2+ and M$+ of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- ions on the electrophoretic mobility of bovine prothrombin tisernent” in accordance with 18U.S.C. Section 1734 solelyto indicate and prothrombin fragment 1. The equilibrium dialysis data associated with these studies indicated that six or seven Ca2+ this fact.

10598

Chemical Modification Bovine Prothrombin of Fragment

1

10599

within approximately 4 h at 37 "C. Given these findings, analytical scale reactions were conducted for 5 h at 37 "C, pH 5.0, followedby adjustment of the pH to 7.4 to promote fragmentation of the Mannich base by loss of carbon dioxide and morpholine. These modifications of the conditions previously reported (15) routinely provide a protein sample containing 9-10 14Cincorporations/fragment 1 molecule. Amino acid analysis of fragment 1 modified under these conditions indicated that 9-10 Gla residues had been converted to ymethylene glutamic acid (y-MGlu) residues, an indication of the high specificity of the modification reaction. SDS-PAGE, non-denaturing PAGE, and size-exclusion HPLC indicated that the protein was homogeneous (data not shown). Upon addition of 10 mM Ca2+the intrinsic Trp fluorescence of the 9-10 y-MGlu-fragment 1was quenched 5%;the overall recovery of the modified protein was 66%. The effect of pH on the fluorescence intensity of bovine fragment 1 and the effect of pH on the Ca2+-inducedfluorescence quenching has been examined (23). The pHdependence of Ca2+quenching indicated an apparent association constant for Ca2+binding to fragment 1, assuming that K, equals 1/ [Ca"], at half-maximal quenching. A plot ofK, uersus pH showed a sharp pH dependence between pH 7 and 8. The apparent KOchanged from approximately 300 M" at pH 5.0 to 9500 M" at pH 8. These observations suggested that very high Ca2+ concentrationswould be required for even minimal Ca2+/fragment 1 interaction under the pH 5 conditions required for the Gla modification reaction. Tb3+ions associate more effectively than Ca2+ ions with fragment 1; the T,,, for the Tb3+ fluorescence transition is approximately 1.8 pM at of approximately 0.2 mM. We pH 6.5 compared with the EXPERIMENTAL PROCEDURES~ reasoned that Gla modification reactions at pH 5.0 in the presence of Tb3+ rather than Ca2+would be more likely to RESULTS reflect the effect of the bound metal ions. Preliminary modification experiments on fragment 1 using Modification Conditions-Price et al. (22) studied the effect [14C]formaldehyde and morpholine (10,000-fold molar exof pH on the rate of y-proton exchange of Gla residues. 5.0, 5-h reaction time, and various Tb3+concencess), at pH Identical tritium exchange rates were observed at pH values of 1.5 and 3.1. The values bracket the first ionization constant trations indicated that essentially complete Gla modification (pKl = 2.0) of the malonyl side chain. A 10-fold decrease in (8-10 14C incorporations/fragment 1 molecule) resulted at exchange rate was observed at pH 5.4 (pK2 = 4.4); however, Tb3+ concentrationsof 2 Kzpand 25 Pzp.Increasing the Tb3+ (molar Tb3+/fragment ratios, at pH 7.4 a 200-fold decrease in exchange rate was observed. concentration to 100-300 13:l and 38:1, respectively) yielded fragment 1 preparations These data suggested that the optimum pH for the reaction of the enolate of a Gla-containing polypeptide with the imi- containing from 3-6 14Cincorporations/molecule. Examination of the pooled protein fraction obtained from nium ion derived from formaldehyde and morpholine (15) would lie in the range of pH 5-6. Preliminary experiments a modification reaction (0.33 mg of fragment l/ml) conducted using bovine fragment 1, [14C]formaldehyde,and morpholine using a molar ratio of Tb3+/fragment1 of 3 8 1 and containing at pHvalues ranging from pH 5-7 confirmed this expectation. 5.3 incorporations of 14C/fragment 1 molecule, by size excluNo 14Cincorporation into fragment 1 occurred at pH 7.0, and sion HPLC (TSK-250 column, 0.02 M Tris. HC1, 0.1 M NaC1, pH 7.4) revealed the modified protein was not homogeneous maximum incorporation was obtained at pH 5.0. With the pH optimum of the modification process estab- (data not shown). Studies of the pooled fraction on SDSlished, we examined conditions for the modification of frag- PAGE using standards of known molecular weight for comment 1 in the absence of metal ions. At pH 5.0 using a 10,OOO:l parison suggested the fraction consisted of monomer, dimer, molar ratio of ['4C]formaldehyde/morpholine to bovine frag- and traces of trimer. Chromatography of the multimer mixment 1, maximum levels of14C incorporation were reached ture on Sephadex G-75 produced an elution profile (data not shown) exhibiting two majorpeaks corresponding to monomer The abbreviations used are: Gla, 4-carboxyglutamicacid; y-MGlu, and dimer/trimer. Each peak contained 14C incorporations; y-methylene glutamic acid; SDS, sodium dodecyl sulfate; PAGE, the monomer fraction contained approximately five I 4 C inpolyacrylamide gel electrophoresis; HPLC, high pressure liquid chrocorporations/fragment 1 molecule, and thedimer/trimer fracmatography. Portions of this paper (including "Experimental Procedures," tion contained approximately four 14C incorporations/fragFigs. 2-4, and Table I) are presented in miniprint at the end of this ment 1 molecule. paper. Miniprint is easily read with the aid of a standard magnifying Using these analytical scale modification experiments as glass. Full size photocopies are available from the Journal of Biolog- guidelines, chemical modification of bovine fragment 1 on a ical Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 85M-3239, cite the authors, and include a check or preparative scale in the presence of 100 K%p Tb3+was exammoney order for $3.60 per set of photocopies. Full size photocopies ined. Modification studies were performed at a fragment 1 are also included in the microfilm edition of the Journal that is concentration of 0.025 PM using Tb3+/fragment 1molar ratios available from Waverly Press. of 22.2 and 11.1, respectively. In these preparative experiion-binding sites are present in fragment 1, in agreement with earlierstudies (2, 5,9). Mixed metal equilibrium dialysis studies indicated that the two high affinity Ca2+ion binding sites are not affected bylow concentrations of Mg2+ ions. Competition between Ca2+and M P ions for a third sitewas suggested as an explanation of the Ca2+ ion-binding data. Finally, Bajaj et al. (9) observed that 6 of 10 Gla residues were, in effect, "protected from thermal decarboxylation when human prothrombin was first incubated with 0.2 mM Ca2+ions. Thus, the metal ion competition and protection studies noted here suggest the existence of distinguishable "classes" of metal ion-binding sites on bovine prothrombin fragment 1. Whether it is possible to attribute function to such sites is a matter deserving further consideration. In this report we describe experiments designed to utilize metal ion binding to bovine prothrombin fragment 1 as means of modulating the accessibility of Gla' residues in theprotein to chemical modification. In view of the extensive evidence supporting the hypothesis that at least two classes of sites exist, it appears likely that, under appropriate conditions, metal ion binding would preferentially protect the Gla residues in one class of sites from chemical modification. Lanthanide ion binding to prothrombin and prothrombin fragment 1 has been demonstrated to occur with approximately 3 orders of magnitude higher affinity than that observed for calcium ion binding (10-14). Thus, we have chosen to investigate modulation of Gla reactivity by Tb3+ ions by utilizing the Gla-specific formaldehyde-morpholine Mannich base fragmentation method that we developed for modification of solutions of Gla-containing peptides and proteins(15).

Pzp

Chemical Modification of Bovine Prothrombin Fragment 1

10600

mentsthe Tb3+concentration was decreased in order to prevent protein precipitation. Identical resultswere obtained from either Tb3+/fragment molar ratio. Purification of the modified fragment 1 was accomplished using a single chromatographic separation on SephadexG-75 (Fig. 1).The modified dimer/trimer fraction was cleanly separated from the monomer fraction using a Tris.HC1 buffer, pH 7.4. Pooled fractions of the modified fragment 1monomer were examined by analytical size exclusion HPLC (TSK-250 column, 0.02 M Tris.HC1, 0.1 M NaCl, pH 7.4) and were homogeneous (not shown). Gel electrophoresis on nondenaturing PAGE (Fig. 1, inset) showed only the modified monomer. The optimized modification reactionconditions have been established as: Tb3+/fragment 1molar ratio, 11:l; formaldehyde-morpholine/ fragment 1 ratio, 10,OOO:l; pH 5.0; 8-h reaction time; adjustment of pH to 7.4 and chromatography on Sephadex G-75. Under these conditions a modified protein, 3 y-MGlu-fragment 1, is obtained in 30% yield. Amino acid analysis of the modified protein obtained under these conditions is given in Table I. Gla content of the modified protein was determined by separate determinations using alkaline hydrolysis (18). Gla analyses indicated that three Gla residues were converted to y-MGlu residues. Since y-MGlu does not yield a detectablefluorescence product with o-phenanthroline/P-mercaptoethanol(l5,17) the presence of these residues could not be quantitated. y-MGlu yields a ninhydrin peakin the standard Spackmann et al. system (19). Analysis of the modified protein using the ninhydrin system did not separate the y-MGlu and Gly peaks. The increased area of the Gly peak is consistent with the presence of yMGlu as a hydrolysis product. Properties of 3 y-MGlu-Fragment 1-The properties of the 3 y-MGlu-fragment 1 obtained by the chemical modification procedure described have been examined by avariety of methods. These include: fluorescence spectroscopy, Ca2+binding studies using 45Ca2+equilibrium dialysis, electrophoretic mobility in the presence of Ca2+ ions, and binding to phos-

025 0.20 0

U

0.15

phatidylserine/phosphatidylcholinephospholipid vesicles in the presence of Ca2+ions. Intrinsic Fluorescence Changes-In these studieswe utilized an excitation wavelength of 290nm andrecorded the emission a t 340 nm in the presence of 10 mM Ca2+added to bovine fragment 1 and 3 y-MGlu-fragment 1 (Fig. 2). Quenching of fragment 1was 50% under these conditions; the quenching of 3 y-MGlu-fragment 1 was 46%. Fragment 1 modified in the absence of Tb3+and containing 9-10 y-MGlu residues exhibited a fluorescence quench of 5%. Fig. 2 indicates that comparable equilibrium quenching values are reached by both fragment 1 and 3 y-MGlu-fragment 1 and thatboth proteins exhibit the biphasic fluorescence quenching curve typical of the bovine protein. Hill plots of the equilibrium Ca2+and the M$+ fluorescence titrations of 3y-MGlu-fragment 1 are shown in Fig. 3. The modified protein was studied at pH 7.4 at Ca2+and M e concentrations from 0.1 to 50 mM (Table 11). Protein concentrations of 1,5, and10 pM were examined; no dependence of the Hill plot slopes on protein concentration was observed. The average T,,, of the Ca2+fluorescence titrations of 3 y-MGlu-fragment 1was 1.8 f 0.2 mM, Hill coefficient (half-response), n = 1.4 f 0.1. This is in sharp contrast to the e2+ value of 0.19 mM reported by Bioom and Mann (4), a value of0.20 mM obtained in our earlier studies on bovine fragment 1 (7) and the average value of 0.34 f 0.09 mM (Table 11). Thus, Ca2+binding to 3 y-MGlu-fragment 1 as represented by the T,,, value of the fluorescence titration has decreased by a factor of 5-10. The Hill coefficients reported for Ca2+ fluorescence titration of bovine fragment 1 are n = 2.4 (4), n = 1.3 (7), and1.6 f 0.3 (Table 11). In contrast to the experiments with Ca2+,the Hill plot of the data from the fluorescence titration of the modified ro tein using Mg2' ion (Fig. 3) exhibited an average 0.40 f 0.03 mM, n = 1.4 (Table 11).These values are comparable to thosereported byBloom and Mann (4), Ep2+ = 0.45mM, and by Nelsestuen et al. (2), T??' = 0.43 mM for bovine fragment 1. These data suggest that the site(s) responding to theCa2+-promotedfluorescence change have been modified but that theoriginal M$+ site has remained intact. A comparison of therate of fluorescence quenching of bovine fragment 1 and 3y-MGlu-fragment 1 was accomplished using Ca2+ and M e ions. The slow fluorescence transition of the biphasic fluorescence quenching curve (Fig. 2) of both proteins was found to follow first-order kinetics. The kinetic measurements were conducted using 5 p~ protein concentrations, 10 mM Ca2+and M$+, in 20 mM Tris buffer, pH 7.4, at 25 "C. The rapid phase of the biphasic equilibrium fluorescence quenching curves (Fig. 2) appear indistinguishable for the two proteins. Similarly the apparent first-order

ep

TABLE I1 T, values (mM) and Hill coefficients for fluoresence titrations of bovine fragment 1 and 3 y-MGlu-fragment I

0.10

O W

Average-

Ca2+ 3 y-MGlu-fragment 1 (n) 20

30

40

50

60 Fraction Number

70

80

90

FIG. 1. Elution profile from Sephadex G-75 (3 X 100-cm column) of bovine fragment 1 (preparative run) modified using 10,000-fold molar excess of formaldehyde/morpholine and 100 K$* Tb3+.Elution was accomplished using 0.02 M Tris. HCl, pH 7.40, buffer. The protein was collected in 3-ml fractions monitored at Am. Inset, gel electrophoresis of 3 y-MGlu-fragment 1. Lane 1, low molecular weight standards; lane 2, fragment 1; lane 3, 3 y-MGlu-fragment 1 (monomer peak); lune 4, modification reaction mixture before chromatography. 5-15% gradient polyacrylamide-SDS gel.

(Tm)

Fragment 1 (n) (T m )

MgZ+ 3 y-MGlu-fragment 1 (n) (Tm)

Fragment 1 (n)

1.4 f 0.2 1.8 f 0.2 1.6 f 0.3 0.34 f 0.09

1.4 f 0.2 0.40 -t 0.04

1.7 f 0.2 0.32 f 0.03 a Average values of determinations for 1,5,and 10 ~ L protein. M (Tm)

Chemical Modification of Bovine Prothrombin Fragment rate constantsobtained from the slow phase of the transition reveal that the3 y-MGlu-fragment 1undergoes the transition at the same rate as is observed with fragment 1 with either Ca2+or Mg2+ ions. Ca2+Binding Studies-The binding of "Ca2+ ions to 3 yMGlu-fragment 1 was studied by equilibrium dialysis. The binding data are shown as a Scatchard plot inFig. 4. Bovine fragment 1 has been demonstrated to bind 6-7 Caz+ions (2, 5 , 8, 9). The Scatchard plot of the dialysis results on bovine fragment 1 (not shown) is concave downward suggesting positive cooperativity in binding of the firsttwo or threeCaZ+ ions. Values of the thermodynamic association constants for binding have been estimated (8)as K1 = 3,455 M-', Kz = 1,104 M-', and K3 = 20,821 M-'. The Scatchard plot of "Ca2+binding to 3 y-MGlu-fragment 1 indicates that modification of the protein hasabolished the positive cooperativity. However, the value of K1 determined by extrapolation from the Scatchard plot as Kl = 2,550 M-' and n = 6-7 remains approximately the same. If an equal equivalent noninteracting site model is assumed, straight line Scatchard behavior is expected. Assuming n = 7 (Fig. 4) we can then compute the site binding constant, k, from

60

10601

1 1

I

54 48

3 42

E 36

O" 30 E

$ 24 2! I 8 12

6 .

.6

.

1.2

118

2.4

-V

3

3.6 4.2 5.4 4.8

6

FIG. 6. Comparison of electrophoretic mobility decrease of bovine fragment 1 (0)and 3 y-MGlu-fragment1 (A) uersw u (Cas+ bound/[proteinJ).Data were obtained from Figs. 4 and 5.

sample migration toward the cathode relative to themigration of bovine serum albumin at thesame Ca2+ion concentration. Fig. 6 shows that bovine fragment 1exhibits a large mobility change when a single Ca2+ion binds. This has been suggested to result from a major change in hydrodynamic friction factor as a result of a conformational change accompanying the binding of a single Ca2+/fragment1molecule (8).Examination thus of the electrophoretic mobility decrease of 3 y-MGlu-fragment 1 (Fig. 6) indicates that this effect is much less pronounced. 7-1+1* K, = k = 2,550 M-'; k = 365 M-'. These data also suggest, in agreement with the *%a2+binding 1 data, that modification has somehow disrupted the initial The overall reduced affinity of the modified protein for Ca2+ binding sites filled at low Ca2+concentrations. observed in the Ca2+fluorescence titration (T,= 1.8 mM) i s Phospholipid Binding Studies-The binding of 3 y-MGlualso apparent from the Scatchard plot of the 45Caz+binding fragment 1 to phospholipid vesicles prepared from a 25:75 data and indicates the modification of one or more of the mixture of bovine phosphatidylserine/l-palmitoyl-2-oleoylCa2+-binding sitesinvolved in the positively cooperative Ca2+ phosphatidylcholine was evaluated. Binding studies were conbinding process. ducted using 5 and 25 mM Caz+ions at pH 7.4. Under these Electrophoretic Mobitity Studies-We have previously re- conditions bovine fragment 1 exhibits a binding constant of ported (8) a study of the decrease in electrophoretic mobility KO= 2.36 X lo6 M" when examined using the light-scattering of bovine fragment 1as afunction of the number of Ca2+ions method described by Nelsestuen and Lim (21). The modified bound at a given Ca2+concentration. A comparison of the protein did not bind to phosphatidylserine/phosphatidylchoelectrophoretic mobility of fragment 1 with 3 y-MGlu-frag- line under these conditions. ment 1 is shown in Fig. 5. A plot of the percentage decrease in electrophoretic mobility versus d (Ca2+bound/protein conDISCUSSION centration) is shown in Fig. 6. The electrophoretic mobility In the absence of added metal ions, the chemical modificadecrease in both Figs. 5 and 6were obtained on nondenaturing of bovine prothrombin fragment 1 with a 10,000-fold tion polyacrylamide gels. Relative mobility is the percentage of molar excess of ['4C]formaldehyde/morpholine at pH 5.0 yields a modified protein containing 9-10 y-methylene glutamic acid residues. In contrast chemical modification in the presence of 100 FZp Tb3+ions provided a homogeneous product which contains three y-methyleneglutamyl residues (i.e. 3 y-MGlu-fragment 1).The binding of Tb3+ions to fragment 1produces a change in the apparent level of chemical modification of the protein, and a specific population of Gla residues seems to have been protected. 244 Nelsestuen et al. (24) demonstrated that bovine prothrombin fragment 1 self-associates in the presence of La3+apparently forming a trimer. Recently Sommerville and Nelsestuen (14)examined Tb3+ binding to fragment 1 at pH 6.5; they observed three Tb3+high affinity sites that were filled noncooperatively. Filling of these sites caused a conformational [c~(II)] change that was detected by a 55% decrease in intrinsic FIG.5. Comparison of electrophoretic mobility decreaseof fluorescence. Thus, the conformational change elicited by bovine fragment 1 ( 0 ) and 3 7-MGlu-fragment 1 (A) in 140 Tb3+is presumed to be the same as that caused by the addition mM Tris.HC1, pH 7.5,6%polyacrylamide gel at various concentrations of Ca" ions (mM).Percentage decrease in mobility is of Ca2+ions. A second class of three lower affinity Tb3+sites the percentage of sample migration (cm) toward the cathode relative was found; Ho3+quenching of T b 3 + emission suggested that to the migration of bovine serum albumin at the same CaZ+concen- these sites were in close proximity to each other and/or the tration. first set of filled sites.

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Chemical Modification of Bovine Prothrombin Fragment

The formaldehyde/mo~holine method for the chemical modification of Gla residues depends on the nucleophilic addition of the y-carbon atom of the Gla side chain to the iminium ion generated from formaldehyde and morpholine. Formation of a chelation bidentate complex between the malonyl side chain and Tb3+ ion (or other bivalent ions) wouldbe expected to reduce the nucleophilicity of the 7carbon atom due to extensive delocalization of the enolate T electrons caused by interaction of T b 3 + with both carboxyl groups. Thus, attackof the enolate onthe iminium ion would be suppressed, and “protection” of the Gla residue by Tb3+ would beobserved. A single Gla carboxylate group interacting with Tb3+ion as a monodentate might be expected to retain a highlevel of y-carbonatom nucleophilicity and should undergo modification by reaction with the formaldehyde/ m o ~ h o l i n iminium e ion. Thus, “protection” of a Gla residue by Tb3+ ions orby divalent metal ions could reflect not only the magnitude of the carboxylate-Tb3+binding constant in the two classes of Tb3+-binding sites(14)but also the geometry of the Tb3+-Gla complex. A distinction between these alternatives cannot be made from data currently available. A model for metal ion binding to bovine fragment 1 is represented by the series of equilibria shown in Scheme I. The A and B forms of bovine fragment 1 represent the equilibrium populations of protein present containing transPro” (A) and cis-ProZ2(B) (25).Slow rate-limiting isomerization of A .+ B ( k )followed by the rapid binding of metal ions (K,-K,) to the cis-ProZ2form of the protein, B, leads to the conformation of the proteinin which fluorescence is quenched. The generation of phospholipid and antibody binding characteristics induced in bovine prothrombin and fragment 1 by metal ions has been shown to follow the same kinetics (1,26). Thus, thephospholipid-binding form of prothrombin probably contains cis-ProZ2. Prendergast and Mann (3, 5) and Bloom and Mann (4) suggested the “two-class-of-sites’’ hypothesis which proposed that filling of the two high affinity siteswith metal ions (Ca2+, Mg2+,Mn2+,Gd3+)was nonselective with regard to themetal ion and resulted in fluorescence transition and circular dichroism spectral changes. The process is positively cooperative with a T,,, of about 0.2-0.4 mM ea2+ions. A second class of three sites of low affinity (T, = 1 mM for Ca2+ions) was suggested by these workers to lead to fragment 1dimerization. These sites are believed to be filled without an associated conformational change. Nelsestuen et al. (24)compared the ion-induced phenomena of fluorescence quenching or circular dichroic spectral changes with protein self-association. They examined the effects of Ca2+,Mg2+,Mn2+, Cd2+, and La3+and concluded that two or three metal ions induce the conformational change and that these sites have higher affinity than the sites causing selfassociation. Nelsestuen et al. (24)noted that the sequence in which the siteswere filled depended on themetal ion, causing overlap or separation of the conformational change as seen by fluorescence quenching and self-association. The physical properties exhibited by 3 y-MGlu-fragment 1 are not entirely consistent with the working model proposed in Scheme I. The 3 y-MGlu protein behaves very much like bovine fragment 1 in terms of equilibrium fluorescence quenching. Both proteins exhibitapproximately 50% quenching of intrinsic fluorescence; both proteins exhibitquenching with either ea2+ or Mg2+ ions. The fluorescence emission k, Kl Kz K3 Kd K6 A=B*BM1=BM2*BM,*BM,*BMs*BMe

SCHEME I

Ke

I

maximum for both proteins is 335 nm in the absence of Caz+ and is shifted to 330 nm for bovine fragment 1 and 332 nm for 3 y-MGlu-fragment 1 in the presence of 10 mM Ca2+.The kinetics of the metal ion-induced fluorescence quenching process are also similar for both proteins. Each protein exhibits the same biphasic kinetic behavior indicating the presence of similar populations of the A and B forms. The apparent first-order rate constants for the slow phase portion of the fluorescence transition are also the same for both proteins with either Ca2+or Mg2+(Table 111). The observed absence of any ea2+-promoted interactionof 3 y-MGlu fragment 1with phosphatidylserine/l-palmitoyl-2oleoylphosphatidylcholine vesicles might, therefore, be interpreted from Scheme I asresulting from loss of the low affinity sites in the modified protein. However, the results of *sCa2+ equilibrium dialysis studies on 3 y-MGlu-fragment 1indicate: (a)the same number of Ca2+ions (6 or 7) bind to themodified protein as bind to bovine fragment 1; ( b )positive cooperativity of Ca2+binding has been lost in the modified protein; (c) the average affinity of the modified protein for the intermediate loading of ea2+ions has been reduced substantially. Thus, the p h o s ~ h o l i binding ~ i ~ ability of the protein has been a ~ ~ ~ h e d without change in the number of Ca2’-binding sites. The loss of three carboxylate groups ( i e . 3 Gla .+ 3 yMGlu) appears to have resulted in modification of the high affinity site(s) with concomitant loss of phospholipid-binding ability without a parallel change in the metal ion-induced fluorescence transition of the modified protein. The fact that a protein form exists which exhibitsCa2+- or M$+-induced fluorescence quenching but not phospholipid binding clearly demonstrates the absence of a direct connection between quenching processes and phospholipid binding. In other words, fluorescence quenching appears to be a necessary but not sufficient condition for phospholipid binding. Deerfield et al. (8) have shown that the binding of a single Ca2+ ion leads to a marked decrease in the electrophoretic mobility of bovine fragment 1. This effect was attributed to a major conformational reorganization which occurs with the binding of one Ca2+ion to theprotein. A similar study of the effect of ea2+ ions on the electrophoretic mobility of 3 yMGlu-fragment 1 (Fig. 6) indicates that theeffect of one Ca2+ ion binding to the modified protein is greatly diminished if not absent. It is tempting to speculate that the binding of a single ea2+ion to bovine fragment 1 establishes the elements of the conformation that are essential for the eventual protein-lipid interaction. Addition of the second and/or third Ca2+ion to sites two and three, established by ea2+binding at the first site, completes the structural reorganization necessary for the protein/phospholipid interaction in the native binding to these would also account for the proteins. ea2+ positive cooperativity observed in the ea2+-bindingprocess. Addition of the remaining three orfour ea2+ions may simply serve to neutralize the charge on the protein sufficiently to permit interaction of the fragment 1-ea2+complex with the TABLEI11 Apparent first-orderrate constants of metal ion-induced fklorescence quenching of bovine fragment 1 and bovine 3 y-MGlu-fragment 1 Protein“

Metal ion

Rate constant

min”

0.29 f 0.02 0.30 f 0.02 MgZ+ 3 y-MGlu-fragment1 Ca2+ 0.24 f 0.02 M g ” L _ . - 0.31 t 0.02 “Measurements were conducted in Tris buffer, pH 7.4, at 25 ”C using a protein concentration of 5 p~ and a metal ion concentration of 10 mM. Fragment 1

Caz+

Chemical ~ o d ~ f i c aoft ~Bovine oProthrombin ~ Fragment

10603

1

and Hiskey, R. G. (1979) J. Biol. Chem. 264, 1026&10275 8. Deerfield, D. W., 11, Berkowitz, P., Olson, D. L., Wells, S., Hoke, R. A., Koehler, K. A., Pedersen, L. G., and Hiskey, R. G. (1986) J. Bwl. Chem. 261,4833-4839 important insight from the current study is that a specific 9. Bajaj, S.P., Price, P. A., and Russell, W. A. (1982) J. Bid. Chem. conformation of the protein generated at low Ca2+concentra257,3726-3731 tions seems to be a prerequisite forrotei in-phosp~lip~ inter- 10. Brittian, H. G., Richardson, F. S., and Martin, R.B. (1976) J. Am. Chem. SOC.98,8255-8260 action. 11. Furie, B. C., Blumenstein, M., and Furie, B. (1979)J. Biol. Chem. Quenching of the intrinsic Trp fluorescence by Ca2+ions is 254,12521-12530 simply a consequence of the addition of the second or third 12. Sarasua, M. M., Scott, M. E., Helpern, J. A., Ten Kortenaar, P. Ca2+ion. In the case of quenching by the addition of other B. W., Boggs, N. T., III, Pedersen, L. G., Koehler, K. A., and Hiskey, R. G. (1980) J. Am. Chem. SOC102, 3413-3419 metal ions the response appears to result from metal ion binding to sites that are distinct from the essential Ca2+ sites 13. Scott, M. E., Sarasua, M. M., Marsh, H. C., Harris, D. L., Hiskey, R. G., and Koehler, K. A. (1980)J. Am. Chem. SOC.102,3413required for eventual phospholipid binding. Implicit in this 3419 argument isthe assumption that theconformation established 14. Sommerville, L. E., and Nelsestuen, G. L. (1985) J. Biol. Chem. by Ca2+binding to thehigh affinity sites is differentby virtue 260,10444-10452 of the geometry of the Ca2+complexes from those generated 15. Wright, S. F., Bourne, C. D., Hoke, R. A., Koehler, K. A., and Hiskey, R. G. (1984) Anal. Bivchem. 139,82-90 by the binding of Mn2+, Mg+, Tb3+, or Eu3+ to the native 16. Mann, K. G. (1976) Methods Enzymot. 45,123-156 protein. It is possible that the three Gla residues that have been 17. Kiapper, D. G. (1982) in Methods of Protein Sequence Analysis (Elzinga, M., ed) pp. 509-515, Humana Press, Clifton, NJ modified in 3 ~ " ~ l u - f r a g m e n t 1comprise portions of several 18. Madar, D. A., Willis, R. A., Koehler, K. A,, and Hiskey, R. G. of the Cat+-binding sites.This result would in fact be antici(1979)Anal. Bwchem. 92,466-471 pated if the high affinity Ca2+ sitesdiffered somewhat from 19. Spackman, D. H., Stein, W. H., andMoore, S. (1958)Anal. Cfrem. 30, 1192-1206 the Tb3+high affinity sites. 20. Chen, P. S., Jr., Toribara, T. V., and Warner, M. (1956) Anal. Chem. 28,1756-1758 REFERENCES 21. Nelsestuen, G. L., and Lim, T.K. (1977) ~ w c ~ 16,4164e m ~ 1. Nelsestuen, G. L. (1976) J. Biol. Chem. 251,5648-5656 4171 2. Nelsestuen G. L., Broderius, M., and Martin, G. (1976) J. Biol. 22. Price, P. A., Williamson, M. K., and Epstein, D. J. (198i) J. Biol. Chem. 251,6886-6893 Chem. 256,1172-1176 3. Prendergast, F. G., Bloom, J., Downing, M. R., and Mann, K. G. 23. Scott, M. E., Koehler, K. A,, and Hiskey, R. G. (1979) Biochem. (19%)) in V i ~ ~ m Ki nM e ~ ~ l i sand m Vitamin K-dependent J. 177.879-886 Proteins (Suttie, J. W.. ed) _VP.- 39-48, University Park Press. 24. Nelsestuen, G. L., Resnick, R.M., Wei, G. J., Pletcher, C. H., Baltimore and Bloomfield, V. A. (1981) 20,351-358 4. Bloom, J. W., and Mann, K. G. (1978) Biochemistry 11, 443025. Marsh, H.C., Scott, M. E., Hiskey, R. G., and Koehler, K. A. 4438 (1979) Biochem. J. 183,513-517 5. Prendergast, F. G., and Mann, K. G. (1977) J. Biol. Chem. 252, 26. Madar, D. A., Hall, T. J., Reisner, H. M., Hiskey, R. G., and 840-850 Koehler, K. A. (1980) J. Biol. Chem. 255,8599-8605 6. Wei, G. J., Bloomfield, V. A., Resnick, R. M., and Nelsestuen, G. 27. Laemmli, U. K. (1970) Nature 227,680-685 28. Malhotra, 0.P., Nesheim, M. E., and Mann, K. G. (1985)J. Biol. L. (1982) Biochemistry 21,1949-1959 7. Marsh, H. C., Robertson, P., Jr., Scott, M.E., Koehler, K. A., Chem. 260,279-287

phospholipid-Ca" surface. The fragment l-Ca'+-phospholipid interaction could occur via Ca2+ bridges, by some sort of hydrophobic interaction, or by a combination of both. The

,

Chemical N o d i f i c a t i o n o f

Supplemental H a t e r i a l To: Bovine P r h r m b i n Fragment 1 In The Presenceof Tbgf Ion8

Steven F. W r i g h t , P o l s s a r k a i t z , Osvid W. D e e r f i e l d . 11, P a t r i c i a A. Byrd, A. Koehler, D u n L. Olson, Richard S. Larson, Gregory C. Hinn,Karl Lee G. Pedersen and Richard G. Hiskey N a t e r i a l s ,l e t h o d s ,

and ExperimentalProcedures

-

A l l chemical6were ofreagentgrade or b e t t e r and Reagents andChemicals w a t e r was d o u b l y d i s t i l l e d and de-ionized. Buffers Were rendered metal ion free by passagethrough a c o l a " packedwithChelex-I00resin(Bio-bd). Norpholine (GoldGrade) was obtained f r w A l d r i e h a n d l p a l d e h y d e (37% aqueous s o l u t i o n s ) v e s obtained f r wN s l l i n c k r o d t . I C1-formaldehyde w a s purchased from NW England Nuclear.Before use. theradioactiveformaldehyde was d i l u t e dt o 5.000 m l (0.2 mCilml) w i t hd i s t i l l e dw a t e r .T e r b i m ( I I 1 ) c a r b o n a t e( u l t r s - p u r e ) n s obtained f r w Alfa. Sephddex 025-150 and were obtained f r w S i p . DUE-cellulose (Type 90) was Sephadex675-150 supplied by S c h l e i c h e r and S c h u e l l . me Koda-Vue gel a t a i n i n g k i t was purchasedfra. Bestmen Kcdek.

-

Bovine P r o t h r m b i nP r a g P e n t Bovine p r o t h r a b i nf r a g m e n t 1 was i a o h t e d " e s s e n t i a l l y as d e s c r i b a d by Hmn (1.6). lhe p r o t e i n e h w r d I s i n g l e b l n d whtn 6ubjeccted t o pcl.ctylamide g e l e l a c t r o p h o r a a i e i n sodiumdodecyl s u l f a t e on g r a d i e n t( 5 - 1 5 t )s l a b si nt h eb u f f e r by L a e m t i (27) andviewed by rod.-vue or CxIwssie B i t s s t a i n i n g . A s i m i l a r~ y y o t . 5 vms v t i l i r e d by Nalhotr. at a l . ( 2 8 )t ou a m i n et h ea c t i v a t i o no f GI.-deficientbovine p r o t h r a b i n . llpLC size-exclurianchrmatagraphywith uv d e t e c t i o n a t 280 m also shovedone peak f o rt h ep r o t e i np r e p a r a t i o n . n t a i n t r i n a i ct r y p t o p h a nf l w r s s c c n c to f Chr p r o t e i n wasqucnehed by 50% upon t h e a d d i t i o n of e a l c i u i o n s (10 mH). Acid- and bare-hydrolyzedsamplesoffragment I warean.lyred forminoacid c o n t e n tb rt h e methodsofKlappar(17) m d Nadar g t a l . (18). P r i o r t o use. fragment I (0.261 mg/ml) wa11 s t o r e d f r o a e n a t -20 C i n a r o l u t i o n of 0.02 W . molacular Tris.RC1, 0.1 N HaGI. 0.02 W e o d i u . a z i d e , pU 7 . 6 0 b u f f e rA weightof 23,500 was w e d f o r bovine fragment 1.

-

UV-Absorbance S p e c t r o p h o t m e q A G i l f o r d model 260 uv-visible spectrophotometer was w e d f o r p r o t e i n q w n t i t s t i o n by uv absorbance. by t h e f o l l o r i n g Absorbancereadings a t 280 w w e r e c o r r e c t e d f o r s c a t t e r i n g equationof Wmn (16).

An e x t i n c t i o n c o e f f i c i e n t o f s c a n t i t a t i o n of f r r m n t I .

1.00 ml/mg'cm

was u s e d f o r s p e c t r o p h o t m e t r i c

-

Nom-denturing Polyacrylamide Gel B l e e t r o p h o r e s i s A Bio-Rad v e r t i c a l s l a b gel a p p a r a t u s was used for a l ~ l e c t r o p h o r e t i c a t u d i e s . G a l s w e r e s t e i n e d w i t h e i t h e r Cwmaa i e Blue or Koda-Vue. E l e c t r o p h o r e t i cm o b i l i t ys t u d i e si n conditions: t h ep r e s e n c e of Ca'* ions were performed u n d e rt h ef o l l w i n g Running gel Stacking gel b u f f e r : 100 mH Tris.HC1. 3% aerylamide, pH 5.5. E l e c t r o p h o r e s i eb u f f e r : 8 mH b u f f e r : 140 M Tris.BC1, 6% a c r y h m i d e , pB 7.5. Tris.HC1, 30 mH HEPES, pH 6.8. SaPpl)+buffer: 50 mI4 Tris.HC1,10% glycerol, Ca i n each b u f f e rf o re a c hg e l were pH 5.5.Varyingeoncentrstionsof used. Protein f o r 2h at roo81 t e m p e r a t u r e w i t h t h e amples were preineubated a p p r o p r i a t e ,zf c o n c e n t r a t i o nt oi n s u r e t h a t any conformational Changes Occurred. A c ' . n s t a n tc u r r e n t (20 mA) was a p p l i e du n t i lt h e sampleentared s 40 m A c o n s t a n t c u r r e n t was a p p l i e d u n t i l t h e d y e f r o n t t h eS t a c k i n gg e l ; 1-2 cm ofthebottamofthegel. The d y ef r o n tm i g r a t e s m i g r a t e dt ow i t h i n on a 6% polyacrylamideslab g e l . a t about 20 cm/h underthesecondition8

-

HPLC Size-Exclusion Chrwatonraphy 1nJectionportequippedwith a 50 250 30 x 7.5 rn s i z ee x c l u r i o n 0 . 1 n NaCl pH 7.hO b u f f e r was set d e t e c t e d a t 280 rm using a Water6 D e t e c t o r o u t p u t was d i g i t i z e d and d e v e l o p e di nt h i sl a b o r a t o r y .

-

-

A W A t e T B model 6000A pump and U6-R p l i n j e c t i o n was coupled t o a Bia-Rad TSRcolmn. P l m - r a t e of t h e 0.02 N Tris.HC1, e t 0.7 m l l s i n u t e .E l u t e dp r o t e i n aw e r e model 450 v a r i a b l e w a v e l e n g t h d e t e c t o r .

procesaed by a m i c r o c w p u t e r u s i n g s o f t v a r e

Fluorescence Weasurmente Before me, b u f f e r e d p r o t e i n sainples 'Yere -de 10 d i a l y z e d a g a i n s t two or t h r e e Changes of 0.02 Tris.HC1, 0.1 b u f f e r a t 4OC. P r o t e i nc o n c e n t r a t i o n was a d j u s t e d t o epproximately 0.07 mg/ml. F l u o r e s c e n c eo e e w r e m e n t s were performed on d. SI," 320 I4 fluorescenceinstrumentcoupledto an SPC-822 m o n o c h r m a t o r c a n t r o l l e r . The ssmplecompartment wen c o n t r o l l e d tlt 25'C using I ) Haake Pli-circulscing constantte2xperatorebath.Excitation and emissionwavelengths were set a t and 340 m r e s p e c t i v e l y .A f t e ra d d i t i o nt ot h ep r o t e i ns o l u t i o n ,t h e no?'':: n t r r r t i o n was 10 mI4. m i s s i o nd e c a y we8 monitored for 50 minutss is expressed as a f t e r e'' ionaddition.Percentagefluorsscencequenched t h ep e r c e n t a g eo fr e d u c t i o ni nf l u o r e s c e n c ec w p s r e dt ot h eo r i g i n a l I vaI ue d 4 i n EDTA and U Wac1 pll 7 . 4 3

.

~ ~

Chemical ~ o ~ i f i cofaBovine t ~ ~ P r o t ~ r o m b ~Fragment n 2

10604

F l u o r e s c e n c e t i t r a t i o n s were p e r f o r m e d w i t h p r o t e i n s o l u t i o n s c o n t a i n i n g I , 5 and 10 pU p r o t e i n i n 2 ml f 0.02 U Tris.HC1, 0.1 H NaC1, pH 7.4. - 50 mU CaZP w e r e f o l l w e d by monitoring A (for Titrationswith0.1 i n n e r f i l t e r c o r r e c t i o n s ) and c h a n g e si nt h ef l u o r e s c e n c e inta899;yA340 Abaorptionreadings and flup$escencephotoncountswerechecked a t 15 min. i n t e r v a l sf o l l w i n ge a c h CS a d d i t i o n . Samples were placed on a Fischer were v e r i f i e d Rotatorbetweenresdings. CaC12 s t o c ks o l u t i o nc o n c e n t r a t i o n s by a t m i c e b s o r p t i o n a n a l y s i s . Amino AcidAnalysis - In p r e p a r a t i o n f o r amino a c i d a n a l y s i s , p r o t e i n a m p l e s were d i a l y z e d a g a i n s t d i s t i l l e d w a t e r . Acid h y d r o l y s i s was performed accordingtotheprocedureofSwckman,Stein and Maare (IS). Alkaline h d a r (18). A f t e r h y d r o l y s i a was performedusingtheprocedureof hydrolysis,sampleswereanalyzedusing LL modifiedprocedureofKlapper(17) employingpost-column derivatizationofminoacids by o-phthsldehyde (OPA) f o l l w e d by f l u o r e s c e n c e d e t e c t i o n o f t h e r e s u l t i n g p r o d u c t s and by t h e ninhydrin post-column derivatization method d e s c r i b e d by Spscluoan e t e l . (19).

"

-

Phos h o l i i d Bindina s t u d i e s l - P a ~ m i t o ~ ~ - 2 - o ~ e o y l p h o s p h ~ t icdhyoll i n e (99%) and ~ - m - & s p h a t i d y l - ~ n e (98-9921) from bovinebrainwerepurchasedfro. AvantiPolarLipids,Inc. and Sigma C h e a i c a lm p a n yr e s p e c t i v e l y . Both sampleswerestoredin e chloroform-methanolBolution(95:5) a t DOC. Buffers were f i l t e r e d t h r o u g h a 0.45 ym M i l l i p o r e f i l t e r p r i o r t o u s e . P h o s p h o l i p i d v e s i c l e s wereprepared by removing theorganic801vent from (1 25:75 m i x t u r e were d i s p e r a e di n ofbovine PS/WPC under a nitrogenatmosphere.Vesicles II b a t h t y p e s o n i c a t o r b u f f e r by v o r t e x i n g a n d s o n i c a t i o n t o c l a r i t y w i t h 190,000 ( h b o r a t o r y Supply Co., I n c . 2 . I h e s o l u t i o n was t h e nc e n t r i f u g e da t g (48,000 rm) f o r I h at 25 C t o remove l a r g e rv e s i c l e s . The phospholipid was determined by a m o d i f i c a t i o n o f concentrationoftheresultingsolution the phosphateassayof Chen e t si. (20). The l i s h t s c a t t e r i n g method d e s c r i b e d by Nelsestuen and Lim (21) was u t i l i z e dt om e a s u r ep r o t h r o m b i n were msde It fragmentI-phospholipidbinding.Lightscatteringmeasurements 90' using an SLH model 8000s s p e c t r o f l u o r i m e t e r . The e x c i t a t i o n and emission wavelengrhswed were 320 om w i t h band passof 2 Experimentswere p e r f o r m e dw i t hs t i r r i n ga t2 5 %i n1 em c u v e t t e s . The i n i t i a l volume f o r t h e t i t r a t i o n s was 1.5 ml of 0.05 H Tria.HC1, 0.1 U NaCl, pH 7 . 4 b u f f e r c o n t a i n i n g 129 pH PSfpC (based on t h e pho*phorous assay) and0.43 til4 of 1. The m o d i f i e dr o t e i ns o l u t i o n and t h es o l u t i o n of modifiedfragment The i n c r e e s e i n l i g h t s c a t t e r i n g phoepholipid Yere both 25 mU i n I n t e n s i t y was converted t o t h e i n c r e a s e i n m o l e c u l a r w e i g h t as d e s c r i b e d by Nelsestuen and Lim (21).

W.

42.'

U Tris.HC1. pH7.40 t o 0.02 M Tris.HC1, 0.50 H NaCl pH 7.40 (30 ml each was determinedusing uv abaorbanct and 14C b u f f e r ) .P r o t e i ne l u t i o np o s i t i o n l i q u i d s c i n t i l l a t i o n c o u n t i n g as d e s c r i b e d i n t h e p r e v i o u s s e c t i o n . F r a c t i o n s c o n t a i n i n g p r o t e i n werepooledand p r o t e i n e o n c e n t r e t i o n and I4C i n c o r p o r a t i o n were determined. The c o n t r o lm o d i f i c a t i o np r o t e i n pool was d i a l y z e d e x h a u s t i v e l y a g a i n e t 0.02 U Tris.HC1 pH 7.40 at 4OC.

-

Sephadex 075 Chromatography A portion of the DUE-cellulose protein pool 1 t h a t hadbeen modifiedinthepresenceofterbium correspondingtofragment ion was p l a c e d i n t o a 6-8K m . w t . c u t - o f f d i a l y s i s bagand c o n c e n t r a t e d u s i n g dry PEG20,OOO t o L fragment 1 c o n c e n t r a t i o na p p r o x i m a t e l y 0.4 mglml a t 4'C. The c o n c e n t r a t i o n was e o n t i n n e d f u r t h e r by removing t h e p r o t e i n a o l u t i o n f r r m t h e d i a l y s i s bag and p l a c i n g it i n t o an Amicon B-15 c a r t r i d g e c o n c e n t r a t o r . I s o l u t i o n wss 2.94 m g / m l . I h e Finalconcentrationofthemodifiedfra-ent c o n c e n t r a t e d p r o t e i n s o l u t i o n was loadedonto a 30 01 x I cm Sephadex 0 7 5 column e q u i l i b r a t e d i n 0.02 U nia.HC1, 0.1 X NaCl pH 7.10 b u f f e r a t 4'6. E l u a t ef r a c t i o n so f 40 dropseachwerecollectedusing s LKB Redi-Rae was monitored by uv absorbance at 280 fractioncollector.Proteinelution Fractionscorrespondingtoproteinpeakswerepooled and q u a n t i t a t e d by *C l i q u i d s c i n t i l l a t i o n c o u n t i n g and uv absorbance.

.

- A 172 p l a l i q u o t of 80.4 d4 t e r b i m f l l l ) PrerativeScaleHodifieation e h l z i d c sol= pH 5.0 YIS added t o 25 ml of a s o l u t i o n c o n t a i n i n g I 2 pH of metal-freebovinefrngmfnt I . The s o l u t i o n was incubated at 37' f o r2h . Po- ldehyde(1.2 x IO- moles, 1057 yl 37% s o l u t i o n ) and s o r p h o l i n e (1.2 x 10Tmoles, 1046 y1)wereaddedto22.8 m l of 0 . 5 sodium a c e t a t e b u f f e r , pH 5.0. Iha f i n a l volume of t h er e a c t i o nm i x t u r e was 50 ml. The pH was a d j u s t e dt o 5 . 0 with con=. H C l . I h er e a c t i o nm i x t u r e was i n c u b a t e df o r 2 h at 37OC b e f o r e a d d i t i o n t o t h e p r o t e i n s o l u t i o n . The reagent and p r o t e i n 37' f o r 8 h. s o l u t i o n s were combined and t h e r e s u l t i n g s o l u t i o n k e p t a t The pH of t h e r e a c t i o n m i x t u r e was a d j u s t e d t o 7.0 (4N NaOH) and t h e s o l u t i o n was s e p a r a t e di n t o NO25 ml a l i q u o t s . One a l i q u o t was immediately a p p l i e d t o a 3 x 100 cm of Sephadax 6 7 5 r e s i n e q u i l i b r a t e d i n 0.02 W T r i s . H C 1 , pH 7.4 b u f f e r . The s e c o n da l i q u o t was s t o r e d a t 4OC o v e r n i g h tf o r a p p l i c a t i o nt ot h e column t h ef o l l w i n gd a y . No d i f f e r e n c ei ne l u t i o n N o a l i q u o t s wss noted. p r o f i l e s ofthe F r a c t i o n s o f 3 m l were c o l l e c t e d u s i n g a t LKB 2070 U l t r o r s c 111 f r a c t i o n collectorwiththeproteinelutionmonitored by Azs0 on a Gilson model 260 uvv i s s p e e t r o p h o t w e t e r as d e s c r i b e d e a r i e r .

Anal t i c a tS t u d i e s .P r e. r a t i o n Of BovineProthrombinFraent1forControl 1 stock s o l u t i o n e q u % e n t t o 7 "8 of U o d i Y f i c a t i o ~ r o t h r ~ b ifragment n n r a t e i n a t 0.261 mnlml was o l a c e d i n t o a 6-8K m . w t . c u t - o f f d i a l y s i s bag and e o n c e n t r a t e d a t :4' using PB020,OOO (Sigma) u n t i l r e s c h i a g a & c e n t r a t i o n ofapproximately 1.5 rnglml. The c o n c e n t r a t e dp r o t e i n s o l u t i o n was then 0.02 M Tris.HC1, 0.1 U NaCl pH 7.40 d i l u t e d t o 1.00 mg fragmentI/mlwith b u f f e r . An a l i q u o to f a 0.2 U EDTA s o l u t i o n was added t o t h e p r o t e i n s o l u t i o n LIO t h a t t h e f i n a l F.DTA c o n c e n t r a t i o n was i0 The p r o t e i n NO or threechangesof 0.02 M Tris.HC1, s o l u t i o n was t h e n d i a l y z e d a g a i n s t 0 . 1 U NaCl pH 7.40 b u f f e r a t a 4OC t o remove EDTA and t r a c e 8 o f a z i d e i o n remaining from t h e s t o r a g e b u f f e r . I h e d i a l y s i s b u f f e r was changed t o 0.05 H After d i a l y s i s against the 5 . 0 0 buffer ( N o sodium a c e t a t e , pH 5.00 a t 4%. or t h r e e c h a n g e s ) , L volumeof thefragment 1 s o l u t i o n c o r r e s p o n d i n g to 5 mg offragment 1 ( 0 . 2 pmolea) was removed f r a ot h ed i a l y e i s bagand p l a c e di n t o a 17 88 x 200 p l a s t i c test tahe.

Amino AcidAwlyaio

~~

-

sequenceb

Residue

mU.

Table I of 3 Y-MGlu Fragment l a

ASD

e

Pre 'rationofBovineProthranbin Pra e n t 1 Uodificatinne 4 " ~ r ~ l - a g m e ~ 1 ( 5 mg) w e prepared as f o r the c o n t r o l reaction abovc. A 104 pU a l i q u o t of a 80.4 mW tarbivm(111)chloride pH 5.0 p r o t e i n s o l u t i o n . The a q u e o u ss o l u t i o n was added t o t h e m e t a l - f r e e , p r o t e i n s o l u t i o n was then a l l w e d t o i n c u b a t e a t 37OC forabout 2 h p r i o r t o combination with the modification reagents.

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moles, 187 yl 352 s o l u t i o n ) Rea e n tP r er s t i o n Fo ldehyde (2.5 x M e o d i m a c e t a t e pH t o 3 ml of0.05 x 10yml)wereadded and'morphol~e(2.5 Ihe pli o ft h es o l u t i o n was lowered t o 5.00 w i t h c o n c e n t r a t e d E l 5.00. ayringe. As a t r a c e r , 1 r n l ( c o r r e s p o n d i n gt o (Fiacher uaing of f 4 m i c r o l i t e r formaldehyde s o l a t i o n W L I B added t o t h e b u f f e r . 0.2 mCi "C) C ftbelled C formaldehydeincomparison t ot h a to fu n l s h e l l e d The mlsr m o u n t of a t o t a lm o d i f i c a t i o nr e a c t i o n volume formaldehyde was i n s i g n i f i c a n t . Tomake of 15.0 ml. a n a p p r o p r i a t e amount of 0.05 M sodium a c e t a t e pH 5.00 b u f f e r vas added t ot h er e a g e n tm i x t u r e . h e pH waa recheckedand, i fn e c e s s a r y , a d ' u s t e d t o pH 5.00. The reagent was a l l w e d to i n c u b a t ef o rt v oh o u r s at 37dC b e f o r e c o m b i n a t i o n w i t h t h e p r o t e i n s o l u t i o n .

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A f t e r N o hoursofincubation a t 37OC. t h e r e a g e n t U o d i f i e a t i o nR e a c t i o n and p r o t e i n s o l u t i o n s were combined. The r e a c t i o nm i x t u r e was thenincubated a t 3 7 O C f o r 8 h i n e Haake F N - c i r c u l a t i n gc o n s t a n tt e m p e r a t u r eb a t h . The r e a c t i o n pH was measuredandthenre-adjustedwith 4N NaOH t o pH 7.00. The m o d i f i c a t i o n r e a c t i o n was a l l w e d t o i n c u b a t e f o r an a d d i t i o n a l 15 min a t 37OC t o f a c i l i t a t e f r a g m e n t a t i o n o f Uannichbaseadducts.

I-UGlu Fragment

14 10 I1 23 10 0 It 10 10 9 1 4 IO

14.3 9.4 IO .7 20.4 8.1 n.d. n.d.

4

2.8

4

3.8 2.7 4.7

10.0

3.4 8.5 0.8

3.5 9.8

2

5

13.1 7.0

15 10

A m *

Acid Analyzerusing ' A n a l y t i c a l d a t a were obtained on a Beckonn 6300 post column ninhydrin (570 and 440 mi d e t e c t i o n . Sequencedatafor 1 (16).Valuasreportedarenotcorrected. aovineprothrombinfragment v a l e n e l u e s GI". G l a , f l n s i a c e r e s i d u e d a t a were obtained by h y d r o l y s i s with 6N HCI, llOo,24h. 7-UGlu and Gly were noteeparated and merged as II s i n g l e peak.fDeterminedinseparateeaprrimentsusingthealkaline h y d r o l y s i s method of Medar e t a l . (18).

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SehadexG25-150 Chr-to ra h A f t e rm e a s u r i n gt h er e a c t i o n volume, two 10 ml Scinti-Verse d u ~ l i c a t e ~ l i q u o wigePrLoved, t s suspendedin ( P i a e h e r ) and countedfor C a c t i v i t y on a PackardTri-Csrb model 300 l i q u i d s c i n t i l l a t i o nc o u n t e r .A f t e rc o r r e c t i o nf o rd i l u t i o nf a c t o r st h e average o f t h ec o u n t so b t a i n e df r a ot h e NOd i q u a t s was used t o d e t e r m i n e t h e t o t a l number o f c o u n t s p r e s e n t i n t h e r e a c t i o n m i x t u r e ( c o r r e s p o n d i n g t o t h e t o t a l number ofmolesof bound end f r e e IormeJdehyde). To s e p a r a t et h ep r o t e i n from t h e r e a g e n t s . t h e r e a c t i o n m i x t u r e was lccaded onto a 50 cm x 2.5 cm i.d. Sepha$epl 025-150 colmn p c e - e q u i l i b r a t e d w i t h 0.02 U Tris.HC1 pH 7.40 b u f f e r a t 20 C. Fractionsof 90 dropseach were takenusing a LKB Redi-Bac f r a c t i o n and r e a g e n tp e a ke l u t i o np o s i t i o n s , c o l l e c t o r . To d e t e r m i n el a b e l l e dp r o t e i n 50 p l a l i q u o t s were removed from eachfraction,suspendedin 10 m l S c i n t i Verse and c o u n t e d .P r o t e i ne l u t i o n was l o c a l i z e d hy nv absorbance were pooled and measurementsofeach f r a c t i o n .F r a c t i o n sc o n t a i n i n gp r o t e i n p r o t e i nc o n c e n t r a t i o nd e t e r m i n e d by uv absorbance.Duplicate 50 p l a l i q n o t a and counted. were removed from thepool,suspendedin 10 ml ofSeinti-Verse n e p r o t e i np o o lo ft h em o d i f i c a t i o nr e a c t i o nc a r r i e do u ti nt h ep r e B e n c eo f t e r b i u m ( I I 1 ) was made 10 mU i n EDTA t o remove protein-boundterbium(lI1). The EDTA-treated p r o t e i n s o l u t i o n was t h e n e x h a u s t i v e l y d i a l y z e d a g a i n s t 0.02 U Tris.HC1 pH 7.40 b u f f e r e t 4'C. The c a n t r o l r e a c t i o n was n o t t r e a t e d w i t h EDTA.

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DUE-CelluloseChromatography The p r o t e i n pool from t h e Sephadex 0 2 5 was l o a d e d o n t o a 20 cp x 1 icm i.d. column of DEA5-celluiose 2OoC. After e o u i l i b r a t e dw i t h 0.02 M Tria.HC1. pH 7 . 4 0 s t a r t i n g b u f f e r a t I d s d i n g , t h e celltmn v a s washed w i t h . f i v e collrma v o l m e s o f s t a r t i n g b u f f e r . F r a c t i o n s of 90 dropseach were c o l l e c t e d . A s a l t g r a d i e n t was run from 0.02

col-

' 1

B I 0

,

, 0

,

, M

,

, 30

,

, 40

,

, M

Tin(Mibd

Fig'Z. Inrrtnsic f l u o r e s c e n c ec h a n g e s a t 340 om of b o v i n e 3 y - f f i l uf r a g m e n t 1 (MBF 1 ) and b o v i n ef r a g m e n t 1 (HF1 a t 25%; the e x c i t a t i o n w a v e l e n g t h YBS 290 nm. Ca2+ (10 a) was added a t t h e p o i n t i n d i c a t e d .

Chemical Modification of Bovine Prothrombin Fragment 1 ColcimTiaofModifi8oviMFmgrrntI

I S

-

12-

10605

-1

a

16 14

ID

-

1.8-

-

0.6 0.4

-x

-0.6-0.8-

-

.

-1 .a

4.2-1.4

-

-1.6-

-1.8-

Fig. 3. Hillplots of equilibrium Ca2* (open points) of 8ndMg2+ (filled points) fluorescence titrations 3 y-lGlu fragment 1.Titration of various concentraA , 1Op~) t i o m of proteinsolutions (0, IPM; 0 ,SIB; were conducted i n 0 . 0 2 M Tris.HC1, 0.1 M NaC1, pH7:40 ConcentTations shown. buffer a t the -tal ion

-V Fig. 4. Scatchard plot of4sCaz+ 3 y-MGlu fragment 1.

binding to