Thermally Denatured BSA, a Surrogate Additive to

Replace BSA in Buffers for High-Throughput Screening

IMANOL PEÑA1 and JUAN MANUEL DOMÍNGUEZ1,2

The use of thermally denatured bovine serum albumin (tdBSA) as an additive in high-throughput screening (HTS) buffers
has been studied with the aim of finding a surrogate to native albumin devoid of its inconveniences, in particular its com-
pound masking effect. The presence of aggregates in the thermally denatured material did not have any negative impact on
common readout technologies used in HTS such as fluorescence intensity (FLINT), fluorescence polarization, time-resolved
fluorescence resonance energy transfer (TR-FRET) and luminescence. tdBSA rendered the same beneficial effects as native
albumin in several assays or even improved its performance due to the lack of specific binding properties. Although tdBSA
still binds compounds nonspecifically as any other protein does, it mitigates the compound masking effect observed with
native albumin and can be postulated as a convenient surrogate to BSA for HTS purposes. (Journal of Biomolecular
Screening 2010:1281-1286)

Key words: BSA, HTS, additive, buffer

INTRODUCTION wide diversity of molecules, from fatty acids to xenobiotics,

A
due to the existence of 4 different binding sites in addition to 2
MONG THE ISSUES THAT A SCREENING SCIENTIST must face small metal binding pockets.2 Such drug-binding properties
when scaling up an assay for high-throughout screening make albumin a critical element influencing the in vivo effects
(HTS), those related to the stability of reagents are particularly of drugs,3,4 and this is precisely the main caveat for using albu-
critical. There are several reasons behind the drop in biological min as an additive in HTS: the effect of compounds in a given
activity observed over time in an HTS platform, but probably the assay may be significantly masked, or even negated, upon
most common ones are deterioration of the reagents and losses binding to albumin. Although other proteins may be used as
in their effective concentrations due to aggregation or to adsorp- surrogates, they are also affected by important drawbacks such
tion to plasticware. To solve this issue, bovine serum albumin as poor solubility (casein) or a considerably higher cost.
(BSA) is commonly added to buffers in µM concentrations given It would, therefore, be desirable to have a protein available
the beneficial effects caused by such an abundant and inexpen- with the beneficial properties of BSA but devoid of its negative
sive protein.1 BSA addition increases significantly the total pro- effects. Since BSA binding ability is dictated by its tertiary struc-
tein concentration, thus stabilizing other biological reagents by ture, thermally denatured BSA (tdBSA) may be a good candi-
making them less susceptible to proteolysis or simply by decreas- date. However, solubility issues inherent to denatured proteins
ing water activity. Furthermore, high BSA concentration pre- may be problematic as the tendency to aggregate may interfere
vents losses of critical reagents caused by plasticware adsorption with the optical detection technologies commonly used in HTS.
as albumin blocks all the possible binding points on the plastic In this article, we describe the best conditions to inactivate albu-
surfaces. In addition, BSA is relatively inert and rarely interferes min by heating, check its performance in a variety of assay
with the biological assay. technologies, and evaluate its effect in real assays with particular
Serum albumin is the most plentiful protein in blood plasma, attention to its possible effects on masking compounds.
where it contributes to osmolarity regulation. It is also widely
recognized as a carrier protein by its unpaired ability to bind a MATERIALS AND METHODS
1
Biological Reagents and Assay Development Department, GlaxoSmithKline, Materials
Madrid, Spain.
2
Current affiliation: Noscira S.A., Research Department, Madrid, Spain. Amplex™ UltraRed was from Invitrogen (Carlsbad, CA).
Received Apr 16, 2010, and in revised form Jun 24, 2010. Accepted for publi- 8-[[2-[(Fluoresceinylthioureido)amino]ethyl]thio]guanosine-3,′
cation Jun 25, 2010. 5′-cyclic monophosphate (8-Fluo-cGMP) was from Biolog
Journal of Biomolecular Screening 15(10); 2010 (Bremen, Germany). Sheep anticyclic GMP polyclonal
DOI: 10.1177/1087057110379768 antibody was from Bethyl (Montgomery, TX). Bright-Glo™

© 2010 Society for Laboratory Automation and Screening   www.sbsonline.org 1281

 Peña and Domínguez

reagents were from Promega (Madison, WI). Europium- plate spectrophotometer (SpectraMax Plus™; Molecular
labeled streptavidin was from PerkinElmer (Waltham, MA). Devices). The assay media (final volume 80 µL) included 70
Allophycocyanin-biotin (APC-biotin) was from Prozyme (San µM crotonoyl-CoA and 50 µM NADH in 50 mM sodium phos-
Leandro, CA). TAMRA-Crosstide and IMAP® beads were from phate (pH 7.4).
Molecular Devices (Sunnyvale, CA). Enoyl-ACP reductase The activity of ICL was followed by coupling isocitrate
(FabI) from Plasmodium falciparum, isocitrate lyase (ICL) production from 300 µM glyoxylate and 500 µM succinate to
from Mycobacterium tuberculosis, human Akt kinase, human isocitrate dehydrogenase (2 IU/mL) and diaphorase (0.2 IU/
D–amino acid oxidase (DAAO), and human fatty acid synthase mL) in the presence of 250 µM resazurin and 1 µM NADH in
(FAS) were cloned, expressed, and purified from suitable vec- 50 mM tricine (pH 7.4) with 5 mM MgCl2 and 0.4% (v/v) glyc-
tors by the Biological Reagents department of GlaxoSmithKline erol in a 20-µL assay volume. The increase of fluorescence
following procedures published elsewhere.5-9 All other reagents, intensity as a consequence of resorufin formation (λex 555 nm,
including dansylsarcosine, fatty acid free albumin from bovine λem 590 nm) was monitored continuously in 384-well polysty-
serum, β-lactoglobulin from bovine milk, and luciferase from rene low-volume black plates (Greiner Bio-One) using a
Vibrio fischeri, were from Sigma-Aldrich (St. Louis, MO). Gemini SpectraMax™ plate reader.
Akt kinase was monitored in the same black plates using 10
Preparation of thermally denatured BSA µM adenosine triphosphate (ATP) and 100 nM TAMRA-
Crosstide in 50 mM HEPES (pH 7.5) with 1 mM dithiothreitol
A 100-µM solution of fatty acid free albumin from bovine (DTT) and 10 mM MgCl2. The 10-µL reaction was stopped at
serum in phosphate-buffered saline (PBS) was heated at 80°C different times by adding 10 µL of IMAP/® beads previously
for 15 min. The solution was then cooled down at room tem- diluted at 1:500 from the commercial stock in assay buffer, and
perature for 2 h and subsequently stored at 4°C up to 1 month. fluorescence polarization (FP) was measured (λex 535 nm, λem
For the studies on thermal denaturation, a 100-µM solution of 580 nm) in an Analyst™ plate reader (Molecular Devices).
BSA in PBS was heated at different temperatures for 30 min DAAO was measured in the same black plates using 2 nM
and then cooled down at room temperature for 2 h. Samples enzyme and varying concentrations of several substrates
were centrifuged and the supernatant filtered (0.45 µm), and (D-Ala, D-Pro, D-Ser, D-Met, D-Trp, and D-Phe) in 50 mM
PBS was added to adjust the protein concentration as deter- Tris buffer (pH 8.0) containing 250 nM flavin adenine dinucle-
mined by absorbance at 280 nm. otide. H2O2 production was monitored continuously with 1 IU/
mL horseradish peroxidase and 100 µM Amplex™ UltraRed
Binding assay measuring resorufin formation as above.
Dansylsarcosine was used as a probe to monitor binding to
Optical readouts
BSA as already described.10 The assay was carried out in 384-
well polystyrene low-volume black plates (Greiner Bio-One, Dynamic light-scattering (DLS) measurements were per-
Gloucestershire, UK) in a final volume of 10 µL, including 2.5 formed on 60-µL samples in a 384-well clear plate (Cliniplate™;
µM BSA (concentration determined spectrophotometrically by Thermo Fisher Scientific, Waltham, MA) using a NEPHELOstar
the absorbance at 280 nm) and 5 µM dansylsarcosine. The Galaxy™ (BMG Labtech, Offenburg, Germany) plate reader
binding of the probe was determined after a 10-min incubation equipped with a laser diode emitting at 635 nm.
period at room temperature, monitoring the increase in the The FP system used to check the effect of tdBSA consisted
fluorescent properties of the dansyl group upon binding to a of a 10-µL mixture of 20 nM 8-Fluo-cGMP and a 1:200 dilu-
hydrophobic environment: the emission at 530 nm was recorded tion of commercial anti-cGMP antibody in 50 mM HEPES (pH
after exciting at 360 nm in a Gemini SpectraMax™ plate reader 7.4) using the same black plates described above and monitor-
(Molecular Devices). Then, 500 µM naproxen was used as a ing FP (λex 485 nm, λem 535 nm). The time-resolved fluores-
control of full dansylsarcosine displacement (Kd for naproxen cence resonance energy transfer (TR-FRET) system consisted
was determined to be 1.6 µM in our lab). of a 10-µL mixture of 10 nM APC-biotin and varying concen-
trations of Europium-labeled streptavidin in 50 mM HEPES
Enzyme assays (pH 7.5). TR-FRET was measured by exciting at 360 nm and
monitoring emission at 620 and 665 nm. Luminescence was
The activity of FabI was determined by monitoring the con- measured with the Bright-Glo™ system following the manu-
sumption of NADH spectrophotometrically through the facturer’s instructions and using several concentrations of
decrease in the absorbance at 340 nm at 25°C in 384-well clear- V. fischeri luciferase. In all these cases, readouts were per-
bottom polystyrene plates (Greiner Bio-One) using a 384-well formed on an Analyst™ reader.

1282 www.sbsonline.org Journal of Biomolecular Screening 15(10); 2010

 Thermally Denatured BSA as an Additive in HTS

A
100
B agreement with the functional data and therefore supporting the
25˚C
80 1 hypothesis that the loss of binding properties is due to confor-
% Binding

50˚C
mational changes. As these data were obtained after having

Fluorescence intensity
60
40 0.8 55˚C
cooled the BSA solution for 2 h, the inactivation can be consid-
20 60˚C
0 65˚C
ered irreversible.
0.6
40 50 60 70 80
Temperature (˚C)
90
70˚C
Kinetics of BSA thermoinactivation at 10 µM BSA followed
C 0.4 75˚C first-order kinetics with a kinetic constant of 3.5 10–3 s–1 (data
0.8 80˚C not shown). More concentrated BSA solutions yielded faster
Ratio em 325/350

0.7
0.2 but more complex kinetic behaviors, with small light scattering
observable in the preparation at wavelengths below 350 nm.
0.6
0 Although no turbidity was visible to the naked eye and no
300 350 400 450
0.5
40 50 60 70 80 90
appreciable sediment was obtained by centrifugation at 14 000
wavelength (nm)
Temperature (˚C) g, the presence of aggregates was evidenced by DLS measure-
FIG. 1. Thermal inactivation of bovine serum albumin (BSA). (A) ments as explained below. The inclusion of N-ethylmaleimide
The ability of the protein to bind dansylsarcosine was evaluated as (to prevent disulfide exchange with the unpaired Cys residue in
described in Materials and Methods and was compared to BSA, which BSA) did not change this behavior, whereas inclusion of urea
had not undergone the thermal denaturation process. (B) Fluorescence to prevent aggregate formation resulted in a more thermostable
emission spectra for excitation at 295 nm of the samples heated at dif- protein that could not be fully inactivated after heating at 80°C
ferent temperatures, recorded at 25°C in a Jobin Yvon Fluorolog®-3
for 30 min. Therefore, it was decided to inactivate BSA by
spectrofluorimeter. Fluorescence emission is expressed in arbitrary
units, with 1 the intensity at the wavelength of the emission maximum
heating a 100-µM solution in PBS at 80°C for 15 min (which,
of the sample heated at 55°C. All samples had identical protein con- according to the kinetics described above, fully abolishes its
centration as judged by their net absorbance at 280 nm. (C) binding properties). This material, referred to as tdBSA hereaf-
Representation of the ratio between the emissions at 325 and 350 nm ter, was investigated for any interference with the optical detec-
for samples heated at different temperatures. tion systems typically used in HTS.

RESULTS AND DISCUSSION Effect of tdBSA on detection technologies

Studies on BSA thermal denaturation DLS experiments have evidenced that both BSA and tdBSA
preparations include aggregates as deduced from the observed
In earlier studies carried out on human and bovine serum albu- light scattering (Fig. 2A). Although these are more prevalent in
mins, aggregation was described as the main event responsible for the latter, their presence in the former is not negligible, and
denaturation only at high temperatures, whereas at mild tempera- indeed the differences in light scattering when comparing BSA
tures, other processes were prevalent.11 Since the presence of with tdBSA are similar to those when comparing BSA with
aggregates may be a nuisance for the optical detection systems plain buffer. Nonetheless, it was considered worthwhile to
typically used in HTS, it was decided to run a thermal stability check the effect of these aggregates on readout technologies
study with BSA with the aim of finding the mildest conditions commonly used in HTS.
leading to full BSA inactivation as determined by the loss of abil- The fluorescent intensity (FLINT) of several fluorescent
ity to bind dansylsarcosine. Figure 1A shows a melting tempera- probes at different concentrations was checked in the presence
ture (i.e., the temperature at which 50% of inactivation is obtained) and absence of 0.5% (w/v) tdBSA or BSA. Cy3B, 7-aminocou-
of 64°C, with full inactivation being achieved at 80°C. Changes in marin, 7-hydroxy-4-trifluoromethylcoumarin (HFC), rhodamine
the intrinsic fluorescence of BSA were also monitored to confirm 110, resorufin, 5-carboxytetramethylrhodamine succinimidyl
that this loss of binding abilities was due to a significant confor- ester (TAMRA), and fluorescein were tested. Both BSA and
mational change and not to any other spurious effect. BSA con- tdBSA caused some decrease in the slopes of the concentration-
tains 2 Trp residues that account for an emission peak with a dependent FLINT curves (data not shown). The magnitude of
maximum at 350 nm when excited at 295 nm, as shown in Figure such decrease ranged from 10% for Cy3B to more severe val-
1B. This peak shifts upon heating to lower wavelengths with lower ues for resorufin and HFC. Interestingly, the decrease was
emission intensities, suggesting a change in the Trp environment similar for both albumin preparations except for the most
to a more hydrophobic one12 due to a gross conformational change severe cases: BSA caused a 70% decrease in the fluorescence
that buries these residues or simply to aggregation following of resorufin and HFC, but the effect of tdBSA was more moder-
denaturation. To calculate the melting temperature from these ate (55% and 40% decrease, respectively). Since DLS values
spectral changes, the ratio of the emission intensities at 325 for tdBSA are higher than those for BSA, it is not possible to
and 350 nm was used to normalize the total loss of intensity. attribute the effect on fluorescence intensity to any optical
From Figure 1C, a melting temperature of 67°C is deduced, in interference caused by protein aggregates.

Journal of Biomolecular Screening 15(10); 2010   www.sbsonline.org 1283

 Peña and Domínguez

A B
600 300

250
400
200

NTU

mP
200 150

100
0
Buffer BSA tdBSA 0.01 0.1 1 10 100
[cGMP] (µM)
C D
10000
140

Ratio em665/em620
120 8000
100
RLU x 10-3

6000
80
60 4000
40
2000
20
0 0
0 200 400 600 800 1000 0.1 1 10 100
[Luciferase] (nM) [EU-Stv] (nM)
FIG. 2. Effect of thermally denatured bovine serum albumin (tdBSA) on optical readouts. (A) Results of dynamic light scattering (DLS) with
phosphate-buffered saline (PBS; ●) and PBS containing 0.1% (w/v) BSA (▲) or 0.1% (w/v) tdBSA (▼). The experiment was performed as
described in Materials and Methods, and data are expressed in arbitrary nephelometric turbidity units (NTU). The lines correspond to the average
values of 8 determinations, whereas error bars denote SD. (B) Titration curve of cGMP using the fluorescence polarization (FP) tandem described
in the text. The background was measured in the absence of antibody. Samples were in buffer with (●) or without (○) antibody, in buffer containing
0.1% (w/v) BSA with (▲) or without (∆) antibody, or in buffer containing 0.1% (w/v) tdBSA with (▼) or without (∇) antibody. (C) Luciferase
titration in plain PBS (●) or in PBS with 0.1% (w/v) BSA (▲) or 0.1% (w/v) tdBSA (▼). The activity of the enzyme was followed using
BrightGlo™. Data are expressed as relative luminescence units, and the lines represent the linear fitting of the experimental points. (D) Titration
of Europium-labeled streptavidin using 10 nM APC-biotin in plain buffer (●) or buffer containing 0.1% (w/v) BSA (▲) or 0.1% (w/v) tdBSA (▼).

To test the effect on FP, the tandem formed by 8-Fluo-cGMP system, as observed in Figure 2C. Noticeably neither BSA nor
and an anti-cGMP antibody was used, and the ability of cGMP tdBSA caused any increase in the background (i.e., the signal
to displace the binding of the fluorescent probe to the antibody, detected in the absence of luciferase). Both preparations of
hence decreasing the resulting FP signal, was evaluated in the BSA also caused a beneficial effect in the performance of the
presence and absence of 0.1% (w/v) BSA and tdBSA. Figure 2B TR-FRET system shown in Figure 2D: a significant increase in
shows that the use of BSA in this system is not permitted as the the ratio of the intensity of emissions at 665 and 620 nm was
signal window disappeared due to the high background likely noticeable, caused by an increase in the emission intensity at
caused by the binding of 8-Fluo-cGMP to BSA, which was not 665 nm, whereas that at 620 nm remained unaffected (data not
prevented by cGMP up to 100 µM. However, tdBSA performed shown). Although at this point it is not possible to justify the
nicely in the assay: although the signal window was slightly reasons for such improved behaviors, it seems likely that both
reduced, the sensitivity window for cGMP remained unaffected preparations of albumin may stabilize some of the components
(from 5 nM to 1 µM). Although in the case concerning this detec- of the assay or simply increase their effective concentration by
tion system, an HTS campaign could be run without any protein preventing adsorption to plasticware. The bell-shaped curves
additive, it may serve to illustrate cases (particularly assays observed are a consequence of the excess of Eu over APC when
based on FP technology) where the inclusion of protein additives the former is above 10 nM, which increased the intensity of the
is necessary but the use of BSA is banned due to its ability to emission at 620 without affecting that at 665 nm.
bind small molecules (e.g., a fluorescent probe). In such cases,
tdBSA seems to be an excellent alternative. Effect of tdBSA on the performance of HTS assays
The effect on luminescence was evaluated by running a
titration of luciferase with the commercial Bright-Glo™ sys- After having proven that tdBSA is not detrimental to the opti-
tem: both BSA and tdBSA improved the performance of the cal detection systems typically used in HTS, we investigated its

1284 www.sbsonline.org Journal of Biomolecular Screening 15(10); 2010

 Thermally Denatured BSA as an Additive in HTS

A B
500 600

400 500

Rate (nM . s–1)

Rate (nM . s–1)
400
300
300
200
200
100 100
0 0
0 5 10 15 20 0 10 20 30 40 50 60 70 80
[Fab I] (nM) [ICL] (nM)
C D

Vmax with BSA or tdBSA

0.1 300
250
Rate (nM . s–1)

0.08
200
0.06
150
0.04
100
0.02
50
0
0 2 4 6 8 10 12 14 16 50 100 150 200 250 300
[Akt] (nM) Vmax without additive
FIG. 3. Effect of thermally denatured bovine serum albumin (tdBSA) on several assays. Titration of (A) FabI, (B) ICL, and (C) Akt in buffer
(●) or buffer with 0.1% (w/v) BSA (▲) or 0.1% (w/v) tdBSA (▼). The assays were performed as described in Materials and Methods, and initial
rates were calculated from the early linear sections of the progress curves. The lines denote the linear regression fits obtained, with the slopes
reporting on the apparent specific activity for each enzyme in each case. Such linear regressions were obtained by including all the points, except
for ICL in plain buffer, where only the data at concentrations above 20 nM were considered. (D) Effect of tdBSA on D–amino acid oxidase
(DAAO). Substrate titrations using 6 different D-amino acids as substrate were run, and Vmax values (expressed as nM H2O2 released per second)
were obtained for them in plain buffer or in buffer with 0.1% (w/v) BSA (▲) or 0.1% (w/v) tdBSA (▼). The dotted line represents y = x, whereas
the solid lines represent the linear fittings of the experimental points.

performance as a BSA surrogate in assays already known to concentration, were compared. As shown in panel Figure 3D,
require albumin. Four assays encompassing different detection there was an increase in the Vmax values when BSA or tdBSA
technologies were tested, all of them involving soluble enzymes was present, and this effect was independent of the substrate
as the screened target: FabI from P. falciparum (signal decrease used, as noticed by the linear correlations obtained. From the
assay monitoring absorbance), human Akt (signal increase slopes of these correlation plots (1.3 for BSA and 1.4 for
assay monitoring FP), and human DAAO and ICL from M. tdBSA), it is deduced that both albumin preparations caused a
tuberculosis (signal increase assays monitoring FLINT). Figure significant increase in the effective concentration of DAAO in
3 shows how both albumin preparations affect the activity of the the assay medium. To summarize, in the 4 cases considered, the
enzymes. The dependency of the initial rate on the concentra- benefits procured by tdBSA were identical to or better than
tion of FabI, ICL, and Akt is shown in Figures 3A,B,C, respec- those procured by BSA and represent an increase of at least 40%
tively: both albumin forms increased the specific activity of the in the “usable” enzyme (or even 600% in the case of Akt), there-
3 enzymes as deduced from the increase in the slopes of the fore providing precious savings for HTS.
plots. In the 3 cases, tdBSA behaved similarly to BSA, suggest- As detailed in the introduction, one of the major concerns
ing that both can be used indistinctly. A closer look at the about using albumin in HTS is based on its potential to mask
changes in the slopes demonstrates that both albumins increased the effect of compounds given its ability to bind them. We have
1.5-fold the observed activity of FabI, circa 2-fold that of ICL therefore investigated the extent of this problem and whether
and more than 6-fold that of Akt. Regarding DAAO, the study the use of tdBSA may contribute to alleviating it. We have
was extended to several substrates that are transformed by the compared the potencies in the presence and absence of BSA
enzyme with different catalytic efficiencies. The enzyme kinetic and tdBSA of several hits found in different assays involving a
parameters were determined for these substrates in the presence diversity of targets: glucose 6-phosphate dehydrogenase, 5
and absence of BSA and tdBSA, and the values of the maximum cytochrome P450 isoforms (1A2, 2D6, 2C9, 2C19, and 3A4),
velocities, which are dependent on the effective enzyme 2 monoamine oxidase isoforms (A and B), and human FAS.

Journal of Biomolecular Screening 15(10); 2010   www.sbsonline.org 1285

 Peña and Domínguez

A B C
7 7 7

pIC50 in plain buffer

pIC50 in plain buffer

β-lactoglobulin
pIC50 with
6 6 6

5 5 5

4 4 4
4 5 6 7 4 5 6 7 4 5 6 7
pIC50 with BSA pIC50 with tdBSA pIC50 with tdBSA
FIG. 4. Effect of protein additives on the potencies of fatty acid synthase (FAS) inhibitors. Seventy-eight FAS hits, identified in a previous
high-throughput screening, were tested against the enzyme as already described13 and their pIC50s determined in plain buffer and compared with
the values obtained in buffer with (A) 0.1% (w/v) bovine serum albumin (BSA) or (B) 0.1% (w/v) thermally denatured BSA (tdBSA). (C)
Comparison between the pIC50 values obtained in buffer with 0.1% (w/v) tdBSA or β-lactoglobulin. The lines in the 3 plots represent y = x.

Interestingly, from all these inhibitors, only those acting on 3. Jusko WJ, Gretch M: Plasma and tissue protein binding of drugs in phar-
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tive proteins in the assay buffer. Consequently, it may well be
5. Muench SP, Rafferty JB, McLeod R, Rice DW, Prigge ST: Expression,
that the deemed compound masking effect is not as widespread
purification and crystallization of the Plasmodium falciparum enoyl
as it is commonly believed, although it is obviously dependent reductase. Acta Crystallogr D Biol Crystallogr 2003;59:1246-1248.
on the chemical features of each individual compound or com- 6. Soh BS, Loke P, Sim TS: Cloning, heterologous expression and purifica-
pound class. Figure 4 shows a significant drop in potencies tion of an isocitrate lyase from Streptomyces clavuligerus NRRL 3585.
for most of the circa 80 FAS inhibitors tested when either BSA Biochim Biophys Acta 2001;1522:112-117.
or tdBSA was included (Figures 4A and 4B, respectively), 7. Kumar CC, Diao R, Yin Z, Liu YH, Samatar AA, Madison V, et al:
although the effect was more pronounced for BSA, probably Expression, purification, characterization and homology modeling of
because tdBSA had lost the ability to specifically bind some active Akt/PKB, a key enzyme involved in cell survival signalling.
compounds. If this reasoning holds true, the drop in potencies Biochim Biophys Acta 2001;1526:257-268.
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Kluwer Academic/Plenum, 1999.
The authors thank María Jesús Vázquez for her help in
13. Vazquez MJ, Ashman S, Ramon F, Calvo D, Bardera A, Martin JJ, et al:
executing and interpreting some of the experiments described Utilization of substrate-induced quenching for screening targets promot-
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1286 www.sbsonline.org Journal of Biomolecular Screening 15(10); 2010