dmd 39:2117–2129, 2011 printed in u.s.a. bovine serum ... · suring drug binding to bsa and to...

Bovine Serum Albumin Decreases Km Values of HumanUDP-Glucuronosyltransferases 1A9 and 2B7 and Increases Vmax

Values of UGT1A9□S

Nenad Manevski, Paolo Svaluto Moreolo, Jari Yli-Kauhaluoma, and Moshe Finel

Division of Pharmaceutical Chemistry (N.M., J.Y.-K.) and Centre for Drug Research (N.M., P.S.M., M.F.), Faculty of Pharmacy,University of Helsinki, Helsinki, Finland

Received June 28, 2011; accepted August 18, 2011

ABSTRACT:

The human UDP-glucuronosyltransferase (UGT) enzymes UGT1A9and UGT2B7 play important roles in the hepatic glucuronidation ofmany drugs. The presence of bovine serum albumin (BSA) during invitro assays was recently reported to lower the Km values of boththese UGTs for their aglycone substrates without affecting thecorresponding Vmax values. Nonetheless, using the specific sub-strates entacapone and zidovudine (AZT) for UGT1A9 and UGT2B7,respectively, and using an improved ultrafiltration method for mea-suring drug binding to BSA and to biological membranes, we foundthat the presence of BSA during the glucuronidation reaction leadsto a large increase in the Vmax value of UGT1A9, in addition tolowering its Km value. On the other hand, in the case of UGT2B7,our results agree with the previously described effect of BSA,

namely lowering the Km value without a large effect on the en-zyme’s Vmax value. The unexpected BSA effect on UGT1A9 wasindependent of the expression system because it was found in arecombinant enzyme that was expressed in baculovirus-infectedinsect cells as well as in the native enzyme in human liver micro-somes. Moreover, the effect of BSA on the kinetics of 4-methyl-umbelliferone glucuronidation by recombinant UGT1A9 was similarto its effect on entacapone glucuronidation. Contrary to the agly-cone substrates, the effect of BSA on the apparent Km of UGT1A9for the cosubstrate UDP-�-D-glucuronic acid was nonsignificant.Our findings call for further investigations of the BSA effects ondifferent UGTs and the inhibitors that it may remove.

Introduction

Human UDP-glucuronosyltransferases (UGTs) play major roles inthe metabolic elimination of numerous endo- and xenobiotics. Theyare membrane enzymes of the endoplasmic reticulum that catalyzeglucuronic acid transfer from the cosubstrate, UDP-�-D-glucuronicacid (UDPGA), to nucleophilic groups of chemically diverse sub-strates. There are 19 functional human UGTs and they are divided intothree subfamilies: 1A, 2A, and 2B (Mackenzie et al., 2005). Closelyrelated enzymes that use somewhat different nucleotide cosubstrateshave recently been discovered and assigned to subfamily 3A (Mac-Kenzie et al., 2011), but they will not be further considered in thiswork. Individual UGT isoforms have distinctive substrate and inhib-itor selectivity (Miners et al., 2010) and are differentially expressed in

various tissues, most notably liver, intestine, and kidney (Ohno andNakajin, 2009).

Because of partial overlaps in the substrate specificity of individualUGTs and the expression of multiple isoforms in each tissue thatexpresses these enzymes, in vitro studies on the UGTs and drugglucuronidation are often performed using recombinant human UGTsthat are either expressed in insect cells (mainly Spodoptera frugiperdaSf9 cells), or in human embryonic kidney (HEK) 293 cells (Radomin-ska-Pandya et al., 2005). Enzyme kinetic constants (e.g., Km andVmax) and inhibition (IC50 or Ki) parameters of drug glucuronidation,determined from in vitro assays, are commonly used to estimate theextent of glucuronidation in vivo (Miners et al., 2010). Because thereis currently no good method to extract the UGTs from the membraneand purify them as fully active enzymes, in vitro glucuronidationassays are performed with different cell fractions rather than withhighly purified enzymes. In such systems, nonspecific substrate bind-ing to the membrane and different proteins within it, as well as thepresence of inhibitors within the membrane, can lead to erroneousestimation of UGT activity. The acquired errors may lead to erroneousestimation of in vivo glucuronidation activity, namely poor in vivo-invitro extrapolation, significantly weakening the ability to predict thepharmacokinetics properties of a drug under development, a problemthat was already faced in the case for many therapeutic drugs that are

This study was supported by the Graduate School in Pharmaceutical Re-search, Academy of Finland (Project Number 120975); the Sigrid JuseliusFoundation; and a Helsinki University Research Foundation grant for youngresearchers.

Article, publication date, and citation information can be found athttp://dmd.aspetjournals.org.

doi:10.1124/dmd.111.041418.□S The online version of this article (available at http://dmd.aspetjournals.org)

contains supplemental material.

ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; AZT, zidovudine (3�-azido-3�-deoxythymidine); BSA, bovine serum albumin; 3D, three-dimensional; HEK, human embryonic kidney; HLM, human liver microsomes; HPLC, high-performance liquid chromatography; 4-MU, 4-methyl-umbelliferone; NSBf, nonspecific binding to the filter device; UDPGA, UDP-�-D-glucuronic acid; UPLC, ultraperformance liquid chromatography;MeOH, methanol; fu, fraction unbound.

0090-9556/11/3911-2117–2129$25.00DRUG METABOLISM AND DISPOSITION Vol. 39, No. 11Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics 41418/3726310DMD 39:2117–2129, 2011 Printed in U.S.A.

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eliminated by glucuronidation (Boase and Miners, 2002; Kilford et al.,2009; Raungrut et al., 2010). Hence, there is a clear need for betterunderstanding of factors that affect the outcome of UGT assays invitro.

In a series of studies, it was found that adding purified bovineserum albumin (BSA) to assays with several recombinant humanUGTs that were expressed in HEK293 cells, or to the native enzymesin human liver microsomes (HLM), can significantly decrease the Km

for drugs that are glucuronidated by UGT1A9 and UGT2B7 withoutaffecting the reaction Vmax (Uchaipichat et al., 2006; Rowland et al.,2007, 2008). These authors suggested that long-chain fatty acids (e.g.,oleic, linoleic, and arachidonic acid) competitively inhibit the UGTs,and that BSA addition reversed that inhibition by binding the inhib-itory fatty acids (Rowland et al., 2007). Because similar inhibition wasnot observed in cultured hepatocytes (Engtrakul et al., 2005), theauthors speculate that the inhibitory fatty acids are released duringmicrosome preparation from either human liver or the cells that wereused for recombinant UGT expression.

Raungrut et al. (2010) have recently studied the effect of BSA oncodeine glucuronidation by the recombinant UGT2B4 and UGT2B7that were expressed in insect cells. However, thus far, this is the onlystudy that examined the BSA effect on UGTs that were expressed ininsect cells, even if most of the research on different aspects of theUGTs is currently conducted using such recombinant enzymes be-cause the commercial UGTs are expressed in insect cells. There aredifferences in lipid composition between the HEK293 cells, insectcells, and HLM (Marheineke et al., 1998), but it is unclear if thesedifferences are “translated” into differences in the BSA effect onindividual UGTs. Our original goal was to investigate the effect ofBSA on the activities of recombinant UGTs that were expressed ininsect cells. As reported below, the obtained results led us also toreexamine the earlier reports about the native UGT enzymes in HLM,particularly the BSA effect on UGT2B7 and UGT1A9. Although theBSA effect does not appear to be dependent on the expression system,the new findings should raise general awareness about factors that caninfluence UGT assays in vitro and the complexity of the BSA effect.They may also be instrumental for better understanding of the gluc-uronidation reaction mechanism and how it may be inhibited as wellas for better predictability of the in vitro assays.

Materials and Methods

Compounds and Reagents. 4-Methylumbelliferone (�99%, CAS 90-33-5), UDPGA (triammonium salt, 98–100%, CAS 63700-19-6), alamethicin(�90%, CAS 27061-78-5), 4-methylumbelliferone-�-D-glucuronide (4-MU;�98%, CAS 6160-80-1), zidovudine (3�-azido-3�-deoxy-thymidine, �98%,CAS 30516-87-1), sodium phosphate monobasic dihydrate (�99%, CAS13472-35-0), and BSA (�96%, CAS 9048-46-8, essentially fatty acid free,�0.004%) were purchased from Sigma-Aldrich (St. Louis, MO). Entacapone(batch 1044842) was a generous gift from Orion Corporation (Espoo, Finland).Entacapone-�-D-glucuronide was synthesized in our laboratory (Luukkanen etal., 1999). Tween 20 (CAS 9005-64-5) and Tween 80 (CAS 9005-65-6) werepurchased from Acros Organics (Fairlawn, NJ). Magnesium chloride hexahy-drate and perchloric acid were obtained from Merck (Darmstadt, Germany).Formic acid (98–100%) was from Riedel-deHaen (Seelze, Germany). Diso-dium hydrogen phosphate dihydrate was purchased from Fluka (Buchs, Swit-zerland). Radiolabeled [14C]UDPGA was acquired from PerkinElmer Life andAnalytical Sciences (Waltham, MA). High-performance liquid chromatogra-phy (HPLC)-grade solvents were used throughout the study.

Enzyme Sources. Recombinant human UGT2B7 and UGT1A9 were ex-pressed as His-tagged proteins in baculovirus-infected Sf9 insect cells asdescribed previously (Kurkela et al., 2007). The collected cells were osmoti-cally lysed and the suspension was centrifuged at 41,000g for 2 h. Theresulting pellets were homogenized, suspended in 25 mM Tris-HCl buffer (pH

7.5) and 0.5 mM EDTA and stored in aliquots at �70°C until use (Kurkela etal., 2003).

Control samples, insect cell membranes without any human UGT, wereprepared by infecting insect cells with baculovirus that does not encode anyhuman UGT and then treating the cells as described above. Pooled HLM (lot18888) and recombinant UGT1A9 “supersomes” (lot 81661; expressed in Sf9insect cells) were purchased from BD Gentest (Woburn, MA). Protein con-centrations were determined by the BCA protein assay (Thermo Fisher Sci-entific, Waltham, MA).

Drug Binding Assays. We developed an ultrafiltration method to measurethe binding of AZT, entacapone, and 4-MU to BSA, control insect cellmembranes, and HLM. The technical component of the assay and basiccalculations to determine the drug fraction that is bound to the device aredescribed under Nonspecific Binding to the Filter Device and Filter Pretreat-ment, whereas the results for the different drugs and the determination of theunbound drug fraction under different conditions are described under Results.The filter devices for the drug binding assays were Amicon Ultra filters with10-kDa Ultracel regenerated cellulose membrane, 500 �l volume, and theywere purchased from Millipore Corporation (Billerica, MA).

Nonspecific Binding to the Filter Device and Filter Pretreatment. Asample of the compound in phosphate buffer (500 �l solution, 50 mM, pH 7.4)was transferred to a filter device and centrifuged twice at 2500g, 1 min eachtime, to collect two separate 50-�l filtrate fractions. In total, a maximal volumeof 100 �l was allowed to pass through the filter, namely �20% of the totalloaded volume. The first filtrate sample was removed, and a 30-�l aliquot fromthe second 50-�l filtrate fraction, as well as a similar sized sample from theprefiltered solution, were collected, each mixed with 60 �l of 4 M perchloricacid/methanol (MeOH) (1:5 mix) and submitted to ultra-performance liquidchromatography (UPLC) analysis. The experiments were performed in tripli-cate, and the nonspecific binding to the filter device was calculated using thefollowing equation:

NSBf �[S]prefilter � [S]filtrate

[S]prefilter

in which [S] is the substrate concentration in the respective solutions.The nonspecific binding to the filter device (NSBf) was significantly low-

ered by the following pretreatment: filter device wash twice with 400 �l of 1%Tween 20 and removal of the remaining detergent solution by 5 min ofcentrifugation at 5000g and a subsequent wash with 500 �l of phosphate buffer(50 mM, pH 7.4).

The integrity of the filter device membrane was tested after the pretreatmentby filling the device with 450 �l of either 2% BSA solution or 1 mg/ml ofcontrol insect cell membranes, centrifuging for 10 min at 5000g, transfer of a200-�l aliquot of the resulting filtrate to a 1.5 ml-centrifuge tube, acidificationof the sample with 20 �l of 4 M perchloric acid, transferring to ice for 20 min,centrifuging for 10 min at 16,000g, and visually inspecting the centrifuge tubefor protein precipitates.

Determination of Substrate Binding to BSA, HLM, and Control InsectCell Membranes. The substrate of interest was first incubated with BSA,HLM, or insect cell membranes in phosphate buffer (50 mM, pH 7.4) in a totalvolume of 500 �l for 60 min at 37°C. The solution was then transferred to thefilter device and centrifuged twice for 1 min at 2500g. In total, a maximalvolume of 100 �l was allowed to pass through the filter (�20% of totalvolume). The 30-�l aliquots from the second filtrate fraction and from theprefiltered solution were each mixed with 60 �l of 4 M perchloric acid/MeOH(1:5 mix), transferred to ice for 20 min, centrifuged for 10 min at 16,000g, andthen submitted to substrate concentration determination by a UPLC analysis.The experiments were performed in triplicates.

Drug Glucuronidation Assays. Stock solutions of AZT, entacapone, and4-MU were prepared in methanol and diluted with methanol to the desiredconcentrations immediately before use. Appropriate amounts of these dilutionswere transferred into 1.5-ml centrifuge tubes and the solvent was evaporated invacuo at ambient temperature. The solid residues were dissolved in the reactionmixture containing phosphate buffer (50 mM, pH 7.4), MgCl2 (10 mM), BSA(0–2%), and an enzyme source (0.02–0.2 mg/ml total protein in the mem-brane, depending on the enzyme source) to a final volume of 100 �l.

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The reaction mixtures for the HLM assays also contained alamethicin at afinal concentration of 5% of the microsomal protein concentrations, and theywere placed on ice for 30 min (Fisher et al., 2000) before continuing as withthe recombinant UGT-containing samples. Alamethicin was not added to theincubations with recombinant UGTs because it has no significant effect on theglucuronidation activity of such samples (Zhang et al., 2011).

The reaction mixtures (in the case of HLM, after the preincubation withalamethicin) were incubated first for 30 min at room temperature, followed by5 min at 37°C, initiated by the addition of UDPGA to a final concentration of5 mM, and performed at 37°C (15–60 min) protected from light. Negativecontrols, including without UDPGA, without substrate, or with control(“empty”) insect cell membranes, were performed for each set of assays. The4-MU glucuronidation reactions were terminated by the addition of 10 �l ofice-cold perchloric acid (4 M). In the cases of AZT and entacapone, thereactions were terminated by the addition of 60 �l of ice-cold 4 M perchloricacid/MeOH (1:5 mix). After reaction termination, the tubes were transferred toice for 30 min and then centrifuged at 16,000g for 10 min. Aliquots of theresulting supernatants were transferred to dark glass vials and subjected toHPLC or UPLC analyses.

Analytical Methods. The HPLC system consisted of an Agilent 1100 seriesdegasser, binary pump, 100-vial autosampler, thermostated column compart-ment, multiple wavelengths UV detector, and fluorescence detector (AgilentTechnologies, Santa Clara, CA). The resulting chromatograms were analyzedwith Agilent ChemStation software (revision B.01.01) on Windows XP Pro-fessional software (Microsoft, Redmond, WA). For separation and detection of4-MU-�-D-glucuronide, we used a Chromolith SpeedROD RP-18e (50 � 4.6mm, 3 �m; Merck) column (at a column temperature of 40°C and injectionvolume of 20 �l). The mobile phase consisted of 80% 50 mM phosphatebuffer, pH 3 (A) and 20% methanol (B) at a constant flow rate of 2 ml/min. Afluorescence detector with an excitation wavelength of 316 nm and emissionwavelength of 82 nm was used for detection. The retention time of 4-MU-�-D-glucuronide under these conditions was 1.45 min. The quantification wasbased on a standard curve prepared using an authentic glucuronide standard.

The UPLC system consisted of a Waters Acquity UPLC (Waters, Milford,MA) system equipped with an Acquity UPLC BEH C18 column (2.1 � 100mm, 1.7 �m; Waters) and a precolumn (column temperature of 40°C), columnmanager, sample manager, binary solvent pump, and photodiode array UVdetector. The UV detector was equipped with high-sensitivity 2.4-�l flow cell.The resulting chromatograms were analyzed with Empower 2 software (Build2154; Waters) on a Windows XP Professional operating system. We developedUPLC methods to separate and detect zidovudine-�-D-glucuronide and enta-capone-�-D-glucuronide on the basis of their UV absorbance as well aszidovudine, entacapone, and 4-MU substrates for analyses of the binding assayresults. The injection volume was 10 �l for all samples.

For the separation of AZT-�-D-glucuronide, the mobile phase consisted of0.1% formic acid (A) and acetonitrile (B) and the flow rate was 0.6 ml/min.UV absorbance at 267 nm was used for detection. The gradient in this methodwas as follows: 0 to 1 min of 5% B, 1 to 4 min of 5 to 30% B, 4.5 to 5 minof 30 to 80% B, and 5 to 6 min of 5% B. The AZT-�-D-glucuronide retentiontime was 2.40 min. The quantification of AZT-�-D-glucuronide was based ona standard curve constructed using the UV absorption of zidovudine.

For the separation of entacapone-�-D-glucuronide, the mobile phase con-sisted of 50 mM phosphate buffer, pH 3 (A), and acetonitrile (B), and the flowrate was 0.5 ml/min. UV absorbance at 309 nm was used for detection, and thequantification was done using a standard curve made with an authentic gluc-uronide standard. The gradient in this method was as follows: 0 to 3 min of 20to 30% B, 3 to 3.2 min of 30 to 80% B, 3.2 to 4 min of 80% B, 4 to 4.1 minof 80 to 20% B, and 4.1 to 6 min of 20% B. The entacapone-�-D-glucuronideretention time was 2.18 min.

A single UPLC method was developed for separation and analysis ofAZT, entacapone, and 4-MU in drug binding assays. The mobile phaseconsisted of 50 mM phosphate buffer, pH 3 (A), and acetonitrile (B), andthe flow rate was 0.6 ml/min throughout. UV absorbance at 267, 309, and321 nm was used for detection of AZT, entacapone, and 4-MU, respec-tively. The run was isocratic with 35% B for 1.5 min. The retention timesof AZT, entacapone, and 4-MU and were 0.72, 0.87, and 1.32 min,respectively. The quantification was based on standard curves preparedwith the respective compounds.

Glucuronidation activities are reported as the average and S.E. of at leastthree replicate determinations. Please note that because of the lack of suitableisoform-specific anti-UGT antibodies, the glucuronidation rates and Vmax

values of the different recombinant UGTs and HLM cannot be compareddirectly.

Enzyme Kinetic Analyses. The protein concentrations and incubationtimes for the kinetic analyses reactions were selected based on preliminaryassays to ensure that product formation was within the linear range withrespect to protein concentration and time, and that the substrate consumptionduring the reaction was less than 10%. The substrate concentration ranges forAZT, entacapone, and 4-MU enzyme kinetic experiments were 50 to 2000, 5to 750, and 5 to 500 �M, respectively. The UDPGA enzyme kinetic assayswere performed with either 75 �M entacapone or 50 �M 4-MU as theaglycone substrate. The incubation times varied from 15 to 60 min.

The enzyme kinetic parameters were obtained by fitting kinetic models tothe experimental data using GraphPad Prism version 5.01 for Windows(GraphPad Software Inc., San Diego, CA). The best model was selected basedon the corrected Akaike’s information criterion, the calculated r2 values,residuals graph, parameter S.E. estimates, 95% confidence intervals, and visualinspection of the Eadie-Hofstee plots. In assays containing BSA, the freesubstrate concentrations (fu, or fraction unbound), were corrected according tothe estimated drug binding to BSA under the specific conditions of eachglucuronidation assay. Data were fitted with the following models:

Michaelis-Menten equation

v �Vmax[S]

Km � [S]

where v is the initial velocity of the enzyme reaction, Vmax is the maximalvelocity, [S] is the substrate concentration, and Km is the Michaelis-Mentenconstant (concentration of substrate at 0.5 of Vmax).

Substrate inhibition equation

v �Vmax[S]

Km � [S]�1 �[S]

Ki�

where Ki is the constant describing the substrate inhibition interaction.Allosteric sigmoidal model (Hill equation)

v �Vmax[S]h

S50h � [S]h

where S50 is the concentration of substrate at 0.5 of Vmax (analogous to Km inthe Michaelis-Menten model) and h is the Hill coefficient.

Two-site biphasic model equation (Korzekwa et al., 1998)

v �Vmax1[S] � CLint[S]2

Km1 � [S]

where Vmax1 and Km1 are estimated from the curved portion of the plot atlower substrate concentrations. The CLint represents the ratio of Vmax2/Km2

and describes the linear portion of the plot exhibited at higher substrateconcentrations.

Two-sites model equation (Houston and Kenworthy, 2000)

v �

Vmax�[S]

Ks�

�[S]2

�Ks2 �

1 �2[S]

Ks�

[S]2

�Ks2

where Ks is a substrate dissociation constant, � describes the change insubstrate binding affinity for the second enzyme site, and � describes thechange in rate of product formation from the substrate-enzyme-substrate (S �

E � S) complex compared with the enzyme-substrate (E � S) complex.

Results

Drug Binding to the Filter Device, BSA and Insect Cell Mem-branes, and HLM. A prerequisite for correct interpretation of the

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BSA effect on enzyme kinetics is being able to determine the con-centration of free substrate (the drug) in the presence of BSA, theso-called fraction unbound (fu). Rowland et al. (2007) have used anequilibrium dialysis device for this, and in the current study, in theabsence of such an instrument, we developed an ultrafiltrationmethod. To obtain values of fu that are as accurate as possible, we tookinto consideration the amount of drug that binds to the filter deviceeven in the absence of BSA, the so-called nonspecific binding (NSBf).The method is detailed in the Materials and Methods, and examplesof the obtained results with an untreated filter device for 20 �M AZT,entacapone, and 4-MU solutions are NSBf values of 20, 99, and 40%,respectively. In an effort to decrease the NSBf, we tested several waysto decrease that nonspecific binding by a suitable filter pretreatment.The best results were achieved by a double wash with a solution of themild detergent Tween 20, 1% final concentration, followed by phos-phate buffer rinse, as described under Materials and Methods. Thispretreatment reduced the NSBf of 20 �M AZT, entacapone, and 4-MUsolutions to the acceptable values of 1, 27, and 7%, respectively.

We further examined the effect of drug concentration on its NSBf

and found that for entacapone (the most lipophilic compound amongthe tested drugs in this study) it is saturable in nature and exponen-tially decreases with increasing entacapone concentration (Fig. 1A,inset). To obtain a good estimation of the NSBf of entacapone at anygiven entacapone concentration, the determined binding values werefitted to the following exponential decay (empirical) equation:

NSBf � (NSBf-max � NSBf-min)e� k[S] � NSBf-min

where NSBf-max is the maximal measured NSBf, NSBf-min is theminimal measured NSBf at the plateau region, k is the exponentialdecay rate constant, and [S] is the concentration of entacapone.

Unlike entacapone, the NSBf of 4-MU was essentially concentrationindependent. Because of this, for calculating its fu, we took the meanvalue of the obtained data points (fu � 7%). The NSBf of AZT topretreated filter devices was negligible.

After the clarification of the NSBf and its dependence on drugconcentration, we turned to the determination of drug binding to BSA.

AA Entacapone binding to 0.1%BSA

0 6

0.8

1.0

one)

Entacapone nonspecific binding0 4

0.2

0.4

0.6

f u(E

ntac

apo g

to filter (NSBf)

0.0

0.1

0.2

0.3

0.4

NSB

f(en

taca

pone

)

0 100 200 300 400 500 600 700 8000.0

[Entacapone], µM

0 200 400 600 800[Entacapone], µM

B B 4-MU binding to 0.1 and 1%BSA

0.8

1.0

)

4-MU nonspecific bindingto filter (NSBf)

0 3

0.4

0.5

MU

)

0.2

0.4

0.6

f u(4

-MU

)

0 100 200 300 400 5000.0

0.1

0.2

0.3

[4-MU], µM

NSB

f(4-

M

0 100 200 300 400 5000.0

fu at 0.1% BSAfu at 1% BSA

[4-MU], µM

FIG. 1. Binding of entacapone to 0.1% BSA (A) and binding of4-MU to 0.1 and 1% BSA (B). The NSBf of entacapone and 4-MUis presented as figure insets. The results are presented as fu andrepresent mean � S.E. (n � 3). The entacapone binding data werefitted to a empirical hyperbolical equation; the entacapone NSBf

data were fitted with an empirical exponential decay equation;4-MU binding data to 0.1 and 1% BSA were fitted with empiricalexponential association equation and linear equation, respectively;and the 4-MU NSBf data were fitted with linear equation (seeMaterials and Methods for all details).


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To avoid impractically high fu values, particularly for entacapone, wetested the effects on enzyme kinetics of lower BSA concentrationsthan 2%, the value used in the previous studies (Rowland et al., 2007,2008). It turned out that the presence of as low as 0.1% BSA in thereaction mixture was sufficient to yield the stimulatory effect (seebelow) and, therefore, for entacapone we mainly used 0.1% BSA. Inthe case of 4-MU, the kinetic experiments were performed in thepresence of either 0.1 or 1% BSA, whereas 1% BSA was used for theAZT studies to make them more comparable to the previously pub-lished studies.

The binding of AZT, entacapone, and 4-MU to BSA and thedifferent biological membranes (enzyme sources) was studied in theconcentration ranges of 5 to 2000, 5 to 750, and 5 to 500 �M,respectively. The fu was calculated by the following equation:

fu �[S]filtrate

[S]prefilter(1 � NSBf)

where [S]filtrate is the drug concentration in filtrate, [S]prefilter is itsconcentration in the prefiltered solution, and NSBf is the nonspecificbinding to the filter in the absence of BSA and/or an enzyme source.

The fu of entacapone in the presence of 0.1% BSA increasedhyperbolically with increasing entacapone concentration and, there-fore, the obtained values were fitted to the following empirical equa-tion that appears to provide a good description of the results:

fu �fu-max[S]

Ks � [S]

where fu-max is the maximal fraction unbound achieved in thepresence of an unlimited amount of entacapone and Ks is theconcentration of entacapone at half fu-max. The binding of entaca-pone to 1% BSA was also tested, but because it was excessive andthe estimated fu was well below 1% at low entacapone concentra-tions (data not shown), all subsequent experiments on the effect ofBSA on entacapone glucuronidation were performed in the pres-ence of 0.1% BSA.

The fu of 4-MU in the presence of 0.1% BSA increased curvi-linearly with increasing 4-MU concentration, and the results werewell described by the following empirical equation of exponentialassociation:

fu � fu-min � (fu-max � fu-min)(1 � e�k[S])

In this equation, fu-min is the minimal measured fraction unbound,fu-max is the maximal fu, k is an association rate constant, and [S] is theconcentration of 4-MU. The fu of 4-MU at 1% BSA increased linearlywith increasing 4-MU concentration.

The binding of AZT to either 0.1 or 1% BSA was negligible(�1%), a result that is in good agreement with the previously reportedbinding properties of this drug (Rowland et al., 2007).

The dependence of entacapone and 4-MU binding to BSA may alsobe presented in the form of a binding plot (supplemental text andSupplemental Fig. 1). These analyses revealed that the binding ofentacapone and, to a lesser extent, of 4-MU is biphasic, suggestingthat two or more binding sites on albumin may be involved. Becauseof this finding, we further tested the binding of entacapone to 0.1%BSA in the presence of 4-MU. However, the results indicated that thefu of entacapone is only modestly increased in the presence of 4-MU,suggesting minor binding competition between the two substrates(Supplemental Fig. 2).

The binding of AZT, entacapone, and 4-MU to the biologicalmembranes that carry the tested UGTs was tested using HLM and

control insect cell membranes. The results indicated that up to 0.2mg/ml (total protein), the highest protein concentration used in this work,the binding of the three tested drugs is very low. However, it was notedthat binding of entacapone to higher than 0.5 mg/ml enzyme source wasconsiderable and should be taken into account if such high concentrationsof an enzyme source are used (data not shown).

We also studied drug binding to BSA in the presence of eitherinsect cell membranes or HLM. It was surprising to note that theresults indicated that the presence of high amounts of an enzymesource lowers entacapone binding to 0.1% BSA, whereas 4-MUbinding to either 0.1 or 1% BSA was not significantly affected by thepresence of insect cell membranes. Although the effect of membranepresence on entacapone binding to BSA was not large, it mightbe significant when more membranes (enzyme source) are added tothe reaction mixture. We thus interpolated an empirical three-dimen-sional (3D) function over the experimental data points (Fig. 2B) thatenables estimation of entacapone binding to 0.1% BSA at any givenconcentration of entacapone and enzyme source used during an invitro assay. Scatchard plots for the binding of entacapone for eitherBSA or insect cell membranes, as well as to different combinations ofthe two, were also drawn from the data presented in Fig. 2. They showthat although the character of entacapone binding to BSA remainsbiphasic, the addition of enzyme source decreases the apparent affin-ity of entacapone for albumin in a concentration-dependent manner(Supplemental Fig. 3).

We also tested if other reaction components, such as MgCl2 andUDPGA, affect drug binding. The results indicated that neither MgCl2nor UDPGA significantly changes the binding of all of the testedcompounds to BSA or the enzyme sources.

Enzyme Kinetics of AZT Glucuronidation with HLM andUGT2B7. After all of the needed experiments and analyses that aredescribed above, we turned to the UGTs and the effect of the presenceof BSA on their kinetics. The studies on the effect of 1% BSA on theAZT glucuronidation kinetics by HLM and recombinant UGT2B7,expressed in insect cells, are shown in Fig. 3, and the derived kineticparameters are presented in Table 1. AZT glucuronidation by HLMand UGT2B7 is best described by the Michaelis-Menten model, andthe addition of 1% BSA significantly decreased the Km values for bothenzyme sources, without affecting the respective Vmax values (thelatter values differ from each other because a different enzyme sourcewas used, and we do not have a good way to determine the UGT2B7concentration within each membrane sample). A further increase ofthe BSA concentration to 2% did not significantly affect the kineticparameters. The obtained kinetic parameter values and the effect ofBSA addition on them are in good agreement with previous resultswith locally made HLMs and recombinant UGT2B7 that was ex-pressed in a different system than the one used in this study, HEK293cells (Uchaipichat et al., 2006; Rowland et al., 2007).

Enzyme Kinetics of Entacapone with HLM and UGT1A9. En-tacapone is a nearly selective UGT1A9 substrate in the liver (Lautala etal., 2000), and we used it as a model compound for studying the effect ofBSA on either recombinant UGT1A9 or the native enzyme in HLMs.Because the preliminary results were significantly different from thepreviously published finding for this enzyme (Rowland et al., 2008), weperformed these studies using two different preparations of recombinantUGT1A9, the one from our laboratory that carry a short C-terminal fusionpeptide (Kurkela et al., 2003) and the commercial UGT1A9 Supersomesthat lack such a fusion peptide. It may be added here that the concentra-tion of active UGT1A9 in the Supersomes sample appears to be signif-icantly higher than in our recombinant UGT1A9 sample, but, as shownbelow, the differences between the two preparations do not much affecttheir kinetics.


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The clear and unexpected result from the experiments with all threedifferent UGT1A9 samples was that in addition to decreasing thereaction Km value, the presence of 0.1% BSA in the reaction mixtureled to a large increase in the three respective Vmax values (Fig. 4;Table 1). Mild substrate inhibition was observed at higher entacaponeconcentrations, and the obtained Ki values were 75- to 1500-foldhigher than the respective Km values (Table 1). Because of the largedifferences between the Km values and the respective Ki values, theobtained Vmax values were not significantly influenced by the Km/Ki

ratio and were not largely underestimated as they would have been ifthe Km/Ki ratio was closer to 1. The presence of BSA also somewhatchanges the apparent enzyme kinetic model of entacapone glucuroni-dation from mild substrate inhibition to partial substrate inhibition.The latter means that on the basis of the expectation from the substrate

inhibition model, the inclusion of BSA led to a less-than-expecteddecrease in the glucuronidation rate in the presence of high entaca-pone concentrations. Therefore, in addition to the empirical substrateinhibition equation, we fitted the experimental data to a mechanistictwo-site model equation (Houston and Kenworthy, 2000). This modelassumes the existence of two identical substrate binding sites and canbe used for sigmoidal and substrate inhibition kinetics. But because noautoactivation was observed, we constrained parameter � to 1 andused parameter � to describe the changes in the rate of productformation from the S � E � S complex in comparison to its formationfrom an E � S complex (Table 1).

Enzyme Kinetics of 4-MU Glucuronidation by UGT1A9. Tofurther explore the effect of BSA on the Vmax of UGT1A9 in entaca-pone glucuronidation (Fig. 4; Table 1) and find out if this is only a

A Entacapone binding to 0.1% BSA and membrane1 0

0.6

0.8

1.0

apon

e)

1.0

0.2

0.4

f u(E

ntac

a

0.00 0.05 0.10 0.15 0.200.0

0.2

0.4

0.6

0.8

f u(E

ntac

apon

e)

0 100 200 300 400 500 600 7000.0

[Entacapone], µM

[Control membrane], mg/mL

B Entacapone binding to 0.1% BSA and membrane(3D plot)

0.8

1.0

ne)

(3D plot)

0.2

0.4

0.6

f u(E

ntac

apon

0.0

200400

600800

0.050.10

] µM

[Control membmg

02000.00

[Entacapone], µmbrane],

mg/mL

FIG. 2. Binding of entacapone to 0.1% BSA without Sf9 controlmembrane (F), and in the presence of 0.032 (Œ), 0.080 (�), and0.16 (�) mg/ml of Sf9 control membrane (A). The results arepresented as fu and represent mean of 3 determinations. The S.E.was very small and for the sake of clarity, in this condensed figurethe S.E. bars were left out. The data were fitted to an empiricalhyperbolical equation. The correlation between measured fu and Sf9control membrane concentration at different entacapone concentra-tions is presented as the inset in panel A. An identical set of data arepresented in the form of a 3D scatter (B). An empirical 3D functionwas fitted to data points (see supplemental material for all details).


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peculiarity of this substrate, we examined the effect of BSA additionon the glucuronidation of 4-MU by UGT1A9. Because 4-MU is not aUGT1A9-specific substrate, the 4-MU glucuronidation assays withUGT1A9 were limited to the recombinant enzymes. One reason forthe selection of 4-MU as the second test substrate for UGT1A9 is thatits use enables a direct comparison of our results with the previousstudy on the effect of BSA on UGT1A9 (Rowland et al., 2008). Theexamination of the two different samples of recombinant UGT1A9that we tested (the locally made and the commercial sample) mostclearly showed that the addition of BSA to either 0.1 or 1% resultedin a significant Km decrease and a concomitant increase in the Vmax

values, with a slightly larger effect on the Vmax by 1% BSA than by0.1% (Fig. 5; Table 1). A detailed kinetic analysis revealed that 4-MUglucuronidation by UGT1A9 also follows the substrate inhibitionequation in the absence and the presence of BSA. It is important tonote that, as in the case of entacapone, the Ki values of UGT1A9 for4-MU were significantly higher than the corresponding Km values,allowing for correct determination of Vmax from the experimental data(Table 1).

Enzyme Kinetics for the Cosubstrate UDPGA with UGT1A9.We also explored the possibility that the presence of BSA affects thekinetics of UGT1A9 with the cosubstrate UDPGA. In these assays, weused either entacapone or 4-MU as the aglycone substrate, and theresults show that, regardless of the aglycone used, the reaction wasbest described by the Michaelis-Menten model (Fig. 6; Table 1). Theinclusion of 0.1% BSA when entacapone was the aglycone substrateresulted in a Vmax increase without a Km change (Fig. 6A). However,when 4-MU was the aglycone substrate, the addition of 0.1% BSA ledto a Km decrease and a Vmax increase (Fig. 6B; Table 1). The reasonfor these differences is currently unclear and should be examined inthe future.

Discussion

Drug glucuronidation rates and kinetics that are determined using invitro assays tend to underestimate the in vivo rates (Boase and Miners,2002; Miners et al., 2006). Rowland et al. (2007, 2008) have foundthat the addition of BSA significantly enhances the activity ofUGT2B7 and UGT1A9 by decreasing their Km for the aglycone

A AZT, HLM, no BSA and 1% BSA

0 3

0.4

0.5

no BSA1% BSA

n/m

g

0.1

0.2

0.3

V, n

mol

/mi

0 0

0.1

0.2

0.3

0.4

0.5

V, n

mol

/min

/mg

0 500 1000 1500 20000.0

[AZT], µM

0.000 0.001 0.002 0.003 0.0040.0

V/[AZT]

B AZT, UGT2B7, no BSA and 1% BSA, ,

0 3

0.4

0.5

no BSA1% BSA

n/m

g

0.1

0.2

0.3

V, n

mol

/min

0.1

0.2

0.3

0.4

0.5

V, n

mol

/min

/mg

0 500 1000 1500 20000.0

[AZT], µM

0.000 0.001 0.0020.0

V/[AZT]

FIG. 3. Enzyme kinetics of AZT glucuronidation by HLM (A) andUGT2B7 (B) without BSA and in the presence of 1% BSA. Thepoints represent an average of three samples � S.E. Glucuronida-tion rates are presented as actual (measured) rates in nmol � min�1 �mg�1 recombinant protein. The derived kinetic constants are pre-sented in Table 1. The data were fitted to the Michaelis-Mentenequation. The Eadie-Hofstee transforms of the data are presented asinsets.


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TA

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5�

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A Entacapone, HLM, no BSA and 0.1% BSA

0 100 200 300 400 500 600 700 800

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

no BSA0.1% BSA

[Entacapone], µM

V, n

mol

/min

/mg

0 1 2 3 4 50.0

4.0

8.0

12.0

V/[Entacapone]

V, n

mol

/min

/mg

B Entacapone, UGT1A9, no BSA and 0.1% BSA

0 100 200 300 400 500 600 700 800

0.0

5.0

10.0

15.0

no BSA0.1% BSA

[Entacapone], µM

V, n

mol

/min

/mg

0 1 2 3 4 50.0

5.0

10.0

15.0

20.0

V/[Entacapone]

V, n

mol

/min

/mg

C Entacapone, UGT1A9 Supersomes,no BSA and 0.1% BSA

0 100 200 300 400 500

0.0

10.0

20.0

30.0

no BSA0.1% BSA

[Entacapone], µM

V, n

mol

/min

/mg

0 2 4 6 8 100.0

10.0

20.0

30.0

V/[Entacapone]

V, n

mol

/min

/mg

FIG. 4. Enzyme kinetics of entacapone glucuronidation by HLM(A), in-house-produced UGT1A9 (B), and commercial UGT1A9(C) without BSA and in the presence of 0.1% BSA. The pointsrepresent an average of three samples � S.E. The concentrations ofentacapone were corrected for nonspecific binding. Glucuronida-tion rates are presented as actual (measured) rates in nmol � min�1 �mg�1 recombinant protein. The derived kinetic constants are pre-sented in Table 1. The data were fitted to the two-site equation (seeMaterials and Methods for details). The Eadie-Hofstee transformsof the data are presented as insets.


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substrate without affecting the Vmax values. They offered an interest-ing explanation for this “BSA effect”—removal of a long-chain fattyacid that competitively inhibits the UGTs. At the start of the studypresented here, we wondered if the proposed inhibitory fatty acids arealso present in the recombinant UGTs that we often use for gluc-uronidation studies—in-house-produced recombinant UGTs that areexpressed in insect cells and carry a C-terminal fusion peptide(Kurkela et al., 2003, 2007). However, the results took us to differentdirections, mainly to validate the initial observation that the BSAeffect in UGT1A9 is different and more complex than in UGT2B7.However, before doing this, we developed the needed methods formeasuring drug binding to BSA and different biological membranes.

Many drugs bind nonspecifically to macromolecules; therefore,determining the fraction of the added drug that is free under theexperimental conditions (the fu) during in vitro assays is importantbecause only the unbound fraction interacts with the target enzyme(Grime and Riley, 2006; Varshney et al., 2010). We measured drugbinding to BSA and two enzyme sources, insect cell membranes andHLM, by a newly developed ultrafiltration assay. Because ultrafiltra-

tion systems often “suffer” from high NSBf values (Lee et al., 2003;Taylor and Harker, 2006), we took care to minimize, determine, andtake into account the nonspecific binding to the filter device, regard-less of whether the binding was to the filter itself or to the walls of thetube. Our results concerning AZT binding to BSA, negligible binding,are the same as in a previous study that used a dialysis system(Rowland et al., 2007). On the other hand, our finding that 4-MUbinding to BSA is concentration dependent to some extent, particu-larly at 4-MU concentrations less than 100 �M 4-MU and in thepresence of 0.1% BSA (Fig. 1B), is not in full agreement with thepublished studies/results (Rowland et al., 2008). A possible reason forthis is that the substrate concentration dependence of 4-MU is mainlyvisible at substrate concentrations less than 50 �M in the presence of0.1% BSA (Fig. 1B), whereas the lowest 4-MU concentration testedin the previous study (in the presence of 0.1, 1, and 2% BSA) was 50�M (Rowland et al., 2007).

Entacapone binds strongly to the filter device and to BSA (Figs.1A and 2). Our analysis clearly demonstrates that, at least for somedrugs, the binding is concentration dependent, and therefore the

A 4-MU, UGT1A9, no BSA, 0.1

3

4

/mg

1

2

V, n

mol

/min

/

0 100 200

0

[4-MU

BB 4-MU, UGT1A9 Supersomes

10

15

in/m

g

1

V, n

mol

/min

/mg

5V, n

mol

/mi

0 100 200

0

[4-MU

1, and 1% BSA

1

2

3

4

V, n

mol

/min

/mg

no BSA

0.0 0.5 1.0 1.50

V/[4-MU]

300 400 500

no BSA0.1% BSA1% BSA

U], µM

s no BSA 0 1 and 1%BSAs, no BSA, 0.1 and 1%BSA

5

0

no BSA0 1% BSA

0 2 4 60

V/[4-MU]

300 400 500

0.1% BSA1% BSA

], µM

FIG. 5. Enzyme kinetics of 4-MU glucuronidation by in-house-produced UGT1A9 (A) and commercial UGT1A9 (B) without BSAand in the presence of 0.1 and 1% BSA. The concentrations of4-MU were corrected for nonspecific binding. The points representan average of three samples � S.E. Glucuronidation rates are pre-sented as actual (measured) rates in nmol � min�1 � mg�1 recombi-nant protein. The derived kinetic constants are presented in Table 1.The data were fitted to the substrate inhibition equation. The Eadie-Hofstee transforms of the data are presented as insets.


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correct fu value for each point on the kinetic curve should be takeninto account. This outcome differs from the simpler view thatemerged from the earlier reports of Rowland et al. (2007, 2008),but our findings are very clear and indicate that the substratedependence of binding to BSA, and perhaps to plasma also, shouldbe evaluated for each drug under study. A further small compli-cation in the case of entacapone binding, and perhaps other highlylipophilic compounds, is possible displacement from BSA by theenzyme source such as insect cell membranes (Fig. 2). This dis-placement was more significant at substrate concentrations lessthan 200 �M and in the presence of higher enzyme source con-centrations (Fig. 2), and although it was not significant in our case,it might turn out to be meaningful with other drugs that exhibitsimilar features but that serve as poor substrates to the tested UGT,perhaps leading to the addition of excessive amounts of recombi-nant enzyme. In such cases, a 3D binding curve, similar to the onewe present in Fig. 2B, may be essential to estimate the fu atdifferent points of the experiment.

The current results on the effect of BSA addition on AZT gluc-uronidation by UGT2B7, either in its native state within HLM or as arecombinant enzyme that was expressed in insect cell membranes andcarries a short C-terminal fusion peptide (Fig. 3), are very similar tothe earlier results for HLM and recombinant UGT2B7 that wasexpressed in HEK293 cells (Rowland et al., 2007). This findingstrongly suggests that, at least with respect to the putative competitiveinhibitor that is removed by the addition of BSA to the reactionmixture, there is no significant difference between the recombinantUGTs, regardless of whether they were expressed in HEK293 orinsect cells, to the native UGT in HLM. Likewise, our results on theBSA effect on UGT1A9 do not give any reason to suspect thatsignificant differences in enzyme kinetics and the interactions ofUGT1A9 with its aglycone substrates exist between the native and therecombinant enzyme that was expressed in insect cells, with or with-out a C-terminal fusion peptide (Figs. 4 and 5). On the other hand,there is a major difference between our results with UGT1A9 andthose from a previous study (Rowland et al., 2008).

A E t UGT1A9 (UDA Entacapone, UGT1A9 (UDno BSA and 0.1% BSA15.0 no BSA

0.1% BSA

mg

5.0

10.0

V, n

mol

/min

/m

0 1000 20000.0

[UD

BB 4-MU, UGT1A9 (UDPGA kno BSA and 0.1% BSA

2.0

2.5

mg

0 5

1.0

1.5

V, n

mol

/min

/m

0 1000 20000.0

0.5no BSA0.1% BSA

[UD

DPGA ki ti )DPGA kinetics),

10.0

15.0

l/min

/mg

0.00 0.02 0.04 0.060.0

5.0

[UDPGA], µM

V, n

mol

0 3000 4000 5000DPGA], µM

kinetics)kinetics),

1

2

3

nmol

/min

/mg

0.000 0.005 0.010 0.015 0.0200

1

V/[UDPGA]

V,n

0 3000 4000 5000PGA], µM

FIG. 6. Enzyme kinetics of UDPGA using entacapone (A) and4-MU (B) as aglycone substrates without BSA and in the presenceof 0.1% BSA. The points represent an average of three sam-ples � S.E. Glucuronidation rates are presented as actual (mea-sured) rates in nmol � min�1 � mg�1 recombinant protein. Thederived kinetic constants are presented in Table 1. The data werefitted to the Michaelis-Menten equation. The Eadie-Hofstee trans-forms of the data are presented as insets.


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We found that the addition of BSA to the entacapone glucuronida-tion reaction, which in HLM is primarily catalyzed by UGT1A9(Lautala et al., 2000), not only lowers the Km value, as previouslyfound for propofol glucuronidation by UGT1A9 (Rowland et al.,2008), but also leads to a large increase in the Vmax value (Fig. 4;Table 1). Moreover, our results for 4-MU glucuronidation by recom-binant UGT1A9 demonstrate that the differences between the currentand previous results are unlikely to be simply due to the use ofdifferent aglycone substrates (something that would have been im-portant by itself). The general importance of our findings stems fromthe fact that they do not fit into the previously suggested schemeaccording to which the BSA effect on UGTs is solely the removal ofa competitive inhibitor, possibly a long-chain fatty acid, from theaglycone substrate binding site of the enzyme (Rowland et al., 2007,2008).

It is currently difficult to explain the differences between the twosets of results, particularly that there is such a good agreementbetween them on the effect of AZT glucuronidation by UGT2B7.Nevertheless, the combined results allow us to construct a theoreticalframework in which the observed BSA effect can be rationalized andfurther studied (Fig. 7). The suggestion is based on the assumptionthat UGT-catalyzed glucuronidation reactions follow a compulsoryordered bi-bi mechanism in which UDPGA is the first binding sub-strate (Luukkanen et al., 2005). Because a Km decrease and a Vmax

increase take place in UGT1A9 catalyzing entacapone glucuronida-tion in the presence of BSA, and because the enzyme affinity toUDPGA was relatively unaffected, it is suggested that the inhibitor(s)that BSA removes binds to either enzyme-UDPGA (E � AX) orenzyme-UDPGA-substrate (E � AX � B) complexes rather than to thefree enzyme, “E” (Fig. 7).

The exact nature of the tentative inhibitor(s) and the reasons for thedifferential effect of BSA toward UGT1A9 and UGT2B7 remainunknown at this stage. One possibility is that UGT1A9 is inhibited bya mixture of different inhibitors, perhaps including competitive, non-competitive, and/or mixed-type inhibitors, all of which are probablyreleased from the cells/membranes during the preparation of the

samples for in vitro glucuronidation assays. Such a mixture willprobably include inhibitors with different affinities to different indi-vidual UGTs and thereby might also explain the small change of thekinetic model for UGT1A9 that was caused by BSA addition (Fig. 4;Table 1).

In summary, our results show that addition of BSA enhances the invitro activities of UGT2B7 and UGT1A9, regardless of whether anative enzyme in HLM was used, or a recombinant UGT with orwithout a C-terminal fusion peptide. Drug binding to BSA differs inthe extent and dependence on drug concentration, indicating that itshould be determined carefully in each case. The BSA effect is (even)more complex than described previously, but its full clarification maylead us to a better and deeper understanding of the full kineticmechanism of drug glucuronidation by the UGTs.

Acknowledgments

We thank Johanna Mosorin for skillful technical assistance.

Authorship Contributions

Participated in research design: Manevski and Finel.Conducted experiments: Manevski and Morelo.Performed data analysis: Manevski, Morelo, and Finel.Wrote or contributed to the writing of the manuscript: Manevski, Yli-

Kauhaluoma, and Finel.

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B

I1

E·AX·I

I2

E·AX·B·I(dead-end complex)

B

E

AXE·AX

BE·AX·B

E·A·BXE·AA

BXB

E·A·B(dead-end complex)

FIG. 7. The proposed mechanism for inhibitor interactions with a compulsory orderedbi-bi mechanism of UGT catalysis (see Discussion for details). E, enzyme (UGTs); AX,UDP-�-D-glucuronic acid; A, UDP; B, substrate (aglycone); I, inhibitor.


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dmd 39:2117–2129, 2011 printed in u.s.a. bovine serum ... · suring drug binding to bsa and to...

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