Phenprocoumon Binding to Bovine and Human α1-Acid Glycoprotein

MICROCHEMICAL JOURNAL 55, 162–168 (1997)ARTICLE NO. MJ961409

Phenprocoumon Binding to Bovine andHuman a1-Acid Glycoprotein

JASON LAURITSEN AND EDWARD C. SHANE

Department of Chemistry, Morningside College, Sioux City, Iowa 51106

The binding of the anticoagulant drug phenprocoumon to bovine and human a1-acid glycopro-tein (AGP) was studied using a fluorescence titration technique. Phenprocoumon was found tohave one moderate strength binding site on bovine AGP with n Å (0.78 { 0.04) and a Scatchardassociation constant, K Å (2.2 { 0.1) 1 104 M01. Phenprocoumon was bound more strongly tohuman AGP; a primary binding site was found with binding affinity, n1K1 Å 4.6 1 105 M01,along with weaker secondary binding, n2K2 Å 1.8 1 104 M01. q 1997 Academic Press

INTRODUCTION

Phenprocoumon is an anticoagulant drug from the coumarin family of compounds.It is used in the treatment of myocardial infarction (heart attack), thrombosis andemboli (blood clots), and after heart valve replacement surgery. Binding levels ofphenprocoumon to blood proteins are of interest for their use in the determination ofproper drug dosage for patients (1) because only the unbound phenprocoumon isphysiologically active.

Phenprocoumon binds to several blood serum proteins including albumin and glyco-protein. In this study we look at binding with bovine and human alpha-1-acid glycopro-tein(a1- AGP). AGP is a large protein (molecular weight Å 44,000) which can beisolated from blood serum (2).

Drug binding to proteins can be examined by several different methods, includingthe fluorescence method used in this study. The phenprocoumon molecule fluorescesweakly when free in solution but fluoresces more strongly when bound to AGP.Therefore, the increase in fluorescence can be used to monitor bound and free phenpro-coumon.

There have been several studies of the binding of phenprocoumon to the bloodserum proteins. These include in vitro studies of binding to human serum albumin(3–5) and in vivo studies of phenprocoumon binding to plasma which showed phenpro-coumon to have a high binding affinity to blood plasma proteins (1, 6–8). Otagiri etal. (9) have investigated the binding of phenprocoumon to human AGP.

The purpose of this study is to determine the number of binding sites and theassociation constant for the binding of phenprocoumon to bovine and human a1-AGP,using a Scatchard analysis.

MATERIALS AND METHODS

Racemic phenprocoumon was a gift of Hoffmann–La Roche, Inc. (Nutley, NJ).Bovine and human a1-AGP were obtained from the Sigma Chemical Company (St.Louis, MO). All other reagents were analytical grade. Solutions were buffered with

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163PHENPROCOUMON BINDING

0.1 M sodium phosphate buffer solution (pH 7.4) and were prepared with glass-distilled water.

Bovine a1-AGP was purified to remove lipids and heavy metals using the methodreported by Gilles et al. (10). We found that unpurified bovine AGP gave variablebinding results while consistent results were obtained with the purified protein. Onlya small amount of human AGP was available for these experiments, so it was usedas obtained from the manufacturer (99% pure).

Fluorescence measurements were taken at 388 nm with a Hitachi F-2000 spec-trofluorometer using 335 nm excitation. Absorbance measurements were taken at 335and 388 nm and were used to correct for inner-filter effects according to the procedurereported by Holland et al. (11) that adjusts fluorescence readings for self-absorbanceat the fluorescence excitation and emission wavelengths.

Titration Method

Phenprocoumon fluoresces at a low level when free in solution, but the fluorescenceincreases significantly when bound to AGP. In this titration method, successive aliquots(5–10 ml) of phenprocoumon were added to three 2-ml samples (high and low [AGP]and buffer) and mixed by a small magnetic stir bar in the fluorescence cell for 5 min;no change occurred in fluorescence for equilibration times between 5 and 60 min, sothe shorter equilibration time was used. Fluorescence and absorbance measurementswere recorded, and the process was repeated.

Data Treatment

For bovine AGP, a calibration curve which related the fluorescence, Fb , of 86%bound phenprocoumon to the total concentration of phenprocoumon was preparedusing 3 1 1004 M bovine AGP. The solubility limit of bovine AGP was reached neara concentration of 3 1 1004 M, so higher concentrations that would have allowed fullbinding were not possible. The 86% binding for the calibration curve was correctedfor in the calculations. A 4 1 1005 M solution was used for the human AGP calibrationcurve; under these conditions binding was greater than 98%. The fluorescence wascorrected for background AGP fluorescence. Plots of Fb versus the total phenprocou-mon concentration were fitted with a least-squares linear regression (Fig. 1). Thefluorescence, Fc , of partially bound phenprocoumon was measured with 2 1 1005 Mbovine AGP or 4 1 1006 M human AGP. The fluorescence of this mixture wascorrected for the background fluorescence of the AGP solution and the fluorescenceof the free phenprocoumon in solution. The corrected fluorescence was then used withthe Fb calibration plot to determine the concentration of bound phenprocoumon.

Binding Model

The binding model (12) for a small ligand bound to a macromolecule is basedon the equilibrium relationship, AGP / PHEN ` AGPrPHEN, where PHEN repre-sents free phenprocoumon, an unbound ligand, and AGPrPHEN represents the protein:bound phenprocoumon complex. For an equilibrium system with multiple, independentbinding sites (Scatchard model) the relationship between bound and free ligand canbe represented as (13, 14)


164 LAURITSEN AND SHANE

FIG. 1. The relative fluorescence of phenprocoumon which is 86% bound to 3 1 1004 M bovine AGP(circles), of phenprocoumon partially bound to 2 1 1005 M bovine AGP (squares), and of free phenprocou-mon (diamonds) at pH 7.4. A linear regression was used to fit the 86% bound data. The x axis representsthe total phenprocoumon concentration.

r Å n1K1F

1 / K1F/ n2K2F, (1)

where r is the number of moles of bound phenprocoumon per mole of AGP, F is theconcentration of free phenprocoumon, n1 is the number of primary binding sites, K1

is the primary Scatchard association constant, and n2K2 is a measure of nonspecificsecondary binding. For an equilibrium system with one primary binding site and nosecondary binding a Scatchard plot of r/F versus r is a reliable device (15) formeasuring n1 and K1 as long as there is significant saturation of the binding sites.

r/F Å n1K1 0 K1r. (2)

RESULTS

As shown in Fig. 1, phenprocoumon fluoresces weakly when free in solution; butits fluorescence is greatly enhanced when phenprocoumon is 86% bound to 3 1 1004

M bovine AGP. The 86% bound curve was linear at phenprocoumon concentrations



FIG. 2. A Scatchard plot for the binding of phenprocoumon to bovine AGP. A linear regression fit (solidline) gave n1 Å (0.78 { 0.04) and K1 Å (2.2 { 0.1) 1 104 M01.

up to 6.6 1 1005 M and was fitted with a least-squares linear regression to prepare acalibration curve which related the fluorescence to the concentration of the 86% boundphenprocoumon: bovine AGP complex. At an intermediate 2 1 1005 M bovine AGPconcentration both bound and free forms of phenprocoumon were present, and thebinding parameters were evaluated from the observed fluorescence of bound and freephenprocoumon. A similar calibration curve was prepared for the phenprocoumon:human AGP system which exhibited a much greater fluorescence enhancement thanthe bovine AGP system. Samples of 4 1 1005 and 4 1 1006 M human AGP wereused for the fully and partially bound phenprocoumon:human AGP experiments, re-spectively.

Under the conditions of this experiment about 50% saturation of the bovine AGPbinding sites was obtained. As Klotz (15) has cautioned, this introduces greater uncer-tainty for the calculated Scatchard parameters, especially the number of binding sites.Given this limitation the linearity of the Scatchard plot (Fig. 2) for the phenprocoumon:bovine AGP system indicates that there is approximately one binding site (n1 Å 0.78{ 0.04) and no cooperativity of binding. The Scatchard association constant, K1 Å(2.2 { 0.1) 1 104 M01 suggests moderate strength binding. Errors reported are onestandard deviation.

Due to limited availability of human AGP, the protein was used without additionalpurification. Scatchard plots for the phenprocoumon: human AGP system exhibitedthe distinct curvature characteristic of a protein with multiple binding sites. A plot of



FIG. 3. The binding of phenprocoumon to human AGP. Experimental data was fit to Eq. (1) using theMarquardt–Levenburg nonlinear curve fitting algorithm (solid line).

bound versus free phenprocoumon (Fig. 3) was found to fit the form of Eq. (1). Thesolid line in Fig. 3 represents a Marquardt–Levenburg (16) nonlinear fit of the data;Gauss–Newton and simplex fits of Eq. (1) produce similar results. The nonlinear fitgave n1K1 Å 4.6 1 105 M01 and n2K2 Å 1.8 1 104 M01 which suggests that there ishigh-affinity primary binding plus weaker secondary binding.

Phenprocoumon binds more strongly to human AGP than to bovine AGP. Whileabout 24% of 5 1 1006 M phenprocoumon was bound to 2 1 1005 M bovine AGP,nearly 97% of 5 1 1006 M phenprocoumon was bound to 2 1 1005 M human AGP.

DISCUSSION

There have been several studies (4, 5, 18) of the binding of phenprocoumon tohuman serum albumin (HSA) and bovine serum albumin (BSA) but only one otherstudy (9) of the binding of phenprocoumon to human AGP and no other study ofbinding to bovine AGP. The linearity of the Scatchard plot (Fig. 2) for the phenprocou-mon: bovine AGP system identifies approximately one primary binding site withmoderate strength binding as determined by the Scatchard association constant, K1 Å2.2 1 104 M01. A Scatchard plot for the phenprocoumon:human AGP system haddistinct curvature with a negative slope indicating that more than one independent



TABLE 1Association Constants and Number of Binding Sites for Phenprocoumon Binding to Macromolecules

Association Number of Bindingconstant, K binding affinity, nKM01 1 1005 sites, n M01 1 1005 Protein Reference

0.22 { 0.01 0.78 { 0.04 0.17 B-AGP This study4.6 (n1K1) H-AGP This study0.18 (n2K2)

7.7 1 7.7 H-AGP (9)0.26 (K1) 4.1 1.0 PVP (17)2.7 (K1) 2 5.4 HSA (3)2.4 (K1) 1 2.4 HSA (18)0.67 (K2) 1 0.679.5 (K1) 1 9.5 HSA (4)0.5 (K2) 1 0.51.2 (K1) 1 1.2 HSA (5)1.2 (K2) 1 1.28.0 (K1) 1 8.0 BSA (4)1.2 (K2) 1 1.2

Note. B-AGP, bovine a1-acid glycoprotein; H-AGP, human a1-acid glycoprotein; PVP, polyvinylpyrroli-done; HSA, human serum albumin; BSA, bovine serum albumin.

binding site is present. The human AGP data was scattered enough that we feel it isappropriate to report only the binding affinity (nK) and not the specific n and K values.With human AGP, phenprocoumon has a strong primary binding site, n1K1 Å 4.6 1105 M01 and weaker secondary binding, n2K2 Å 1.8 1 104 M01.

Binding parameters for the interaction of phenprocoumon with several macromoleculesare summarized in Table 1. Our binding affinity (nK) for bovine AGP is of moderatestrength and is similar to that found by Otagiri et al. (17) for binding to polyvinylpyrrolidoneor to the secondary binding affinities for phenprocoumon binding to HSA and BSA (4, 5,18). Our primary binding affinity (nK) for human AGP is about half that reported by Otagiriet al. (9). In addition, Otagiri et al. found one primary binding site and no secondary bindingwith human AGP while we found weak secondary binding. Otagiri et al. proposed that thestrong phenprocoumon binding to human AGP is hydrophobic; such a strong bindinghydrophobic site appears to be absent in bovine AGP which suggests that there are structuraldifferences between the bovine AGP and human AGP molecules.

In summary, the fluorescence of phenprocoumon is enhanced when bound to humanand bovine AGP and other macromolecules. This enhanced fluorescence can be ex-ploited in a fluorescence titration method as long as care is taken to correct for inner-filter absorption of the excitation beam and the emitted fluorescence. The fluorescencetitration method offers an alternative to the commonly employed equilibrium dialysismethod for measuring binding parameters.

ACKNOWLEDGMENTSWe thank MidAmerican Energy for their support of summer undergraduate research at Morningside

College and the NSF for partial funding of the spectrofluorometer under ILI Grant USE-9152550. Thanksalso to Molly Foster for her valuable contributions as a high school summer research assistant.



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Phenprocoumon Binding to Bovine and Human α1-Acid Glycoprotein