THE JOURNAL OF CHEMISTRY Vol ,266, No. 10, Issue April 5 ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Vol

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol ,266, No. 10, Issue of April 5, pp. 6353-6364,1991 Printed in U.S.A.

Predominant Contribution of Surface Approximation to the Mechanism of Heparin Acceleration of the Antithrombin-Thrombin Reaction ELUCIDATION FROM SALT CONCENTRATION EFFECTS*

(Received for publication, August 27, 1990)

Steven T. OlsonSB and Ingemar Bjorky From the $Henry Ford Hospital, Division of Biochemical Research, Detroit, Michigan 48202 and the YDepartment of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, T h e Biomedical Center, Box 575, S-751 23 Uppsala, Sweden

Heparin has been shown to accelerate the inactivation of a-thrombin by antithrombin I11 (AT) by promoting the initial encounter of proteinase and inhibitor in a ternary thrombin-AT-heparin complex. The aim of the present work was to evaluate the relative contributions of an AT conformational change induced by heparin and of a thrombin-heparin interaction to the promotion by heparin of the thrombin-AT interaction in this ternary complex. This was achieved by compar- ing the ionic and nonionic contributions to the binary and ternary complex interactions involved in ternary complex assembly at pH 7.4, 25 ‘Cy and 0.1-0.35 M NaCl. Equilibrium binding and kinetic studies of the binary complex interactions as a function of salt concentration indicated a similar large ionic component for thrombin-heparin and AT-heparin interactions, but a predominantly nonionic contribution to the thrombin-AT interaction. Stopped-flow kinetic studies of ternary complex formation under conditions where heparin was always saturated with AT demonstrated that the ternary complex was assembled primarily from free thrombin and AT-heparin binary complex at all salt concentrations. Moreover, the ternary complex interaction of thrombin with AT bound to heparin exhibited a substantial ionic component similar to that of the thrombin-heparin binary complex interaction. Comparison of the ionic and nonionic components of thrombin binary and ternary complex interactions indicated that: 1) additive contributions of ionic thrombin-heparin and nonionic thrombin-AT binary complex interactions completely accounted for the binding energy of the thrombin ternary complex interaction, and 2 ) the heparin-induced AT conformational change made a relatively insignificant contribution to this binding energy. The results thus suggest that heparin promotes the encounter of thrombin and AT primarily by approximating the proteinase and inhibitor on the polysaccharide surface. Evidence was further obtained for alternative modes of thrombin binding to the AT- heparin complex, either with or without the active site

* This work was supported by Grants HL-39888 from the National Institutes of Health (to S. T. O.), a n Established Investigatorship of the American Heart Association (to S. T. O.), and Swedish Medical Research Council Grant 4212 (to I. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Div. of Biochem- ical Res., Education and Research Bldg., Rm. 3126, Henry Ford

3196. Hospital, 2799 W. Grand Blvd., Detroit, MI 48202. Tel.: 313-876-

of the enzyme complexed with AT. This finding is consistent with the ternary complex encounter of thrombin and AT being mediated by thrombin binding to nonspecific heparin sites, followed by diffusion along the heparin surface to a unique site adjacent to the bound inhibitor.

Heparin, a highly sulfated glycosaminoglycan, acts as an antagonist of blood coagulation by accelerating the inactivation of the procoagulant proteinases of this system by their natural plasma protein inhibitor antithrombin 111 (AT)’ (1, 2). The mechanism of heparin’s accelerating effect on AT- proteinase reactions has been demonstrated to involve antithrombin binding to a specific pentasaccharide site on the polysaccharide (3-9). This interaction induces a conformational change in the inhibitor (10, ll), which has been suggested to be an important contributor to heparin rate enhancement (3, 8, 12). In the case of antithrombin’s reaction with proteinases such as factor Xa and plasma kallikrein, this proposal is supported by the ability of the pentasaccharide or small oligosaccharides containing this specific sequence to produce a rate-enhancing effect comparable to that of natural length heparin chains (6, 13-15). However, such oligosaccharides possess negligible ability to accelerate antithrombin inhibition of other proteinases, such as thrombin or factor IXa. A minimum heparin chain length of 18 saccharides is thus necessary to significantly enhance the rates of these latter proteinase reactions (4, 15, 15). This observation has suggested that proteinase binding to heparin, in addition to AT binding to the polysaccharide, is essential for heparin rate enhancement of the latter class of AT-proteinase reactions. Support for this proposal has come from: 1) chemical modification studies in which the heparin accelerating effect could be selectively abolished by modification of basic residues of the proteinase presumably involved in heparin binding (16, 17); 2) kinetic studies that have shown inhibition of the rate enhancement at high heparin concentrations which correlate with the binding of inhibitor and proteinase to separate heparin chains (7-9, 14, 18-20); and 3) binding studies that have shown that the smallest heparin chain capable of rate enhancement corresponds to the smallest heparin that can bind both AT and the active-site blocked proteinase (21).

Despite this evidence, some investigators have emphasized a primary role for heparin activation of AT in mediating

I The abbreviations used are: AT, antithrombin 111; T, a-thrombin; H, heparin; P, p-aminobenzamidine; PEG, polyethylene glycol.

6353

6354 Heparin-Antithrombin-Thrombin Reaction Mechanism

heparin rate enhancement for all proteinases, with proteinase binding to heparin being of lesser importance (3, 8, 12, 14, 22-24). Thus, the larger heparin chain length requirement for accelerating the reaction of AT with thrombin and other proteinases has been rationalized as being due to: 1) a further conformational change induced in the inhibitor by the longer heparin chain (22, 23) or 2) the need to neutralize the charge of the inhibitor and proteinase for the activated inhibitor to interact with cationic proteinases (24). Spectroscopic evidence for an additional conformational change in the inhibitor induced by natural length but not oligosaccharide heparins (22, 23), together with the observation of a substantial residual rate enhancement at high heparin concentrations for antithrombin reactions with thrombin and factor IXa (8, 14) has been offered in support of these proposals. However, the interpretation of the spectroscopic changes induced in antithrombin by larger heparins in terms of an additional inhibitor conformational change, as well as the true extent of the heparin rate enhancement remaining at high heparin concentrations, have been disputed by other investigators who have conducted similar experiments (20, 25). Moreover, the observation that the extent of the heparin-induced AT conformational change can be diminished by chemical modification of the inhibitor without affecting the extent of heparin rate enhancement with thrombin as the proteinase (26) has suggested that the AT conformational change may not be required to accelerate the inactivation of all proteinases. In- stead, approximation of AT and proteinase on the heparin surface may suffice to explain heparin rate enhancement in such cases (4,9, 15, 18-21, 26).

Rapid reaction studies have established that heparin accelerates the thrombin-AT reaction by promoting the initial encounter of proteinase and inhibitor in a ternary complex with heparin, without affecting the rate at which this intermediate complex is converted to a stable thrombin-AT complex (27). Heparin promotion of the ternary complex interaction was observed to be mediated by AT binding to heparin, resulting in an enhanced binding of the inhibitor to thrombin to form the ternary complex, as evidenced by a nearly 1,000- fold increase in the AT-thrombin binding affinity (9, 27). However, the contribution to this enhanced binding affinity of the AT conformational change and of thrombin binding to heparin could not be determined from these studies.

The contributions of the AT conformational change and a proteinase-heparin interaction to heparin promotion of ternary complex formation can be distinguished by the involve- ment of heparin binding to only the inhibitor in the former case, but heparin binding to both proteinase and inhibitor in the latter case. We thus hypothesized that the sensitivity of protein-heparin interactions to the solution ionic strength could be used as a probe of the relative contribution of the two mechanisms to ternary complex formation. This hypoth- esis has been examined in the present study by analyses of the salt-concentration dependence of the protein-protein and protein-heparin binary and ternary complex interactions which participate in the heparin enhanced thrombin-AT reaction by the use of saturation kinetics and equilibrium binding techniques. These studies have shown that the large enhancement of the binding affinity of thrombin for AT resulting from heparin binding to the inhibitor can be accounted for by the additional binding energy provided by thrombin interacting with heparin in the ternary complex. In contrast, the AT conformational change is shown to make a relatively minor contribution to this enhanced affinity. Ad- ditional evidence is provided to show that ternary complex formation from AT-heparin complex and thrombin involves both a productive mode, in which thrombin is bound to both

0 0 .2 * 4 .6 .a

C A T I ~ / ( ~ + C P I ~ . / K T . P ) (PM) FIG. 1. NaCl concentration dependence of the second-order

rate constant for the uncatalyzed thrombin-AT reaction. Ob- served pseudo first-order rate constants (kobr) for the thrombin-AT reaction were measured in the presence of 700 WM p-aminobenzamidine as a function of AT concentration (2.5 to 10 WM) a t molar NaCl concentrations of 0.1 (O), 0.15 (O), 0.2 (m), 0.25 (A), 0.3 (A), and 0.35 (0) in sodium phosphate buffer, pH 7.4, a t 25 “C, as described under “Materials and Methods.” Second-order rate constants were obtained from the slope of linear regression fits of the dependence of kohs on effective AT concentration (i.e. corrected for the competitive effect of p-aminobenzamidine with the use of a value of 65 FM for the thrombin p-aminobenzamidine dissociation constant (KT.p)). The average linear regression fit for all data sets is shown (solid line).

heparin and AT with the inhibitor occupying the proteinase active site, and an alternative binding mode, in which thrombin is bound to just heparin with its active site free. This evidence is consistent with the ternary complex encounter of thrombin with AT being mediated by thrombin binding to nonspecific heparin sites, followed by diffusion of thrombin to a unique heparin site adjacent to the inhibitor.

MATERIALS AND METHOD$

RESULTS

NaCl Concentration Dependence of Thrombin, Antithrom- bin, and Heparin Binary Complex Interactions-The NaCl concentration dependence of the protein-heparin and protein- protein binary and ternary complex interactions involved in the heparin (H)-accelerated thrombin-AT reaction were evaluated as a means of elucidating the relative contributions of the AT conformational change induced by heparin binding and of a thrombin-heparin interaction to heparin rate enhancement (1,2). The effect of salt on the initial equilibrium interaction between AT and thrombin that precedes stable thrombin-AT complex formation (Scheme 1) was first evaluated. This was determined from the kinetics of AT inactivation of thrombin in the absence of heparin as a function of NaCl concentration at pH 7.4, 25 “C (Fig. 1). Thrombin-AT reactions were continuously monitored by the decreased fluorescence accompanying the displacement of p-aminobenzamidine (P) from the active site of the proteinase by the inhibitor under pseudo first-order conditions ([AT]”, [PI0 >> [TIo), as in past studies (9, 27, 29). NaCl had no effect on the dissociation constant for the thrombin-p-aminobenzamidine interaction over the range examined (0.1-0.35 M, Table I), SO

that observed pseudo first-order inactivation rate constants

Portions of this paper (including “Materials and Methods” and “Appendix”) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

Heparin-Antithrombin-Thrombin Reaction Mechanism 6355

TABLE I NaCl concentration dependence of dissociation constants for

thrombin, antithrombin, heparin, and p-aminobenzamidine binary complex interactions

[NaCIl KT,P‘ KT,AT~ KAT.H‘ K T , H ~ K4.n‘ M P M

0.10 64 f 5 1200 0.014t- 0.003 l o + 1 0.91 0.15 5 8 + 6 1200 0.045 t-0.004 44 k 6 4.0 0.20 66 k 6 1200 0.10t- 0.01 150+ 10 14 0.25 64 + 1 1200 0.23 f 0.01 370 f 110 34 0.30 62 & 1 1100 0.44-CO.02 950+ 60 86 0.35 61 k 2 1100 0.70 f 0.03 1600 + 150 140

a From equilibrium binding titrations of thrombin with p-aminobenzamidine monitored by fluorescence as described under “Materials and Methods.” Average values from 2 titrations except at 0.1, 0.15, and 0.25 M NaCl where 4, 11, and 18 titrations were averaged, respectively.

* Calculated from the second-order rate constants ( k / K T . A T ) of Fig. 1 assuming a NaCl concentration independent value of 10 s-l for k determined previously at Z 0.3 (27). Standard errors in second-order rate constants were on the order of 1%.

‘ From equilibrium binding titrations performed as described under “Materials and Methods.”

Average values from equilibrium binding titrations of 1.5-7 pM thrombin with a low affinity heparin preparation having the same molecular weight as t.he high affinity heparin used in this study. Similar values of 9.0, 42, and 230 p~ were measured for KTJ at NaCl concentrations of 0.1, 0.15, and 0.25 M, respectively, with the high affinity heparin (Fig. 6). All values are taken from the preceding manuscript (28).

“Calculated from the ratio of KT,” to the number of nonspecific thrombin-binding sites on a heparin chain of M, 7,900 (i.e. -11 sites). The values are indistinguishable from thrombin-heparin dissociation constants measured at low thrombin binding densities (28).

( k o b s ) measured at different salt concentrations, but at iden- tical inhibitor and probe concentrations, could be directly compared. Fig. 1 shows that an indistinguishable proportional increase in kobs with “effective” AT concentration (i.e. corrected for probe competition (37)) was found at each salt concentration examined. Linear regression analysis of these data indicated little or no dependence of the second-order rate constant on the NaCl concentration with an average second-order rate constant of 8.7 f 0.3 X lo3 M” s-’ (range 8.3-9.2 X 10:’ M” s-’). These results suggest that the dissociation constant of -1 mM for the initial thrombin-AT binding interaction (KT,A.r) and the intrinsic rate constant of -10 s” for the subsequent formation of the stable proteinase-inhibitor complex ( k , Scheme l ) , which were measured previously at I 0.3 (27), are also independent of NaCl concentration (Table I).

- 60 pM KT.A.r - 1 mM P . T t ” - - - - - P + T + A T Y

T.AT __if

k - 10 s-l

T-AT

SCHEME 1

The minimal dependence of the thrombin-AT and thrombin- p-aminobenzamidine interactions on NaCl concentration suggests that the anionic binding site within the active center specificity pocket of the proteinase (38) is shielded from bulk solvent. This would be in keeping with previous studies in which the specificity site was characterized as a hydrophobic crevice (29, 38).The effect of NaCl on the AT-heparin binary complex interaction was examined by equilibrium binding titrations of fixed concentrations of AT with an M , - 7,900 heparin at the same NaCl concentrations used above (i.e. 0.10-0.35 M , equivalent to ionic strengths of 0.15-0.4). Bind- ing was monitored from the 40% enhancement in protein

.5

.I

. 3

0 0 .Z . 4 . 6 .8 I

[AT-Heparinl/(l*[P1, /K7.,l (nW) [AT-Heprrinl/(l*F’L /KT,* w) FIG. 2. NaCl concentration dependence of the second-order

rate constant for the heparin-catalyzed thrombin-AT reaction. Observed pseudo first-order rate constants ( kobs) were measured for single turnover reactions of AT-heparin complex with thrombin as a function of AT-heparin complex concentration (500 nM AT and 20 to 100 nM heparin in panel A and 10 p~ AT and 1-5 p~ heparin in panel B ) at NaCl concentrations of 0.1 M (a) in panel A, or 0.15 (O), 0.25 (A), and 0.35 M (0) in panel B. All reactions were done in pH 7.4 sodium phosphate buffer at 25 “C. Reactions were monitored by assays of residual thrombin activity with a chromogenic substrate (panel A ) or by p-aminobenzamidine displacement in the stopped- flow fluorometer (panel B ) at 0.25 (O), 1 (A), or 5 (0, .) mM p - aminobenzamidine, as described under “Materials and Methods.” AT- heparin complex concentrations were calculated from measured values of KAT,H (Table I), and second-order rate constants were obtained from the slope of linear regression fits (solid lines), as described in the legend to Fig. 1.

fluorescence which accompanies the interaction (10, 11, 39). Similar to past results (11, 39), a marked dependence of this binding on the salt concentration was found (Table I), consistent with ionic interactions making a significant contribution to the binding energy. All titration curves were satisfactorily fit by nonlinear least squares analysis to a model which assumes that AT binds to specific sites on heparin (about one per heparin molecule). These analyses indicated NaCl concentration-dependent dissociation constants (KAT,H) which increased -50-fold from 0.1 to 0.35 M NaCl (Table I).’3

Equilibrium binding of thrombin to M , 7,900 heparins with either low or high affinity for AT, monitored by a -16% quenching of the fluorescence of enzyme-bound p-aminobenzamidine as a function of NaCl concentration, also indicated a marked sensitivity of this interaction to salt (see the preceding paper (28)). As demonstrated in the preceding paper, thrombin binding to heparin predominantly involves a nonspecific electrostatic association of the proteinase with any three contiguous disaccharide units of the polysaccharide chain (28). Analysis of binding data by a nonspecific binding model which accounts for the statistical effect of overlapping binding sites on the polysaccharide (28) indicated intrinsic dissociation constants (KT,H) which increased from 10 to 1600 pM (160-fold change) when the NaCl concentration was increased from 0.1 to 0.35 M (Table I). The intrinsic affinity of this interaction was thus 1000-2000-fold weaker than the AT- heparin interaction over the NaCl concentration range examined (Table I).

NaCl Concentration Dependence of the Ternary Thrombin- Antithrombin-Heparin Complex Interaction-The strong de-

”’ The dissociation constants reported here for the M , 7,900 heparin

tion of higher molecular weight ( M , -14,000) (11). These differences are somewhat tighter than values previously reported for a prepara-

may he the result of the lower and higher M , heparins having been isolated from a saturated uersus an unsaturated AT affinity column, respectively. Selection for a higher affinity heparin may thus have resulted in the case of the M, 7,900 heparin (7).


pendence of the protein-heparin interactions but independence of the protein-protein interaction on the NaCl concentration suggested that the contributions each of these interactions makes in the ternary thrombin-AT-heparin complex could be evaluated from a kinetic analysis of the NaCl concentration dependence of ternary complex formation. Since previous studies at 10.3 established that the ternary complex was assembled predominantly from an intermediary AT-heparin binary complex and free thrombin (9), it was first nec-

0 s I O I S 2 0 2 1 CAT-Heparin1 (wM)

FIG. 3. NaCl concentration dependence of ternary complex and stable complex formation steps in the heparin-catalyzed thrombin-AT reaction. Observed pseudo first-order rate constants (kobs, panel A ) and reaction amplitudes (panel B ) were measured for single turnover reactions of AT-heparin complex with thrombin, monitored by p-aminobenzamidine displacement in the stopped-flow fluorometer, as in Fig. 2. Reactions were performed at varying AT- heparin complex concentrations (0.5-30 PM heparin and a 1.1-6-fold molar excess of AT (1-32 p ~ ) ) and at a fixed p-aminobenzamidine concentration of 70 I.LM in pH 7.4 sodium phosphate buffer containing molar NaCl concentrations of 0.15 (E), 0.2 (a), 0.25 (A), 0.3 (A), and 0.35 (0), at 25 "C. Reaction amplitudes are expressed relative to the thrombin concentration and are normalized to the initial fluorescence measured for the bound probe. Rate constant data were fit to Equation 3, whereas amplitude data were fit to the untransformed Equations 5 (straight lines) or 11 (curued lines). In the fits to Equation 11, AFo was allowed to vary as a parameter. The fits to Equation 5 appear to weight the data at low AT-heparin complex concentrations only because the reciprocal plot magnifies the small amplitudes observed at high concentrations.

essary to determine whether this pathway was affected by the NaCl concentration. The kinetics of the heparin-accelerated thrombin-AT reaction was therefore studied from 0.1 to 0.35 M NaC1, either by p-aminobenzamidine displacement in the stopped-flow fluorometer or by discontinuous assay of residual thrombin enzymatic activity. These studies were done under conditions resulting in a pseudo first-order inactivation of thrombin in a single heparin turnover and at concentrations of AT-heparin complex that were either well below saturation of the ternary complex or in a range where ternary complex saturation would occur. Such conditions were achieved as in past studies by use of a large molar excess of AT-heparin complex and p-aminobenzamidine over thrombin (9) ( i e . [ATIo > [HIo >> [TIo << [PIo). To insure that the NaCl concentration dependence of the AT-heparin interaction would not contribute to that of the observed pseudo first- order rate constant ( k o b s ) , a molar excess of AT over heparin was also used in these experiments so as to always saturate the polysaccharide with the inhibitor, as was confirmed from measured values of KAT,H. This latter condition further allowed us to neglect the inhibitory effect of free heparin on the reaction rate (9). The results of these kinetic studies, shown in Figs. 2 and 3, were consistent with a reaction pathway involving a bimolecular association between AT- heparin complex and free thrombin at all NaCl concentrations (Scheme 2 ) .

KT.P P.T - P + T + AT.H T.AT.H % T-AT + H

KT,ATH

SCHEME 2

Equations 3-6 describe the dependence of kobs and reaction amplitudes on AT-heparin complex concentration for Scheme 2:

which reduces to:

kobs = (kH/KT.ATH)[AT.H1/(1 + [PIO/KT.P) (4)

under conditions where AT-heparin complex concentrations are much lower than the apparent dissociation constant for ternary complex formation (9) ( i e . [AT-H] << KT,ATH(~ + [p]O/KT,P)).

AF KT.ATH(~ + [p]O/KT,P) a F o KT.ATH(~ + [PIo/KT,P) + [AT.Hl " - (5)

where AF is the observable reaction amplitude and A F o is the total amplitude due to the bound probe. In reciprocal form, this equation becomes:

TABLE I1 NaCl concentration dependence of ternary complex dissociation constants and limiting rate constants for the

heparin-accelerated thrombin-AT reaction Ternary complex dissociation constants (KT,ATH) and limiting rate constants ( k ) were determined by fitting data

in Figs. 3 and 4 to the simple competitive model of Scheme 2 or the nonproductive binding model of Scheme 3 (in parentheses). The latter assumed that K&,ATH was equal to K&,H for NaCl concentrations of 0.25-0.35 M.

[NaCl] KT,*TH' K T , A T ~ * k k/KT.ATHC M P M S-I PA." s"

0.10 (0.12 ? 0.05) (4.8 k 0.5) (40 3~ 20)

0.20 1.2 f 0.2 (1.5) 1.4 f 0.2 (1.0) 5.4 -C 0.2 (6.7) 4.6 f 0.9 0.15 0.54 f 0.14 (0.62) 0.64 k 0.15 (0.43) 5.1 -C 0.2 (5.9) 9.5 f 2.8

0.25 2.7 f 0.2 (3.3) 4.6 & 0.4 (3.5) 5.6 5z 0.2 (6.7) 2.0 & 0.2 0.30 5.6 & 0.4 (6.4) 7.5 k 0.8 (6.5) 5.1 k 0.1 (5.9) 0.91 ? 0.08 0.35 10 f 1 (12) 13 f 3 (11) 4.9 -c 0.2 (5.7) 0.48 & 0.07

From rate constant data. From reaction amplitude data. Calculated second-order rate constants from rate constant data.

Heparin-Antithrombin- Thrombin Reaction Mechanism 6357

hFo- l+ [AT.H]

"

AF KT.ATH(1 + [P]O/KT,P) (6)

Fig. 2 shows that koba was linearly dependent on the effective AT-heparin complex concentration (i.e. [AT - H]/(l+ [P]o/ KT,p))) at each salt concentration when AT-heparin complex concentrations were subsaturating with respect to ternary complex formation, as predicted by Equation 4 for Scheme 2. This was consistent with a second-order reaction between AT-heparin complex and free thrombin in all cases. Such kinetic behavior is also in keeping with the substantially greater affinity of AT than of thrombin for heparin observed at all salt concentrations examined (9). Fig. 3A shows that a saturable dependence of kobs on AT-heparin complex concentration, reflecting intermediary ternary complex formation, was found at NaCl concentrations ranging from 0.15 to 0.35 M when AT-heparin complex concentrations were extended to -25 PM at a fixed probe concentration of 70 p ~ . The satisfactory nonlinear least squares fits of these data to the rectangular hyperbolic function of Equation 3 indicated that Scheme 2 is sufficient to describe the heparin-accelerated thrombin-AT reaction from 0.15 to 0.35 M NaCl.

In contrast to the independence of the second-order rate constant k/KT,AT for the uncatalyzed thrombin-AT reaction (Scheme 1) on NaCl concentration, second-order rate constants determined for the heparin-accelerated reaction from the slopes of the linear plots of Fig. 2 ( i e . ~H/KT,ATH) were strongly dependent on the NaCl concentration. Progressively decreasing values for ~H/KT,ATH of 37 k 1,8.0 f 0.5, 1.7 k 0.1, and 0.41 f 0.01 p W 1 s" were thus obtained at 0.1,0.15, 0.25, and 0.35 M NaC1, respectively, amounting to a 90-fold decrease over this range of salt concentrations. The salt concentration dependence of the second-order rate constant for the heparin- accelerated reaction might be due to NaCl affecting either the ternary complex dissociation constant for the initial interaction of AT-heparin complex with thrombin (KT,ATH) or the rate constant for subsequent formation of the stable thrombin-AT complex (kH). The computer fits of the dependence of kohs on AT-heparin complex concentration in Fig. 3A to Equation 3 (9, 27) revealed that all saturation curves reached an indistinguishable limiting first-order rate constant, kH, of -5 s-' (range 4.9 to 5.6 s-l, Table 11), similar to that previously reported at Z 0.3 (27, 31). This indicated that the salt concentration dependence of kohl resided completely in the ternary complex dissociation constant, KT,ATH. Fitted values for this parameter indeed increased about 20-fold from 0.54 to 10 p~ over the NaCl concentration range covered (Table 11).

This conclusion was confirmed by independent analyses of ternary complex formation from the increasing dead time displacement of p-aminobenzamidine which accompanied the approach of kobs to a limiting value. This biphasic displacement of the probe reflects an initial rapid equilibrium binding of heparin-bound AT to the active site of thrombin to form the ternary complex prior to stable complex formation (Scheme 2) (27). The reaction amplitude Equations 5 and 6 quantitatively describe this behavior (27). These equations predict that saturation of the ternary complex by increasing AT-heparin complex concentrations will be accompanied by a hyperbolic decrease in observable reaction amplitudes which asymptotically approach a value of zero. This behavior is reflected in the linearity of a reciprocal plot (27). Fig. 3B shows that the reciprocal of the observed fractional fluorescence amplitude increased linearly with AT-heparin complex concentration at each salt concentration at least up to a -70% loss in reaction amplitude. Moreover, in all cases the values extrapolated to an ordinate intercept indistinguishable from the expected value of 1. Deviations from linearity at residual

1

2t 1 1 1 1

0 1 2 3 4 5 6

[AT-Heparin1 $I41

FIG. 4. Effect of p-aminobenzamidine on the limiting inactivation rate constant and reaction amplitudes for the heparin-catalyzed thrombin-AT reaction. Average values of koba ( A ) and reaction amplitudes ( B ) were measured for single turnover reactions of AT-heparin complex with thrombin as described in the legends to Figs. 2 and 3 as a function of AT-heparin complex concentration (0.6-6 PM heparin and a 1.2-1.5-fold molar excess of AT (0.75-7 PM)) a t p-aminobenzamidine concentrations of 200 (0) or 1,000 (0) PM. Reactions were conducted at 25 "C in sodium phosphate buffer, pH 7.4, containing 0.1 M NaCl. Data are also shown from Fig. 5B at 4.9 PM AT-heparin complex and probe concentrations of 25 (A), 50 (A), 100 (0) p ~ . Solid lines show a global fit of all rate constant data to Equation 8 and all amplitude data to Equation 11, assuming K T . ~ = 64 PM. Fits of amplitude data also assumed R = 0.83, based on fluorescence measurements in the stopped-flow fluorometer, and the value of KT,ATH determined from fits of the rate constant data. Amplitudes are expressed relative to the thrombin concentration and are normalized to the best fit value of AFo. Pre- dicted hyperbolic curves for all p-aminohenzamidine concentrations studied in panel A (increasing from top to bottom) are shown only to illustrate the approach of these curves to a limiting rate constant similar to that in Fig. 3.

amplitudes less than 30%, which were attained only at the lowest salt concentrations, reflect nonproductive binding of thrombin to AT-heparin complex, as shown below. The solid line fits of the amplitude data of Fig. 3B to the untransformed Equation 5 indicated values of KT,ATH in fair agreement with those determined from the k& dependence (Table 11), thus confirming that we were measuring the ternary complex encounter of heparin-bound antithrombin with the active site of thrombin at each NaCl concentration.

Evidence for Nonproductive Binding of Thrombin to Anti- thrombin-Heparin Complex-While the kinetic data for the heparin-accelerated thrombin-AT reaction in the presence of p-aminobenzamidine appeared to be adequately described over the NaCl concentration range of 0.15-0.35 M by the simple competitive model of Scheme 2 (with the exception of the amplitude data at lower ionic strength), this model was insufficient to account for similar kinetic data obtained at 0.1 M NaCl (Z 0.15). Thus, Fig. 4A shows that when higher p - aminobenzamidine concentrations were used to increase the apparent ternary complex dissociation constant, KT,ATH X (1 + [P]o/KT,p), at this salt concentration, so that it would be in a measurable range (see Equation 3), saturation kinetics were observed, but the limiting inactivation rate constant was considerably lower than that obtained at higher salt concentrations and was dependent on the probe concentration (-1-


* ' ~ ~ z z Y L J 0 o SI l o o 150 mo m u a m a m ~ m ,

Cp-AMINOBENZAMIDINEI (pM) FIG. 5. p-Aminobenzamidine concentration dependence of

kobs for the heparin-catalyzed thrombin-AT reaction as a function of NaCl concentration. kobs was measured for single turnover reactions of AT-heparin complex with thrombin as described in the legends to Figs. 2-4 as a function of p-aminobenzamidine concentration and at molar NaCl concentrations of 0.1 (0, panel B ) , 0.15 (0, panel A ) , and 0.35 (0, panel A ) . Reactions were performed in pH 7.4 sodium phosphate buffer, 25 "C, using fixed AT-heparin complex concentrations of 4.9 @I (5 p M heparin, 6 FM AT) (e), 2.3 FM (2.5 p M heparin, 3 FM AT) (a), or 2.2 FM (2.5 p~ heparin, 7.5 pM AT) (0). Solid lines are linear regression fits.

3 s-'). Moreover, the amplitude dependence (Fig. 4B) indicated that an increasing fraction of enzyme-bound p-aminobenzamidine was not displaceable in the dead time reaction phase at saturating levels of AT-heparin complex. Analysis of the dependence of kobs on the p-aminobenzamidine concentration by a reciprocal plot (Fig. 5B), which from Equation 3 has the linear form,

1 / h = (1 + KT,ATH/[AT.H])/~H ( 7 )

+ (KT.ATH/(~H[AT.H~KT,P))[P~~ yielded a satisfactory fit to a straight line. However, the intercept/slope of this line together with the value for KT,ATH

predicted at this NaCl concentration from its salt concentration dependence (Fig. 6), indicated a value for the thrombin- p-aminobenzamidine dissociation constant (KT,~) of 12 f 2 p ~ , substantially different from the value determined by equilibrium binding (Table I) . This contrasted with the good agreement between kinetic and equilibrium values for KT,P found at higher salt concentrations (52 k 10 uersus 58 -+ 6 p ~ , respectively, at 0.15 M salt and 69 -+ 3 versus 61 -+ 2 pM, respectively, at 0.35 M salt; cf. Fig. 5A and Table I), which affirmed the simple competitive model of Scheme 2. This problem was not due to heterogeneous binding of p-amino-

v B n

d

u)

0

Y

m - I I 1 I I

-. B -. 6 -. 4 -. 2 0

log [No+] (MI

FIG. 6. Dependence of log on log [Na+] for binary and ternary complex interactions. Values of KD,obs for the indicated interactions, given in Tables I and 11, were measured in this or the preceding study (28) as a function of NaCl concentration in 20 mM sodium phosphate, pH 7.4, 25 "C. Sodium ion concentrations were calculated from the concentrations of salt and buffer components. Open circles and triangles shown for KT,H represent measurements with low affinity and high affinity heparins, respectively, of the same molecular weight. Solid lines are linear regression fits.

in which thrombin was bound to heparin but not to AT (Scheme 3).

KT.P P .T -P + T + AT.H I T.AT.H"*T-AT + H

KT.ATH kH

+ + AT.H AT.H

* I

SCHEME 3

In this scheme, KT,ATH represents the dissociation constant for the productive binding between thrombin and AT-heparin complex that results in displacement of the probe from the enzyme active-site, while K+,ATH represents the dissociation constant for the nonproductive binding of these components which does not result in probe displacement. The independence ofp-aminobenzamidine and heparin binding to thrombin implied in this model is supported by equilibrium binding studies of these interactions (9, 28). The dependence of kobs

and reaction amplitudes on AT-heparin complex concentration predicted by Scheme 3 is given by Equations 8-12 (derived in the "Appendix"):

kH[AT. HI kobs =

KT.ATH(~ + [P]o/KT,P)(~ + [AT.H]/K~,ATH) + [AT.H] (8)

- - (kH/(1 + KT,ATH(~ + [P]O/KT.P)/K~,ATH))[AT.H] KT,ATH(~ + [PIO/KT,P)/(~ + KT.ATH(~ + [P]o/KT,P)/K~,ATH) + [AT.Hl

(9)

benzamidine to thrombin at the lower salt concentration, since the nonfluorescent inhibitor, benzamidine, was able to + (KT.ATH(~ + [AT.H]/K~.ATH)/(~H[AT.HIKT.P))[PIo completely displace p-aminobenzamidine (up to 96%) from thrombin under these conditions, in the manner predicted by - KT,ATH(l + [PIO/KT,P)(l + [AT.HI/K+,ATH) + ,AT.Hl - (11)

Equation 5 for a pure competitive inhibitor with a KO similar to the KI previously determined by steady-state kinetics (29, 9 - K+.ATH + (1 + K~,ATH/KT.ATH(~ + [PIO/KT.P))[AT.H] (12) not shown). These observations suggested that binding of p - aminobenzamidine and AT-heparin complex to thrombin was not strictly competitive, as implied by the simple model of where R is the ratio of the fluorescence yields of P - T . H . A T Scheme 2. Such behavior could be rationalized by a nonpro- and P.T complexes of Scheme 3 (measured to be 0.83 under ductive mode of thrombin binding to the AT-heparin complex, the conditions of the kinetics experiments). Comparisons of

l/k,t,, = (1 + KT.ATH(~ + [AT.H]/K;.,ATH)/[AT.H])/~H (10)

KT,ATH(~ + [P]o/KT,P)(~ + R[AT.HI/K+.ATH)

- K+,ATH + R[AT.Hl


Equations 8 and 11 with Equations 3 and 5 indicates that nonproductive binding of thrombin to the AT-heparin complex adds a competitive factor (1 + [AT-H]/K&,ATH), analo- gous to the competitive factor, (1 + [H]~,,/K&,H), previously shown to describe nonproductive binding of thrombin to free heparin chains. In the latter expression K+,H is the ratio of the intrinsic KT,H to the number of nonspecific sites on the free heparin chain (i.e. the apparent KO for the binding of a single thrombin molecule to the free heparin chain; see Ref. 9 and the preceding paper). The rearranged form of Equation 8 given by Equation 9 (see “Appendix”) reveals that nonproductive binding still results in a rectangular hyperbolic dependence of kobs on AT-heparin complex concentration, but with the limiting inactivation rate constant kH, and the apparent dissociation constant for productive ternary complex formation, KT,ATH (1 + [p]o/K~.p), both reduced by a factor (1 + KT,ATH(l + [P]o/KT,~)/K~,ATH) which increases as the ratio of productive to nonproductive dissociation constants increases. Nonproductive binding is thus not evident when AT- heparin complex concentrations are well below saturation with respect to ternary complex formation, since both Equa- tions 9 and 3 reduce to Equation 4 in this case. The reciprocal of Equation 8 (Equation 10) indicates that l/kobs remains linearly dependent on [PIo, but with the intercept and slope of this dependence being a function of both productive and nonproductive dissociation constants. Equation 12 indicates that nonproductive binding results in a nonlinear dependence of reciprocal reaction amplitudes on AT-heparin complex concentration that approaches a limiting fractional amplitude of R/(1 + K&,ATH/KT.ATH(~ + [P]O/KT.P)) when [AT.Hl >> K&,ATH. This limiting nondisplaceable fraction of enzyme- bound p-aminobenzamidine is also predicted to increase as the ratio of productive to nonproductive dissociation constants increases.

A global fit of the data of Fig. 4A and 5B to Equation 8 and of Fig. 4B to Equation 11, in which KT,p was fixed at the equilibrium value of 64 p ~ , indicated that nonproductive binding satisfactorily accounted for the deviations from the simple competitive Scheme 2 (solid lines). Thus, the intrinsic rate constant for stable thrombin-AT complex formation of 4.8 k 0.5 s-l obtained from this fit (Table 11) agreed favorably with the NaCl concentration-independent value obtained at higher salt concentrations. Moreover, the dissociation constant obtained for nonproductive binding of thrombin to the AT-heparin complex (K&,ATH) of 0.7 f 0.4 and 1.3 f 0.4 p~ from rate constant or amplitude data, respectively, was similar to values of 0.8 to 1.1 pM measured for the apparent dissociation constant for thrombin binding to free heparin chains (K&,H) by equilibrium binding (Table I).4 This suggested that the nonproductive binding of thrombin to AT-heparin complex involved the binding of thrombin to a site on heparin that was not contiguous with bound antithrombin. The ratio of kH/KT,ATH of 40 f 20 pL”l s-’ obtained by fitting to the nonproductive binding model further agree with the second- order rate constant derived from the data of Fig. 2A by use of Equation 4, as predicted. Assuming a value for K&,ATH equal to K’;.,H also generated the expected equilibrium value of 70 f 30 pM for K T , ~ from the intercept/slope of the plot of Fig. 5B. The nonproductive binding model also accounted for the observed curvature in reciprocal amplitude plots of Fig. 3B at the lower NaCl concentrations of 0.15 and 0.2 M (Equation 12). Thus, a fit of these data to the untransformed Equation 11 indicated values for the nonproductive dissociation constant, K+,ATH, of 6.4 f 1.4 and 10 f 5 p~ at 0.15 and 0.2 M salt, respectively, similar to values of K+,H (Table I).4 Values

Since AT covers maximally -6 disaccharides in the AT-heparin complex (28), a t least half of the -13-disaccharide heparin used in

of the productive dissociation constant, KT,ATH, obtained from these fits were not distinguishably different from those determined by assuming Scheme 2. The absence of curvature in the amplitude plots at NaCl concentrations higher than 0.2 M precluded a determination of the nonproductive binding constant at these salt concentrations. However, an estimate of the effect of nonproductive binding on the measured values of KT,ATH was made by assuming that K&,ATH was equal to K&,H (Table I) and then fitting the kinetic data of Fig. 3 to Equations 8 and 11. This indicated at most a 10-20% error in KT,ATH and kH values measured assuming the model of Scheme 2 (Table 11), consistent with the ratio of productive to assumed nonproductive dissociation constants.

Comparison of the NaCl Concentration Dependence of Bi- nary and Ternary Complex Interactions-Fig. 6 summarizes the NaC1-concentration dependence of all the binary and ternary complex interactions determined in this study as a plot of log KD,obs versus log [Na’]. According to polyelectrolyte theory (33-36), this plot can be used to estimate the ionic and nonionic contributions to the binding energy of a protein- polyelectrolyte interaction, as well as the number of ionic interactions involved, from the equation:

1% KD.ohs = log + zIc.lOg [Na’] (13)

where Kn represents the dissociation constant for the nonionic component of the binding interaction at 1 M Na+, 2 is the number of ionic interactions involved in the binding, and $ is the fraction of a counterion which is bound to the polyelectrolyte per ionic charge and which is released upon the binding of the protein. The ionic strength-independent value of $ calculated for heparin from its axial charge density is -0.8 (28). The dependence of log on log [Na’] for the two protein-heparin binary complex interactions conformed to the linear relationship of Equation 13. The slopes of these lines (4.8 f 0.2 for KT,H and 3.8 4 0.1 for KAT,H), together with the value of $, indicated that 5-6 and 4-5 ionic interactions contribute to thrombin and AT interactions with heparin, respectively. The latter value is in agreement with a previous study (39). The intercepts at 1 M Na’ further suggested a minimal nonionic component for the thrombin-heparin interaction (log KD -0.8 f 0.1) but a substantial nonionic component for the AT-heparin interaction (log Kn -4.6 f O. l ) , consistent with the known specificities of these binding interactions (1-9, 28). The dependence of log K I ~ , ~ ~ ~ on log [Na’] for the ternary complex interaction of thrombin with AT- heparin complex was similar to that of the thrombin-heparin binary complex interaction. Since the ternary complex interaction reflects the binding of free thrombin to antithrombin already bound to heparin, it should contain no contribution of the AT-heparin interaction (see “Discussion”). The similarity of the sodium ion concentration dependence of the ternary complex interaction with that of the AT-heparin binary complex interaction must therefore reflect the coinci- dental similarity of the sodium ion concentration dependence of the two protein-heparin interactions. The dependence of log KD,obs on log [Na’] for the thrombin ternary complex interaction fit well the linear relationship predicted by Equa- tion 13. From the slope of 4.2 f 0.2, a substantial ionic component involving -5 ionic interactions was indicated, in reasonable agreement with that of the thrombin-heparin binary complex interaction. The nonionic component of the thrombin ternary complex interaction obtained from the intercept at 1 M Na+ (log KD -3.3 f 0.1) was similar in

these studies would still be available for thrombin to bind. This suggests that K+.ATH measured kinetically should be comparable in magnitude (i.e. a t most 2-fold greater) to KG,,, measured independ- ently by equilibrium binding.


magnitude to the NaC1-independent binding affinity determined for the thrombin-AT encounter complex interaction (log KO -2.9-3.0, Table I). These results suggest that the binding energy of the thrombin ternary complex interaction is approximately accounted for by the sum of the binding energies of the thrombin-heparin and thrombin-AT binary complex interactions.

DISCUSSION

Previous rapid-reaction studies have shown that heparin accelerates the inactivation of thrombin by AT by binding to AT and thereby promoting the initial encounter between the inhibitor and proteinase in a thrombin-AT-heparin ternary complex without significantly affecting the rate a t which this intermediate complex is converted to a stable thrombin-AT complex (9, 27). Two mechanisms were considered in these studies as explanations of how heparin binding to AT may promote the subsequent binding of AT to thrombin to form the ternary complex: 1) the conformational change induced in AT by heparin binding may make the inhibitor more complementary to the thrombin active site, thereby resulting in a greater affinity of the inhibitor for the proteinase (3, 8, 12, 14, 22-24); or 2) both AT and heparin may interact with thrombin in the ternary encounter complex, thereby providing additional binding energy for the proteinase-inhibitor interaction (4, 7-9, 14-21, 26, 27, 40). The purpose of the present study was to distinguish the contributions of these two mechanisms to heparin rate enhancement from the different salt concentration dependencies of ternary complex formation predicted by the two mechanisms. Thus, since mechanism 2 postulates that an ionic thrombin-heparin interaction is re- sponsible for facilitating the interaction between proteinase and inhibitor in the ternary complex, this mechanism predicts that ternary complex formation from thrombin and AT- heparin complex should exhibit a strong dependence on salt concentration. Moreover, this dependence should parallel the salt concentration dependence of the thrombin-heparin binary complex interaction. In contrast, mechanism 1, which postulates that the AT conformational change promotes the interaction between proteinase and inhibitor in the ternary complex, predicts that only a protein-protein interaction is involved in forming the ternary complex from thrombin and AT-heparin complex. Therefore, a weaker dependence on salt concentration that is different from that of the thrombin- heparin binary complex interaction should be observed in this case.

Implicit in the above predictions is that the salt dependence of the ternary complex interaction reflects that of thrombin binding to antithrombin that is already bound to heparin and contains no contribution of antithrombin binding to heparin. Kinetic studies conducted under conditions where heparin was always saturated with antithrombin thus showed that kOhs

was proportional to the AT-heparin complex concentration over a range that was subsaturating with respect to ternary complex formation and showed saturation behavior at higher AT-heparin complex concentrations, indicating that an intermediate ternary complex interaction between thrombin and AT bound to heparin was being measured. A rapid and saturable binding of the heparin-bound inhibitor to the active site of the enzyme was more directly evident from the fractional displacement of active site-bound p-aminobenzamidine from thrombin by AT-heparin complex in the stopped-flow dead time which paralleled the saturation curve for kohs. Analysis of ternary complex formation under conditions where heparin is saturated with AT and as a function of the AT-heparin binary complex concentration thus resolves the contribution

to ternary complex formation of thrombin binding to AT- heparin complex from that of AT binding to heparin.

In accordance with the prediction of mechanism 2, the ternary complex interaction between thrombin and AT-heparin complex was observed to be strongly dependent on NaCl concentration, and this dependence was found to parallel that of the thrombin-heparin binary complex interaction (Fig. 6). This observation together with the salt independence of the thrombin-AT binary complex interaction therefore clearly supports the conclusion that the salt dependence of the thrombin ternary complex interaction is due to thrombin interacting with heparin in the ternary complex. Because the ternary complex interaction should not contain any contribution from the AT-heparin binary complex interaction, the parallel salt dependence of the ternary complex interaction with that of the AT-heparin interaction must be regarded as a fortuitous consequence of the similar salt dependence of the two protein-heparin interactions. That the salt dependence of the ternary complex interaction, measured under conditions where heparin is saturated with antithrombin, is solely due to a proteinase-heparin interaction and contains no contribution of the AT-heparin interaction, is supported by similar studies of the heparin pentasaccharide. Thus, this small heparin fragment, which specifically binds AT and is not large enough to bind the proteinase, accelerates thrombin or factor Xa inactivation in a salt-independent manner when saturated with AT (40).

Because the thrombin-heparin binary complex interaction is essentially all ionic, whereas the thrombin-AT binary complex interaction is predominantly nonionic, the contribution of thrombin-heparin and thrombin-AT interactions to the thrombin ternary complex interaction must be represented by the ionic and nonionic components, respectively, of the ternary complex intera~t ion.~ The nonionic component of the ternary complex interaction is given by the extrapolated at 1 M Na' (0.5 X M). The similarity of this value to the &,oh$ for the nonionic thrombin-AT interaction measured in the absence of heparin (1 X M ) thus indicates that the binding energy provided by the thrombin-AT interaction in the ternary complex approximates that measured for the proteinase-inhibitor interaction in the absence of heparin. This implies that the AT conformational change does not significantly alter the affinity of AT for thrombin in the ternary complex. The ability of heparin to promote the binding of AT to thrombin in the ternary complex and thereby accelerate the thrombin-AT reaction thus can be mainly accounted for by the additional binding energy contributed by an ionic thrombin-heparin interaction.

This conclusion also follows from the observation that the binding energy of the thrombin ternary complex interaction is well accounted for by the sum of the binding energies of the thrombin-heparin and thrombin-AT binary complex interactions (Fig. 6). This is evident by noting that log KT.AT approximates and even somewhat exceeds the difference between log KT,H and log KT,ATH over the range of NaCl concentrations examined. Because the binding energy derived from the thrombin-heparin interaction results from an ion-exchange process (28), the contribution this interaction makes

This conclusion assumes that the salt concentration independence of the thrombin-AT interaction measured in the absence of heparin is unaffected by the conformational change induced in antithrombin by heparin in the thrombin-AT ternary complex interaction. This assumption is supported by the salt concentration independence of the 1.7-fold thrombin-AT reaction rate enhancement produced by the specific heparin pentasaccharide, which binds to AT and induces a conformational change in a manner similar to that of natural length heparins (40).


to the binding energy of the thrombin ternary complex interaction is expected to be additive to that of the thrombin-AT interaction (33-37).6 The demonstration of such additivity shows that no additional contribution of the AT conformational change to stabilization of the ternary complex need be postulated. The fact that the sum of the binding energies of the binary complex interactions somewhat exceeds the ternary complex binding energy can be accounted for by: 1) the experimental uncertainty in the weak affinity measured for the thrombin-AT binary complex interaction by saturation kinetics (27); 2) the possible underestimation of the statistical number of thrombin-binding sites on heparin, which would result in the intrinsic affinity of the thrombin-heparin binary complex interaction being too high (28); or 3) the small difference in slopes of the log Ko,,b./log [Na’] plots observed for the thrombin-heparin binary and ternary complex interactions reflecting a smaller number of ionic interactions involved in forming the ternary complex as compared to the binary complex, due to orientational constraints imposed by the protein-protein interaction.

The quantitative contribution shown here of thrombin- heparin and AT-heparin interactions to heparin’s accelerating effect on the thrombin-AT reaction, and the insignificance of the contribution from the AT conformational change, supports earlier proposals of the essential role of inhibitor and proteinase binding to heparin (1 ,2,4, 15-21, 26). The results are thus consistent with heparin acting primarily as a surface to approximate the proteinase and inh ib i t~r .~ Previous proposals of this mechanism were based on: 1) the minimum heparin chain length of 18 saccharide residues required to accelerate the thrombin-AT reaction as well as to bind both AT and active site-blocked thrombin (4, 15, 21, 25); 2) the evidence for distinct heparin-binding regions in thrombin and AT essential for heparin rate enhancement (1, 2, 16, 17); and 3) the observation that heparin rate enhancement can be diminished by excess heparin or active site-blocked thrombin which decrease the probability of heparin bridging AT and active thrombin in a ternary complex (7-9, 14, 18-20). Our results cannot exclude the possibility, however, that the AT conformational change plays a role in orienting the reactive bond of AT so that thrombin is able to interact with both AT and heparin in the ternary complex. In this case, both the AT conformational change and a thrombin-heparin interaction would be required to promote the thrombin ternary complex interaction, even though the thrombin-heparin interaction would still be the predominant source of binding energy to promote this interaction. Other evidence, however, does not favor such a mechanism. Thus, a series of chemically modified antithrombins, whose affinity for heparin and ability to undergo the conformational change was reduced to varying extents, nevertheless all had their reactions with thrombin accelerated to the same extent by saturating concentrations of heparin (26). This result suggests that the AT conformational change is not required for the inhibitor to optimally interact with the proteinase in the ternary complex.

The evidence which has supported a primary role for the AT conformational change in mediating heparin’s rate en-

This additivity presumably is a consequence of the electrostatic binding of thrombin to heparin not being associated with the loss of entropy which accompanies a site-specific interaction due to the protein or condensed counterions being free to diffuse in a restricted volume surrounding the polyelectrolyte chain (33). ’ The surface approximation mechanism has also been labeled the

“template” mechanism (18-20). We prefer the former designation since the term template suggests a specific mode of interaction between heparin and the reacting proteins, which is true for AT but not thrombin.

hancing effect on the thrombin-AT reaction has included 1) the substantial rate enhancement remaining at high heparin concentrations, at which AT and thrombin are presumed to be bound to separate heparin chains (8, 14); and 2) unique spectroscopic changes in AT which are induced by the larger heparin chains required to accelerate the thrombin-AT reaction and which are indicative of a further conformational change (22, 23). However, the magnitude of the heparin rate enhancement remaining at high heparin concentrations, as well as the interpretation of the unique spectroscopic changes produced in AT by larger heparin chains in terms of an additional conformational change rather than a change in the electrostatic environment of surface AT residues, has been disputed in subsequent investigations (20, 25, 40). Moreover, evidence for two distinct binding sites on thrombin for heparin (28,41) suggests that an approximation of AT and thrombin on the same heparin molecule may still be possible even when thrombin is also bound to another heparin chain. This could account for the heparin rate enhancement observable at high heparin concentrations. Finally, the similar affinities of natural length and oligosaccharide heparins for AT (14,25, 40) are inconsistent with an additional conformational change being induced in AT by natural length heparin chains, since such a conformational change would be expected to produce a substantial increase in heparin binding affinity (11).

Another proposal which has emphasized a primary role for the AT conformational change in mediating heparin rate enhancement has suggested that thrombin binding to heparin plays a secondary role by neutralizing the cationic charge of thrombin, so that an unfavorable interaction with the posi- tively charged inhibitor is avoided (24). Our results suggest, however, that thrombin or AT charge neutralization plays no role in promoting the interaction between AT and thrombin in the ternary complex. Thus, if this were so, the interaction between thrombin and AT in binary and ternary complexes should show a strong dependence on salt concentration, due to the screening effect of salt on the charges of the interacting proteins. Instead, our results indicate that the inhibitor-proteinase interaction is independent of the salt concentration in the absence of heparin and that the salt dependence of the ternary complex interaction is completely accounted for by that of the thrombin-heparin interaction. These results are therefore consistent with the interaction between thrombin and AT not being influenced by the surface charge of the two proteins either in the absence or presence of h e ~ a r i n . ~

Although our results suggest that the AT conformational change plays little if any role in promoting the interaction of AT with thrombin in the ternary complex, it nevertheless plays an important role in promoting the high affinity binding of AT to heparin (11, 26). Moreover, the loss of this conformational change which accompanies stable thrombin-AT complex formation facilitates the release of heparin from the reaction product and allows the polysaccharide to recycle as a catalyst (31). In the case of the heparin-accelerated factor Xa-AT reaction, however, the A T conformational change may additionally act tc, promote the initial proteinase-inhibitor interaction (6, 14, 15, 37, 40). Thus, the ability of the pentasaccharide which comprises the AT-binding region of heparin to produce a rate enhancement of the factor Xa-AT reaction comparable to that of natural length heparins (6), together with the independence of these rate enhancements on the NaCl concentration (40), is consistent with this conclusion.

The present study has also provided evidence for nonproductive binding of thrombin to the AT-heparin complex. This behavior was not unexpected based on the nonspecific electrostatic mechanism of thrombin binding to heparin demonstrated in the preceding paper (28). The ability of thrombin


to interact nonspecifically with any 3-disaccharide unit along the heparin chain (28) thus indicates that many nonproductive binding sites for thrombin will exist in the AT-heparin complex in addition to the productive thrombin-binding site adjacent to the bound i n h i b i t ~ r . ~ Kinetic experiments de- signed to measure ternary complex saturation of thrombin by increasing AT-heparin complex concentrations must thus account for the competitive effect of nonproductive sites, whose number is increased concurrently with productive sites. Nonproductive binding of thrombin to AT-heparin complex was evident from the incomplete displacement of the fluorescence probe p-aminobenzamidine bound at the thrombin active site by saturating levels of AT-heparin complex and from the reduction in the limiting first-order rate constant for stable thrombin-AT complex formation a t high probe concentrations. These effects result from: 1) thrombin forming productive and nonproductive ternary complexes in a constant ratio as AT-heparin complex concentrations are increased, and 2) high probe concentrations reducing the competitive advantage of thrombin binding to the productive site in the AT-heparin complex as compared to nonproductive sites in this complex. The magnitudes of these effects depend on the ratio of productive to nonproductive ternary complex dissociation constants, given by KT,**” (1 + [PIO/KT,p)/K&,ATH,

which is a function of probe concentration. It follows that the simple competitive model of Scheme 2 satisfactorily describes kinetic data when the ratio of productive to nonproductive dissociation constants is much less than 1 or when AT- heparin complex concentrations do not reach saturating levels, as was the case at higher NaCl concentrations.

Because all of the complexes involved in assembling the productive ternary complex are in rapid equilibrium, it is possible that the nonproductive ternary complex is actually an intermediate on the pathway to the productive ternary complex, as depicted in Scheme 4, rather than the dead end complex implied by Scheme 3. Thus, a direct pathway from the nonproductive to the productive ternary complex is ther- modynamically equivalent to an indirect pathway in which the nonproductive ternary complex dissociates and then reas- sociates productively. The former possibility would be in keeping with established mechanisms for the binding of proteins to specific sites on polyelectrolyte surfaces, in which an intermediate binding of the protein to nonspecific sites is followed by diffusion of the protein on the polyelectrolyte surface to the specific site (20, 42).

To summarize, the results of the present and past studies support the reaction mechanism of Scheme 4 for heparin’s accelerating effect on the reaction between thrombin and AT, with dissociation constant and rate constant values given in Scheme 4 referring to pH 7.4, I 0.15, and 25 “C. Thrombin, AT, and heparin are first assembled by a series of rapid equilibrium binding steps into a productive ternary complex, in which both thrombin and AT are bound to heparin, and an active site-dependent interaction between the inhibitor and proteinase is established. This assembly occurs primarily by AT binding to heparin prior to thrombin due to the much greater affinity of AT for heparin, although a pathway to the ternary complex through a thrombin-heparin binary complex intermediate is possible (9).’ The greater affinity of AT for

The ordered binding of first AT and then thrombin to heparin to form the ternary complex intermediate follows from the large difference in AT. H uersus T.H affinities because these affinities dictate that the concentration of the AT.H binary complex will in most cases greatly exceed that of the T.H binary complex, given that all complexes remain equilibrated during the reaction. Assuming similar diffusion-limited rates of association of all complexes, ternary complex formation will thus occur preferably from the predominant binary complex intermediate (9).

AT-H

+ H T-AT

SCHEME 4

heparin results from the inhibitor binding to a specific polysaccharide-binding site (4-6), which causes AT to change from a low affinity to a high affinity heparin binding conformation (11). Thrombin subsequently binds to the AT-heparin complex, but may do so in a nonproductive mode as well as in a productive mode. In the former mode, thrombin binds to any one of a number of nonspecific sites on the heparin surface, which are not contiguous with the bound inhibitor. In the latter mode, thrombin binds to a “specific” site adjacent to AT that facilitates an interaction between the proteinase and inh ib i t~r .~ Diffusion of thrombin along the heparin surface may result in the direct transformation of the nonproductive to the productive ternary complex. The net result of this process is the promotion by heparin of an intermediate proteinase-inhibitor interaction, mainly through an approximation of the two proteins on the heparin surface. The conformational change induced in AT by heparin contributes insignificantly to the intrinsic affinity of this interaction. The productive ternary complex is subsequently converted to the stable thrombin-AT complex at a rate of 5 s-’, which is relatively unaffected by heparin. This step is accompanied by a return of AT to a conformation with low affinity for heparin that promotes the concomitant release and catalytic recycling of heparin (31). According to this mechanism, heparin rate enhancement is achieved mainly through an ordered assembly of AT and thrombin on the heparin surface mediated by a specific inhibitor “receptor” site on the polysaccharide. Such a mechanism may provide a model for other receptor-mediated processes which involve the assembly of multiprotein complexes on surfaces.

1. 2.

3.

4.

5.

6.

7.

8.

REFERENCES

Bjork, I., and Lindahl, U. (1982) Mol. Cell. Biochem. 48, 161-182 Bjork, I., Olson, S. T., and Shore, J. D. (1989) in Heparin:

Chemical and Biological Properties, Clinical Applications (Lane, D. L., and Lindahl, U., eds) pp. 229-255, Edward Arnold Ltd., London

Rosenberg, R. D., and Damus, P. S. (1973) J. Biol. Chem. 248, 6490-6505

Laurent, T. C . , Tengblad, A., Thunberg, L., Hook, M., and Lindahl, U. (1978) Biochem. J . 175, 691-701

Thunberg, L., Backstrom, G., and Lindahl, U. (1982) Carbohydr. Res. 100, 393-410

Choay, J., Petitou, M., Lormeau, J-C., Sinay, P., Casu, B., and Gatti, G. (1983) Biochem. Biophys. Res. Commun. 116, 492- 499

Jordan. R.. Beeler, D., and Rosenberg, R. (1979) J. Biol. Chem. 254,’2902-2913

. .

Jordan. R. E.. Oosta. G. M.. Gardner. W. T., and Rosenberg, R. D. (1980) J.’ Biol. Chem. 255, 10081-10090

9. Olson, S. T. (1988) J. Bid. Chem. 263, 1698-1708 10. Olson, S. T., and Shore, J. D. (1981) J. Biol. Chem. 256,11065-

11. Olson. S. T.. Srinivasan. K. R., Biork, I., and Shore, J. D. (1981) 11072

J. Biol. Chem. 256, 11073-11079 12. Carrell, R. W., Christey, P. B., and Boswell, D. R. (1987) in

The productive site is specific not in terms of structure but only as a consequence of its localization adjacent to the AT-binding site.

13. 14.

15.

16.

17.

18. 19. 20.

21.

22.

23. 24.

25.

26.

27.


Thrombosis and Haemostasis (Verstraete, M., Vermylen, J., Lijnen, H. R., and Arnout, J., eds) pp. 1-15, Leuven University Press, Leuven, Belgium

Olson, S. T., and Choay, J. (1989) Thromb. Haemostasis 62,326 Oosta, G. M., Gardner, W. T., Beeler, D. L., and Rosenberg, R.

Lane, D. L., Denton, J., Flynn, A. M., Thunberg, L., and Lindahl,

Pomerantz, M. W., and Owen, W. G. (1978) Biochim. Biophys.

Machovich, R., Staub, M., and Patthy, L. (1978) Eur. J . Biochem.

Griffith, M. J. (1982) J. Biol. Chem. 257, 7360-7365 Nesheim, M. E. (1983) J. Bid. Chem. 258,14708-14717 Hoylaerts, M., Owen, W. G., and Collen, D. (1984) J. Biol. Chem.

Danielsson, A, Raub, E., Lindahl, U., and Bjork, I. (1986) J. Biol.

Stone, A. L., Beeler, D., Oosta, G., and Rosenberg, R. D. (1982)

Gettins, P., and Choay, J. (1989) Carbohydr. Res. 185, 69-76 Beresford, C. H., and Owen, M. C. (1990) Znt. J . Biochem. 22,

Lindahl, U., Thunberg, L., Backstrom, G., Riesenfeld, J., Nor- dling, K., and Bjork, I. (1984) J. Biol. Chem. 259,12368-12376

D. (1981) Proc. Natl. Acad. Sci. U. S. A . 78,829-833

U. (1984) Biochem. J. 218,725-732

Acta 535,66-77

83,473-477

259,5670T5677

Chem. 261, 15467-15473

Proc. Natl. Acad. Sci. U. S. A. 79, 7190-7194

121-128

Peterson, C. B., and Blackburn, M. N. (1987) Jececec Biol. Chem. 262, 7559-7566

Olson, S. T., and Shore, J. D. (1982) J. Biol. Chem. 257, 14891-

28.

29.

30. 31.

32. 33. 34.

35.

36.

37.

38.

39.

40.

41.

42.

14895 Olson, S. T., Halvorson, H. R., and Bjork, I. (1991) J. Bid. Chem.

266,6342-6352 Evans, S. A., Olson, S. T., and Shore, J. D. (1982) J. Biol. Chem.

257,3014-3017 Latallo, Z. S., and Hall, J. A. (1986) Thromb. Res. 43, 507-521 Olson, S. T., and Shore, J. D. (1986) J. Biol. Chem. 261, 13151-

Duggleby, R. G. (1984) Comp. Bid. Med. 14, 447-455 Manning, G. S. (1978) Q. Reu. Biophys. 11, 179-246 Record, M. T., Jr., Lohman, T. M., and deHaseth, P. (1976) J.

Record, M. T., Jr., Anderson, C. F., and Lohman, T. M. (1978)

Lohman, T. M., deHaseth, P. L., and Record, M. T., Jr. (1980)

Craig, P. A., Olson, S. T., and Shore, J. D. (1989) J. Biol. Chem.

Mares-Guia, M., and Shaw, E. (1965) J. Biol. Chem. 240, 1579-

Nordenman, B., and Bjork, I. (1981) Biochim. Biophys. Acta 672,

Olson, S. T., Bjork, I., Craig, P., Shore, J. D., and Choay, J.

Naski, M. C., Fenton, J. W., 11, Maraganore, J. M., Olson, S. T.,

Winter, R. B., Berg, 0. G., and von Hippel, P. H. (1981) Biochem-

13159

Mol. Bid. 107, 145-158

Q. Reu. Biophys. 11, 103-178

Biochemistry 19, 3522-3530

264, 5452-5461

1585

227-238

(1987) Thromb. Haemostasis 58, 8

and Shafer, J. A. (1990) J. Bid. Chem. 265, 13484-13489

istry 20, 6961-6977

SUPPLEMENTARY MATERIAL

FOR

I IFPARIN ACCELERATION OF THE ANTTHROMBIN/THROMBIN REACTION. Elucidation from I'II1.DOMINANTCONTRlBUTlON OF SURFACE APPROXIMATION TOTHE MECHANISM 01.

Salt Concentration Effects

Steven T. Olson and lngemar Bjock

Heparin-Antithrombin-Thrombin Reaction Mechanism Continuous kinetic measurements monitored byp-minabemmidine displacement were performed

in a stopped-flow fluorometer which was interfaced to a Northstar computer and operated with commercial software (OLIS, Jefferson. Georgia), as described previously (27.31). Thrombin (0.05.1 pM) andp-

vmmohenzamidine (25 DM-5 mM) in one syringe were mixed with heparin (0.5-30 PM) and a I.I- l0-fold malar cxccss o f A T over the polysaccharide (0.75-32 $M) in the other syringe (final concentrations after

included in the A T syringe and the heparin in the thrombin syringe with no effect on the resulu. In both

mixing) following tcmperature equilihratmn for at least 5 minuter. In some experiments. the probe was

complex andp-aminobenramidine over thrombin in the final reaction mixture (9). AT-heparin complex

cases. pseudo-first-order conditiom were achieved by having at least an 8-fold molar excess of AT-heparin

concentrations were calculated from measured dissociation constants by the quadratic formula (9). whhch showed that heparin was 80.98% saturated with AT, Reduction of the heparin-accelerated reaction rate due

due to the free heparin concentralion being at l e u t IO-fold lower than the apparent dissociation constant

to residual free heparin competing with AT.heparin complex far the available thrombin could he neglected

WPS ealmlhtcd to contribute as much as 17% to robs i n the experiment o f figure 2. bared on measured

for the nonspecific thromhin-heparin hinary complex interaction under these conditions (9). The CXECSI A T

second-order rate constants for the heparimindependent reaction, and this contribution war therefore

subtracted (9). In all other experiments. the contribution by excess A T was at most 7% and was thus

filter having a cutoff at 350 nm and 50% transmission at 357 nm. Reaction traces were fit to a single

neglected. The exponential decrease in probe fluorescence was monitored at Lex 330 nm with an emision

exponential function to give kObs and the reaction amplitude (9), with at least four duplicate runs averaged for each set of reactant concentrations. A t the lowest thrombin concentrations (0.05-0.3 pM). where the

signal WPI noisy. 2-4 reaclion traces were averaged. and at least four rueh averaged traces were used for

fitting to the exponential function. Thhe starting fluorescence due to throllhin-boundp-aminohenlamidinc

was determined by recording the voltage after mixing the prohe in one syringe with buffer or increasing

ccmcentrations of thrombin in the other syringe. The extent ofquenching of Ihromhwbaund probe

syrlngc (5 pM in 10.1s buffer). Because of the ohrerved linear relationship between voltage and thrombin fluorescence by heparin was similarly measured by including saturating levels of heparin in the probe

concentration at a Constant probe concentration (fheparin). reaction amplitudes obtained at different

thrombin concentrations could be compared by normalizing them to a 1 pM thrombin concentration. Amplitudes were not analyzed in experiments where the probe concentration was varied due to variable inner filter effects arising from the absorbance of the probe. Nonlinear least squares fitting of kinethc daln l o

equations in Ihe text was performed with a published program (32) with reporred errors representing t2 S.E.

Analysis of the NaCl €mcsnmtion Deoendenre of P r o t e # n - H c w m h m a C m i The NaCl concentration

dependence of A T and thrombin interactions with heparin in binary and ternary complexes was analyzed

according to the polyelectrolyte theory developed by Manning and Record and coworkers for protein interilctions with linear polyelearolyter (33-35), as described in the preceding manuscript (28). Briefly. the

theory states that the interaction of a Z-valent paritivcly charged site on a protein (Pzt) with 2 adjacent

negative charges on a linear polyelectrolyte such as heparin (H) i n the presence Of a monovalent salt

involves the stoichiometric displacement of bound c o ~ n t e r i ~ n s ( M + ) according 10 the ion-exchange equilihrium:

PZt + H 7 P.H + Z#M+ (1)

%,here # represents the fraction of B monovalent countemn bound per heparin charge due to condensation

and screening effects. This fraction is a function only o f the axial charge density of the polysaccharide and i5

independent of the soluLion ionic Strength. The above equilihrium neglects anion release from the prolein.

which is not expccrcd to make a rignlficant contribution in comparison with M I release (28.34-36). The dissociation constant for this hinding equilihrium is given by:

. .

where KD obs represents the measured dissociation constant for the piotcin-polyelectrolyt~ interaction. For a nonspecific electrostatic protein-polyelectrolyt~ interaction. where there is overlap of protein binding rite, on the polyelcarolyte. is the intrinsic dissociation constant for the bindingof the protein to a single

polyclettrolyte site and can he obtained by methods described ~n the preceding manuscript (28). Taking

logarithms of both sides of this equatmn and rearranging yields the linear dependence of log KD,,,~~ on lrlg

[M'] given by e q u a t m 13 in "Results" for the case where M I is the sodium ion. The slope of this dependence thus provider the numher of ionic interactions involved in the binding of the protein 10 the

polyelcetrolyte, given the value for $ (0.80 for heparin (28)). and the intercept ut 1 M M' yields the true

KD for the ion-exchange equilihrium governing this intcrsction. The ionic contribusion to the binding

energy is therefore a function of the solution counterion concentration and given by RTZd In [M'l. The

nonionic contribution to the hinding energy is then given hy R T l n K D (at 1 M M'). K D should approximate a value of I (i.e.. log KD -0) far a protein-polyelectrolyte interaction governed completely by

nonspecific elccmstatic interactions. hut h a w a value l c s than 1 for a sequence-specific internclion (28).

APPEN!NX

Equrlionr 8 and 9 which dsrcrihe the nonproductive binding model of scheme 3 are derivcd as follows. The differential equation for the formation of the product o f the heparin accelerated thrombin/AT

reaction. Le., the stable T - A T complex. is:

d[T-AT] - = k H [T.AT.H]

dt From the mass action equation for the ternary T.AT.H complex. equation 1A can be written:

d[T-AT] k H IT] [AT.H] -=- ( 2 4

dt Thhe free thrombin concenfiafion m equation 2A can be expressed in terms of the slable thromhin-AT

KT.ATH

complex concentration and constant terms by using the conservation equation for the thrombin-containing species of scheme 3

[TI, = [T]+[P.T]+[T.H.AT]+[P.T.H.AT]+[T.AT.H]+p.AT] (3A)

where the zero subscript denotes the total concentration. From the mas action equations for each of the

noneovalent complexes of equation 3 4 this equation an be witten as:

[ T 1 ~ = [ T ] 1 ( 1 + [ ~ 1 o / K ~ . p + I A T . H I / ~ ~ , ~ m + [ P l o [ A T . H I / K ~ , p K ~ , ~ ~ ~ + [ A T . H I / K ~ , ~ m ) + [ T ~ A 7 1 ( 4 A )

where the sum in parentheses remains essentially constant under the pseudo-first order conditions of the kinetics experiments, [AT],,, [HI,, > > VI0 < < [PIw Solving equation 4A for the free thrombin

concentration and substituting hack into the differential equation 2A then yields:

d [T-AT] B - t - [T-AT] = ! [TIo dt (1 0

where a and Bare constants defined by the sum i n parentheses in equation 4A and the expression,

~HIAT.HI/KT,ATH, respectively. Equation SA is a first-order differential equation with the solution:

[T-Q = [T],,(l. e.kobst)

where (6.4)

~ H [ A T . H I / K T , A ~ koba = 8/11 =

( ~ ~ [ P I ~ / K T , ~ + [ A T . H I / K ~ , A ~ + [ ~ ~ ~ ~ A T . H I / ~ T , ~ ~ ~ , A ~ + [ A ~ . ~ I / ~ T , A ~ ) (7.4)

denominator yields equation 8 of the text. Equation 9 of the text is obtained by expanding and collecting

Multiplication of numerator and denominator o f the expression for kObs by K T , ~ ~ and factoring the

coefficient of the AT.H complex concentration term in the denominator. Differentiation of the comervation

terms i n the denominator o f equation 8 followed by division of bath numerator and denominator by the

equation 4A with respect to time followed by substitution of the mass action expression. [T.P]KT,~/[P]~, for

[T I and rearrangement yields the differential equation:

Equatlon 8A indicates that the loss of the observable fluorescence of thrombin-boundp-aminobenlamidine is proportional to the rate of stable thrombin-AT complcx formation and therefore decays with an identicol

kohn (9). The amplitude equation 11 of the text that applies to scheme 3 i s derived by first noting that the

observable fluorescence change which accompanies the inactivation of thrombin by antithrombin in the presence of heparin and the fluorescence probc.p-aminobcnramidine. under pseudo-first order conditions.

resuks ent~rely from the loss of the enhanced fluorescence of thrombin-probe complexes (9). The maximum

fluorescence change possible for this reaction is thus equal to the initial fluorescence of the thrombin-p-

amlnobenzamidine complex in the absence ofanrithrombin and heparin:

AFo = *p[T.P]i (9.4)

where e p is the fluorescence yield of the T.P (or P.7) complex under the conditions of measurement and

[he subscript, i, denotes the inital concentration of this complex. Addition of AT.H complex to the

([AT],, [HI,) ~esults in an immediate loss of a fraction of the slarting enzyme-bound prohe fluorescence.

thrombin-probe ~olut ion under conditions where the free heparin concentration is insignificant

This 8s due to the rapid equilibrium formation of productive and nonproductive complexes between T/T.P

species and the AT.H complex that reduce the initial concentration ar well as the fluorescence yield of

thrombiwprobe complexes (scheme 3) (9.27). The fluorescence of residual thrombin-probe complexes.

whch subsequently decays in an observable exponential phase due to the irreversible formation of the

m h l e thrombin-ATcamplex, is then given by:

AF = ~ T ~ [ T . P ] + ~ ~ H ~ T [ P . T . H . A T I ( 1 0 4

where +WHAT is the fluorescence yield Of the P.T.H.AT complex. The observed fluorescence change expresed relative to the total fluorescence change attainable (;.e., the limitingvalue as the e~ncentrations

of AT.H complex tend to zero) then becomes

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THE JOURNAL OF CHEMISTRY Vol ,266, No. 10, Issue April 5 ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Vol