Characterization of the interaction between prostacyclin and human serum albumin using a fluorescent analogue, 2,6-dichloro-4-aminophenol iloprost

74

Biochtmica et Btophysiea A eta, 993 (1989)74-82

Elsevier BBAGEN 23203

Characterization of the interaction between prostacyclin and human serum albumin using a fluorescent analogue,

2,6-dichloro-4-aminophenol iloprost Ah-lim Tsai, Ming-Jo Hsu and Kenneth

K. W u

Department of Internal Medicine, Division of Hematology/Oncology and Center for Vascular and Thrombosis Researcli. The Unit,ersi(v of Texas Health Science Center at Houston. Houston, TX (U.S.A.)

(Received 31 January 1989) (Revised manuscript received6 July 1989)

Key words: Prostacyclin;Prostaglandin; Fluorescentprobe; Dichloroaminophenoliloprost We synthesized a fluorescent probe, 2,6-dichloro-4-aminopbenol iloprost or dichlorohydroxypbenylamide of iloprost (DCHPA-iloprost) by reacting the stable prostacyclin analog, iloprost (ZK 35 374), with 2,6-dichloro-4-aminophenol with a yield of 60%, This probe exhibited an optical spectrum which overlapped with the emission spectrum of the sole tryptophan of human serum albumin (lISA). Energy transfer from the tryptophan residue to the phenol moiety of DClIPA-iloprost was observed. We utilized this donor-quenching phenomenon to quantitate the binding stoichiometry and affinity as well as the association rate o~ DClIPA-iloprost binding to HSA. As DCHPA-ilnprost showed similar binding characteristics similar to those of iloprost and prostacyciin and competed with iloprost for lISA binding sites, we used DCI-gPA-i|,~prost as a probe to locate the binding domain of prostacyclin (PGIz) in HoSA. The distance between the tryptophan indole and the phenol group of DCHPA-tgoprost was estimated to be 15-18 A. Because iloprost binding to HSA was competitive with warfarin and not with free fatty acid, we propose that PG| z binds to the 'domain 2' of HSA molecules. A possible molecular mechanism by which HSA reduces the chemical degradation of PGI z and stabilizes its activity could be derived from this model.

Introduction Prostacyclin (PGI2) is an important arachidonate metabolite involved in modulating physiological functions including hemostasis and vascular thrombosis. It has vasodilatory action on the vascular smooth muscle [1] and is a potent platelet suppressant agent [2]. These effects are most probably mediated by stimulation of adenylate cyclase [3,4]. PGI 2 also has a moderate fibrinolytic action [5] and cytoprotective effect against noxious stimuli [6,7]. The biological activity of PGI 2 is regulated by serum proteins. A full understanding of the regulation mechanism has been hampered by its chemical instability. Prostacyclin easily hydrolyzes to a

Abbr,;viations:PGI z. prostacyclin(prostaglandin I); (c)HSA, (crystalline) human serum albumin: DCHPA-iloprost, 2,6-dichlero-4aminophenol iloprost; Correspondence:A.-L. Tsai, Department of Internal Medicine Division of Hematology/-Oncology, The University of Texas Health Science Center at Houston, P.O. Box 20708, Houston, TX, 77225 U.S.A.

stable but inactive compound, 6-keto P G F ~ , in few minutes at physiological pH [8]. However, in the presence of serum or plasma, the half-life of the chemical degradation of PGI2 is extended for at least an order of magnitude [9,10]. We have investigated the mechanism of the chemical stabilization by binding-kinetic measurements using a stable PGI 2 analogue, iloprost [11]. Iloprost exhibited serum-binding properties almost identical to those of PG12, without the complication of rapid chemical hydrolysis. The kinetic data indicate that prevention of chemical hydrolysis of PGI 2 is provided mainly by direct binding with HSA [11,12]. In order to obtain detailed information concerning the interaction between PGI z and HSA for further elucidation of the protection mechanism, we prepared a fluorescent probe, 2,6-dichloro-hydroxyt~henylamide of iloprost (DCHPA-iloprost) to characterize the binding of prostacyclin to HSA. We demonstrated competitive binding between iloprost and its fluorescent analog. We used this probe t,: determine the binding stoichiometry, quantitate the binding parameters and measure the distance between the sole tryptophan residue in HSA and the phenol ring of the fluorescent probe based on the

0304-4165/89/$03.50 © 1989 ElsevicLScience Publishers B.V. (Biomedical Division)

75 mechanism of energy transfer. Together with bindingcompetition experiments using ligands with known binding sites in HSA, we were able to locate the possible binding domain of PGI 2 in the HSA molecule.

Experimental procedures Materials

Human serum albumin, crystalline (cHSA) and defatted, were purchased from Sigma and used directly without further treatments. 4-Amino-2.6-dichlorophenol was the product of Aldrich. Free acid itoprost was a generous gift of Dr. E. Schillinger, Schering Laboratories, F.R.G. The trometamol salt of iloprost and [ l i 3H]iloprost (14.8 C i / m m o l ) were purchased from Amersham, Chicago, IL, Coupling agent, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride, warfarin, 3-(a-acetonylbenzyl)-4-hydroxycoumarin and palmitic acid were obtained from Sigma. Other chemicals were reagent grade. Methods Synthesis of DCHPA-iloprost.

0.5 ml of iloprost (trometamol salt, 1.03 mM) was first mixed with 95% ethyl alcohol at 1 : 1 volume ratio, and 6 ~mol of fresh coupling agent was added and mixed well. 100 p,l of 100 mM 4-amino-2,6-dichlorophenol was introduced into the reaction mixture and the pH was adjusted to 5.0 by dilute hydrochloric acid. [3H]Iloprost was added at this point when percentage product conversion of the reaction was followed. The reaction mixture was covered by tin foil and incubated overnight at 24°C. A sma!! aliquot of the reaction mixture was removed and checked for product formation by thin-layer chromatography (Whatman K5F plate, silica gel 150A; F-254, 5 × 20 cm, thickness 250 /Lm) using a solvent system of chlorof o r m / m e t h a n o l (90:10). R v values for iloprost and DCHPA-iloprost were 0,16-0.18 and 0.41-0.43, respectively. The fluorescent probe, DCHPA-iloprost, was purified by a reverse phase HPLC C18 column (RadialPak Cartridge contained in a Z module radial compression separation system, Waters. MA) using a solvent system containing acetonitrile, acetic acid and water [13]. DCHPA-iloprost was eluted at 55% acetonitrile. To confirm that the collected fractions contained a single compound, reinjection was performed using the same solvent system or a methanol-based solvent system. The product was stable for at least 1 month if not exposed to light. Spectrometry. Optical spectra of iloprost and various aminophenol derivatives were determined using an IBM 9430 UV-VIS spectrophotometer. Fluorescence spectra were acquired on a SLM SPF 500C spectrofluorometer with built-in microprocessor control and thermostated observation chamber. Digitized data were temporarily otored in the volatile memory units in the fluorometer

and downloaded to an IBM-PC microcomputer for storage and further analysis. Mass analysis was conducted on a Finnlgan MAT TSQ 70 by direct ~;ast atom bombardment (FAB) ionization. Fast kinetic measurements. These were performed on a Gibson-Durrum stopped-flow instrument maintained at 24°C using a deuterium lamp as light source cad cut-off filters to select the wavelength range of the fluorescent light, l~,inetic data were collected with OLiS model 3820 data acquisition system connected to the stopped-flow apparatus through a North Star microcomputer. The kinetic data were fit to several exponentials using standard nonlinear least-squares methods

[141. Nonradiative enerKv transfer between H S A and DCHPA-iloprost. Due to a low quantum yield of

DCHPA-iloprost it was not possible for us to measure the energy transfer by the mechanism of ':,ensitized emission' [15]. We measured the energy transfer through the mechanism of donor quenching and evaluated the quantum efficiency of the sole albur-:: ':,r",~",t~"~r• in the presence and absence of the t,c~_~:,~: .,,." ,.. iloprost according to the method described by Fairclough and Cantor [15]. The tryptophan emission spectra of 30 !,tM cHSA were recorded in the presence and absence of DCHPA-iloprost. Both spectra were corrected for possible light scattering and conformational changes due to ligand binding by subtracting from each the spectrum from the mixture of photodecomposed cHSA and iloprost under identical experimental conditions and concentrations as the authentic donor-acceptor pair. The inner-filtering effect was minimized by using a microcuvette which was placed offcentered in the cell holder to provide a shorter light path and therefore a linear relationship between cHSA concentration and fluorescence intensity, The ratio of the quantum efficiency was then calculated as follows: g~l)a/@l) = [fDA(~1), X,)/fl)(XI),

X, )],,,

(1)

where iDA( X D" X, ) and fl:,( X 1), z~i ) are the fluorescence intensity (with or without acceptor, respectively) at wavelengths within a selected wavelength interval, J ~ , containing donor emission but not aeceptor emission. This ra~io was used to calculate the corrected 'transfer efficiency', E~: E,,h, = 1 - ( ~i)A/ ~i) )

12)

t:',l = E,,h,/fa

(3)

where f~ is the fraction of HSA molecules associated with acceptor. Calculation of critical distance, R o. attd attual distance, R. R o was calculated according to Dale and

Eisinger [16]: R~ = (8.79. lO2~)KZn "~i)JDA

(4)

JDA is the spectral overlap integral:

JDA = [fo~fD(~ )(A (X )~4 dx l /[ fo~'fD(~ ) d)~]

(5)

It was calculated from the absorption spectrt;m of DCHPA-iloprost and the emission spectrum of cHSA. The orientation factor, 2 was assigned a value of 2 / 3 by assuming that the transition dipoles are free to reorient during the lifetime of the donor. An average value, 1A, was used as the refractive index, t., of the donor-acceptor ensemble. ~D is the quantum yield of the donor, i.e., HSA. This value was calculated by the method of Chen [1"/] using quinine sulfate as standard according to: qbD= ~b. FD q~" A~ F,

qo

AD

(6)

w h e r e F is the relative fluorescence,

q is the r e l a t i v e

photon output of the source at the wavelength employed for excitation. A is the optical absorbance at the cxciting wavelength. The actual distance between the two transition dipoles was calculated according to the Frrster's relationship [18]: E=

(, Roo + Rf') or Ro/(

R = (I/E-

1)t/°Ro

(7,~

Results

Preparation and characterization of synthesi'.ed probe, 4-amino-2,6-dicklorophenol iloprost (DCHPA-iloprost) As shown in Fig. 1, DCHPA-iloprost was well re-

solved from iloprost by the reversed-phase HPLC. Because the original iloprost was a mixture of two stereoisomers, the synthesized probe also existed as a mi×ture. The two DCHPA-iloprost isomers were, however, not as clearly resolved as the original iloprost isomers (Fig. 1) due to the attachment of the aminophenol group to the iloprost molecule. Time-dependent experiments indicated that a 24 h reaction period resulted in a maximal yield. The overall conversion of iloprost to DCHPAiloprost was around 60%, as estimated by either TLC separation of HPLC method. Re-injection of the pooled product fractions onto the second C18 column did nor increase the purity of the compound further even using another methanol-based solvent system. The samples corresponding to fraction No. 40 and fraction No. 43 as shown in Fig. 1 were further tested for their susceptibility to alkali hydrolysis. Both samples were incubated in 5 M KOH at 100°C for 5 min, then cooled to 2 5 ° C and incubated for another h. The reaction mixture was checked by TLC together with the ilop~c~t standard. The synthesized product of fraction No. 40 (Fig. 1) was resistant to alkali hydrolysis, indicative of an amide formation between the 4-amino group of the aminophenol moiety and the carboxylate of iloprost, while the product of fraction No. 43 was readily hydrolysed to iloprost, indicative of an ester formation between the phenoxyl hydroxyl and the carboxylate of iloprost. The optical spectrum showed distinct features below 340 nm, i.e., 260 nm (12.9 m M - t - c m - I ) , 280 nm (9.2 m M - t. c m - ]), and 300 nm (5.5 m M - 1. c m - ~). The parent compound, iloprost, was essentially featureless between 340 and 240nm (Fig. 2a). When KOH was

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20 30 40 50 I:E'rENTIDN TiME train) Fig. 1. HPLCprofileof the reactiea mixtureof iloprostand 4-amino-2,6-dichlorophenol.50 p,l of reaclion mixture was injectedinto a C18 reversed phase column. An aceton/trile-basedsolventgradient identical to that describedin Ref. 13 was used to elute differentcompounds.Radioactivity (~olid line) and absorbaneeat 300 nm (broken line) were followed.Retention times for different compoundswere: iloprost isomers, 19-20 min: DCHPA-iloprost,amidederivative,39-41 rain: dichloroaminophenoi-iloprost,ester derivative,45 min.

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added to the DCHPA-iloprost preparation, there was an instant bathochromic shift and enhancement of the intensity,i.e., 268 nm (14.5 m M - ~ - cm ~). These changes typify the behavior of an aromatic hydroxyl group. Addition of HCI to the alkali-treated sample led to resumption of the original spectrum. These findings confirmed that the synthesized product was an amide rather than an ester, because the reversible change of the ,~pectrum by pH change cerrelates with the dissociation and association of the phenoxyl proton. Analysis of this probe by mass spectrum showed clearly the main molecular ion (519, re~z) and the main fragment with deletion of one molecule of hydrochloride (483, m/z) (Fig. 2b)

Fig. 2. (a) Optical spectrum of iloprost ( . . . . . ), DCHPA-ilaprez, ( ) and DCHPA-iloprost plus alkali ( . . . . . ). 48 g M DCHPiloprost in 0.1 M Tris ~pH 7.6)/1 mM EDTA was used for this measurement. 4 M K O H ,vas added to a final concentration of 4 mM K O H Jn the alkali-treat,.'d sample. (b) Mass spectrum of D C H P A iloprost obtained by the direct probe method (FAB-MS analysis).

HSA binding with DCHPA-iloprost Due to the significant overlap between the absorption spectrum of DCHPA-iloprost and emission spectrum of lISA, binding of DCHPA-iloprost to HSA could be followed by the albumin fluorescence quenching. As shown in Fig. 3, the albumin tryptophan t'luorescence was monitored during a titration experiment using DCHPA-iloprost as the ligand. After constructir, g the relative fluorescence amplitude change and the corresponding ligand,/cHSA molar ratio, the binding stoichiometry was quantitated as the intercept of the extrapolation of the initial linear portion of the titration curve, with the amplitude coinciding with the end-point of the titration (thc horizontal broken line in Fig. 3).

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Fig. 3. Determination of binding parameters of iloprost ,Jsing DCHPA-iloprost. Fluorescence quenching by DCHPA-iloprost (1 mM stcck in 50% ethanol) was recovered by stoichiometric titration in szmples containing fixed concentrations of cHSA (1,10, 30,63 #M) in 0.1 M potassium phosphate (pH 7.4) in a microfluorescence cell. Data were corrected for any dilution due to addition of the probe. The final ethanol concentration was also checked, which had no effect on the spectral property of albumin tryptophan. The end point of the titration was determined as the point at which the current spectrum coincided with the preceding spectrum before further addition of the probe. The titration curve of 30/LM cHSA was used as the 'reference" to calculate the concentrations of bound and free ligand for data obtained from lower cHSA concentrations according to Chignell [21]. For example, the data of 1 p,M eHSA aualy~ed based on one or two independent sites by nonlinear regressior, are shown in the inset. Excitation wavelength = 294 nm. Emission spectrum was recorded from 300 nm to 350 rim. Slit width = 4 nm for both excitation and emission.

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Fig. 4. Binding displacement of DCHPA-iloprost and warfarin by iloprost. For the displacement by iloprost, 0.3 ml of 30/aM cHSA in 0.02 M potassium phosphate (pH 7.4) was first mixed with 110/~M DCblPA-iloprost to quench more than g07~ of tile quenchable trytophan fluorescence. lloprosl (55 mM stock in pure ethanol or 10 mM in 50% ethanol) was added quantitatively, and bi~ading competition was followed by the rest~mption of the fluorescence signal amplitude. For the biading competition of warfarin with iloprost, 1.5 : 1 molar ratio of warfarin was added to the same HSA solution as above and the enhanced warfarin fluorescence (about 9-fold enhancement with excitation at 320 nm and emission spectra taken at the waveleogth range of 330-530 nm) was recorded, lloprost was then added stoichiometrically and the competition was followed as the decrement of the warfarin fluorescence. All data were corrected for volume changes due to addition of iloprost. The final concentration of ethanol was less than 5% and was tested to have no obvious effect on the tryptophan emission spectrum. Hill plot analysis was done according to Chignell [21]. ( f -, f a ) / ( f c - f ) was plotted against the concentration ratio of the competing ligand, iloprost, and the displaced ligand, i.e., DCHPA-iloprost or warfarin in a log-log scale, f, and f are the initial fluorescence and the final residual fluorescence, respectively. The warfarin displacement data were analyzed by a linear regression yielding a straight line with a slope of 2.5. The DCHPA-iloprost displacement curve appeared curvilinear. further analysis was deemed unnecessary.

79 The binding stoichiometry was close to 1. The binding affinity of this probe for HSA was determined through serial titrations at increasing levels of cHSA till the final titration curve almost coincided with its preceding titration curve (as shown in Fig. 3 by the data of 30 and 63 /tM cHSA). At these HSA levels, all the added ligands were consid,.'red to be associated with protein. We used the 30 p.M cI-ISA titration curve as the reference index to calculate the concentraticn of free and bound ligands of titra'ion curves obtained from lower HSA concentrations [19]. A nonlinear regression analysis was conducted to obtain Bmax and K D, using models of one or two independent binding classes. The data were decently fit by one binding-class model, yielding a KD of 0.18 #M and a Bm~x of 1.01 m o l / m o l cHSA (Fig. 3, inset). As cHSA usually has 1-2 equivalents of associated free fatty acid [32], we chose to look into the binding of DCHPA-iloprost with defatted HSA. A similar binding stoichiometry was obtained, implying that the primary PGI 2 binding site is not identical to the primary fatty acid binding site. We then carried out experiments to determine the displacement of DCHPA-iloprost binding to HSA by iloprost. Percentages of displacement were measured by following the resumption of the albumin tryptophan fluorescence. We also performed parallel experiments to determine the displacement of warfarin-binding by iioprost. The binding site of warfarin is known to be at the domain 2 of HSA [19,20]. The reported binding affinity of warfarin for cHSA [21] appeared to be close to that of DCHPA-iloprost. As binding of warfarin to HSA results in an enhancement of warfarin fluorescence, displacement of warfarin could be measured by

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following the decline of warfarin-induced fluorescence enhancement. Dose-response curves of binding displacement are shown in Fig. 4. Displacement of DCHPA-iloprost and warfarin by iloprost occurred at similar concentration ranges, indicative of similar binding affinity for HSA. However, displacement of DCHPA-iloprost followed a steeper dose response than warfarin displacement, implying a different displacement mechanism for these two ligands. Since prostaglandins are derived from arachidonic acid and share common structure features with long-chain fatty acid, it is important to know whether prostacyclin binds to the primary binding site of fatty acid. In separate experiments similar to that shown in Fig. 4, we used p~dmitic acid and oleic acid to titrate 30 /~M cHSA or defatted HSA pretreated with 30 t~M DCHPA-iloproq. l'he quenched fluorescence of albumin did not start to resume until a fatty a c i d / H S A ratio reached 5:1 for defatted HSA, or 2:1 for cHSA. These results supported that PGI 2 and free fatty acid have different primary binding sites.

Distance measurements between the phenol ring of DCHPA-iloprost and the tryptophan indole Distance measurements between two transition dipoles were carried out based n the nonradiation encrgy transfer mechanism. The spectral overlap integral, JDA, was calculated directly from the absorbance spectrum of DCHPA-iloprost (Fig. 2a) and the emission spectrum of albumin tryptophan as 1.976.10 -t5 cm 2- M -l, according to Eqn. 5. Corrected transfer efficiency was calculated to be 0.622 according to Eqns. 1-3 using 30 ~tM HSA and 10/tM DCHPA-iloprost. '¢/ith the calcu-

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Fig. 5. Association of DCHPA-iloprost with cHSA. Binding association of DCHPA-iloprost was monitored kinetically on a fluorescence stopped-flowapnaratus at 24 + 1° C. Excitationwavelengthwas 27g nm and emissionwas monitoredby a 90 o fluorescenceusing a glass cut-off filter at 350 nm. 2/aM cHSAin 0.1 M potassiumphosphate(pH 7.4) was mixedwith 10- and 20-fold molar excessof DCHPA-iloprostto assumea pseudo-first-ordercondition. Data were shown as ~ realized signal change. Both kinetic data exhibited similar observed huorescencechanges. Each uat,', point was the averageof fiveconsecutiveshots.

lated 'critical distance', R 0, 16.6 .~., and the quantum efficiency of tryptophan, 0.07, we obtained the distance. R, to be 15.2 A. Utilization of DCHPA-iloprost in binding rate-measurement and mechanistic study. Because the albumin fluorescence quenching is the consequence of the binding of DCHPA-iloprost, measurement of the time-course of this signal change could serve as the index for measuring binding rate. We conducted the mixing experiments at pseudo-first-order conditions, i.e., [ligand] >> [HSA]. None of the "kinetic data we obtained showed simple one-exponential quenching. Each kinetic tracing was fit minimally by two exponentials, indicating a complicated binding mechanism. Each phase appeared to make similar contributions to the overall fluorescence change. The obsera,ed fluorescence change appeared to be constant, and the rate exhibited a linear dependence on the ligand concentration (Fig. 5). The second-order rate constants estimated for these two binding phases were 0.42 • l0 t, and 0.034- 10' M - i. s- ~, respectively. Discussion

In this study, we prepared a stable prostacyclin analog from iloprost, which proves useful for probing the PGl2-binding domain structure in HSA. As neither prostacyclin nor iloprost binding to HSA led to any useful spectral signal change, we produced an an'fide probe by reacting 4-amino-2,6-dichlorophenol with iloprost. This probe competes with iloprost for the binding site in HSA, and interacts with the albumin sole tryptophan residue through dipole-dipole energy transfer. Taking advantage of this fluorescence quenching phenomenon, we have been able to quantitate the binding stoichiometry and various binding parameters including K D, Bin, , (Fig. 3) and the association rate constant (Fig. 5). This probe has enabled us to measure the spatial separation between the phenol-ring of DCHPA-iloprost and the tryptophan indole group. Such an analog will ~~e very useful in further characterizing the binding domain :',f prostacyclin in HSA and can be a powerful tool for clelineating the binding defects in disease states exhibiting reduced PGI2-binding [22,23]. In an attempt to develop useful fluorescence probes, we initially explored the possibility of conjugating iloprost to a series of aminophenol analogs, hoping to obtain compounds with favorable spectral properties. The aminophenol analogs tested were: 4-aminophenol, 4-amino-2,3-dimethylphenol, 4-amino-2-chlorobenzoic acid and 4-amino-2,6-dichlorophenol. Among these compounds, only the last two displayed significant overlaps of their optical spectra with the emission spectrum of albumin tryptophan. However, since the 4-amino-2chlorobenzoic acid derivativc possessed unstable optical properties, we decided to focus on the 4-amino-2,6-di-

chlorophenol iloprost. The purity of the HPLC-purified DCHPA-iloprost was checked by reinjecting the compound for further HPI..C separation and by mass analysis. The compound appeared to be homogeneous. Based on our studies with iloprost [13], this homogeneously purified compound contains two isomers. Due to the close proximity of these two DCHPA-iloprost isomers on the HPLC chromatograms, despite use of different solvent systems, we did not carry out farther experiments to purify these two stereoisomers. Although DCHPA-iloprost interacts efficiently with the sole tryptophan residue through nonradiation energy transfer (Fig. 3), it is only weakly fluorescent in the wavelength range between 350 and 500 nm (data not shown). Therefore, it is virtually impossible to obtain information about the close surroundings of the probe interacting with the binding site by the measurement of fluorescence anisotropy. The binding stoichiometry obtained by linear extrapolation and nonlinear regression agrees with each other indicating one specific binding site of DCHPAiloprost in HSA. However, our equilibrium binding data by a gel partition method using [3H]iloprost as ligand yielded a K o of 94 + S.D. 19 p.M and a Bma~ of 2.0 _+.0.6, iloprost : cHSA, for the primary binding site [24]. Therefore, only one of the two binding sites was detected b~ the fluorescence method at our experimental conditions. In a separate experiment, we used DCHPA-iloprost to displace the [3H]iloprost which was bound to cI-!SA. We found that the fluorescence probe displaced only one of the two iloprost binding sites in a noncompetitive fashion, i.e., the binding curve shewed a decrease in the apparent Bmax but not in the K D. This result must due to the big difference of the binding affinity between iloprost and DCHPA-iloprost. The affinity of DCHPA-iloprost is about 500-.times stronger than iloprost. Attachment of 4-amino-2,6-dichlorophenol group to iloprost apparently leads to a significant increase in the binding affinity. Therefore, it binds to HSA very efficiently and became independent of the presence of iloprost. Use of DCHPA-iloprost facilitates the binding quantitation because th~ quantity of t'ae protein as well as the ligand required is reduced tremendously. Although we can not exclude the possibility thin 4-amino-2,6-dichlorophenol moiety might recognize additional binding sites in HSA, the fact that the protein-associated DCHPA-iloprost can be completely displaced by iloprost supports a direct competition of the same binding site on HSA (Fig. 4). The displacement curve, however, showed cooperative behavior. On the other hand, the displacement curve of warfarin appeared to exhibit typical competition with iloprost, i.e., the ligand concentrations corresponding to 90% displacement was about 80-90-fold of that corresponding to 10% displacement. Both warfarin and DCHPA-

iloprost, which have similar K D for HSA, displayed similar ilop-ost concentration d-.pendence, ~.arfarin being somewhat weaker than diehioro-iloprost. Further analysis of tho displacement ,'ata to obtain the binding parameters such as K o e l ;:~e ligand and K~ of the competing ligand is, howevel, difficult. The experimental conditions for binding did not satisfy the simplified case described by Cheng and Prusoff [25], namely, the amound of total ligand was not always present in great excess of the amount of HSA. Analysis of the data of dichloro-iloprost binding displacement by a generalized formula recently described by Horovitz and Levitzki [26] was also hampered by the apparent cooperativity. Furthermore, successful analysis of the data according to this method requires (1 - y ) 2 > y where Y is the fraction of the receptor occupied by the tagged ligand (DCHPA-iloprost) in the presence of the competing ligand (iloprost). Hill plot analysis as described by Chignell [21] was conducted for comparison between the binding displacement of warfarin and DCHPAiloprost (Fig. 4, right). Displacement of DCHPA-iloprost by iloprost is curvilinear with the initial portion, showing a simple displacement followed by a positive cooperative behavior. Displacement of warfarin, although linear in appearance, exhibits a slope of 2.5 instead of 1. Therefole, neither of the two binding displacements follows a simple competitive model, a typical behavior of HSA binding with many other ligands [20,27]. Distance measurements determined from the efficiency of nonradiative single-singlet energy transfer between pairs of fluorescent dyes have been widely used to study the macromolecular assembly and to localize the ligand binding site [28,29]. The outstanding feature of this energy transfer is that the rate of transfer is proportional to the inverse sixth power of th distance between a given pair of chromophores. In this work, we calculated the distance between the phenol in DCHPA~loprost and the indole of tryptophan by this principle. We had to make certain assumptions in these experiments which require consideration. First, the quantum yield, t/, D, of the tryptophan was measured as 0.07 bs' ti;e method of Chen [17]. This value is similar to the deteHnination of Chignell [301. However, a detailed study by Bursein et al. on a number of tryptophan-containing proteins, either native or denatured, indicated three discrete spectral classes for tryptophan [3!]: one buried in nonpolar regions of the protein (hm~, = 330--332 rim, AX = 48-49 n m , • = 0.11) and two on the surface. One of the latter is completely exposed to water (hm,~ = 350-353 rim, AX = 59-61 nm, if} = 0.2) and the other has only limited contact with water (~.m~= 3",0--342 nm, Z11~=53-55 nm, {/}=0.3). Careful exam!nation of our HSA emission spectrum revealed that the tryptophan appeared to be completely exposed to the water (~..,,~ = 350 nm and AX = 60 nm). Therefore, a quantum yield of 0.2 would be predicted based on the

analysis by Burstein et al. Given a quantum yield of 0.2, the R 0 and R values would be 19Y ,~, and 18.2 A., respectively, instead of 1'5.6 ,'~ and 15.2 .A when 0.07 quantum yield was used. These spectral features were the same for both cryst llline and defatted HSA samples obtained from Sigma. Second, the orien~:ation factor, ~2, which can have values ranging from 0 to 4 [16], was given a value of 2 / 3 in this study because we believe that both the donor and the acceptor transition dipoles have sufficient, although limited, freedom to assume most of the possible orientations during the excited-state lifetime of the donor in our buffer systems and temperature. Furthermore, statistical weighing of relative donor-acceptor geometries described by Hillel and Wu [33] significantly lowers the upper limit of the critical distance. The sixth power relationship between ~2 and Ro further cut down the possible severe deviation of the estimated distance. Because the binding of DCHPA-iloprost to HSA is a relative weak interaction, the:re was a compromise in selecting the concentration of HSA in order to avoid the possible inner filtering effect. As shown in the binding studies (Fig. 3), a protein concentration greater than 30 p.M guaranteed a complete association of the added ligand. We therefore kep t the ttSA concentration at around 30 t~M. However, this high concentration of HSA introduced severe inner filtering effect during fluorescence measurements which could not be totally removed by using a microcell or by adjusting its position relative to the measuring beam. To minimize this interfering factor, we used a ligand concentration corresponding to the initial linear portion of the binding curves as shown in Fig. 3 in combination with lower cHSA concentrations, i.c., 10 and 15 /tM, and calculated the transfer efficiency at each protein level. We found less than 10% increase in the transfer efficiency when compared to the data obtained at 30 ttM cHSA concentration, The biphasic nature of the stopped-flow kinetic data may be the consequence of the presence of two stereoisomers. These two isomers interact with HSA at somewhat different rates (i.e., 0.42.10 6 VS. 0.034.106 M • s -1) which coincide with the molar ratio of these two isomers in the original iloprost mixture [34]. They contribute similarly to the total fluorescence change (Fig. 5). The biphasic property of the stopped-flow kinetic data can not be the result of con|ormational changes following the ligand binding, becaus{ the changes ir both phases are proportional to the ligand concentra tion change, an indication of bimolecular interaction for each phase. It will be helpful to synthesize DCHPAiloprost from purilied iloprost isomers. Use of Ihe resultant DCHPA-iloprost, which exists as a single :aereoisomer, in the binding experiments will cer,aimy simplify the data interpretation. Based on these data, we propose a model for the

prostacyclin binding sites in HSA. Each HSA molecule has one primary binding site and a secondary site with lower affinity. The primary binding site is very likely to be localized in domain 2 of the HSA molecule [27] because of the following reasons: (a) Palmitic acid and oleic acid do not compete with DCHPA-iloprost binding until the stoichiometry of f~.tty ~ c i d / h S A is above 5 : 1 for defatted HSA and is above 2 : 1 for cHSA. Therefore the primary binding site,, of prostaeyclin could not be located m domain 3, which contains a specific and strong binding, site for free fatty acid [32,35]. (b) That the distance between the phenol of DCHPA-iloprost and the indole of t r y p t o p h a n is 15-18 is comp,~tible with the notion that the binding site of iloprost is located in domain 2. (c) Ilopr6st is cotnpetitive with warfarin for H A binding. Warfarin i,~ known to bind to d o m a i n 2 of HSA with a spatial separation from tr3'ptophan of 34/~ [36]. As the distance, between the albumin t r y p t o p h a n and theoPhenol of DCblPA-iloprost was measured to be 15-18 A, it appears that the phenol ring of DCHPA.. i!oprost is located near the midpoint between warfarin ar~a. u y p t o p h a n . Wynalda a~id Fitzpatrick first reported that purified HSA stabilizes P G I : but ac,;eierates The decomposition of prostaglandins containing a fl-hydroxyketone (e.g. P G E or P G D series) [37,38]. They proposed that a basic mieroenvironment bearing an amino-acid sequence LysAla-Trp-Ala-Val-Ala-Arg is involved in the binding of various prostaglandins. This alkaline environment enhances the base-catalysed decomposition of D and E type prostaglandins but decreases the acid-catalysed degradation of PGI 2. A similar mechanism for the stabilization of t h r o m b o x a n e A , and leukotriene A 4 was proposed by these authors [39,40]. O u r experimental results concur with this model. D C H P A - i l o p r o s t appears to be best or:cnted in a way so that its two five-r,aembered ring s~ructures are adjacent to the trypt o p h a n and occupy the so-called "alkaline mlcroenvironment '. Such conformation will provide the best protection for the labile vinyl ether moiety from chemical degradation. Acknowledgements We wish 1o thank Ms, Ida G o r d o n for preparing this manuscript. References 1 Fruchgon. R.F. and Zawadzki, J, (1980) Nature 288, 373-376. 2 Mottcada, S.. GryglewskL R.J.. Bunting S. and Vane, J.R. (1976) Nature 263. 663-665. 3 Dembmska-Kiec. A.. Rucker. W. and Schonhofer, P.S. (1980) Nauayn-Schmiedebergs Arch. Pharmacol. 311, 67-70. 4 Ito, TT.. Ogawa. K.. Enomoto, J.. Hazimoto. H., Kai. J. and Satake. T. (1980) in Advances in Proslagtandin Thromboxane

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