This document describes a method for selectively staining proteins with hydrophobic surface sites on native electrophoretic gels. The authors demonstrate that soluble globular proteins with exposed hydrophobic residues can be detected by staining under nondenaturing conditions. They propose establishing a subproteomic library of proteins characterized by hydrophobic surface sites, which are often involved in important interactions. Selective staining of native gels may help elucidate new protein functions in proteomic studies.

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Article
Selective Staining of Proteins with Hydrophobic
Surface Sites on a Native Electrophoretic Gel
Martina Bertsch, and Richard J. Kassner
Journal of Proteome Research, 2003, 2 (5), 469-475• DOI: 10.1021/pr025579+ • Publication Date (Web): 16 August 2003
Downloaded from http://pubs.acs.org on April 2, 2009
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2. Selective Staining of Proteins with Hydrophobic Surface Sites on a
Native Electrophoretic Gel
Martina Bertsch† and Richard J. Kassner*
Department of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, Suite 4500,
Chicago, Illinois 60607
Received October 14, 2002
Chemical proteomics aims to characterize all of the proteins in the proteome with respect to their
function, which is associated with their interaction with other molecules. We propose the identification
of a subproteomic library of expressed proteins whose native structures are typified by the presence
of hydrophobic surface sites, which are often involved in interactions with small molecules, membrane
lipids, and other proteins, pertaining to their functions. We demonstrate that soluble globular proteins
with hydrophobic surface sites can be detected selectively by staining on an electrophoretic gel run
under nondenaturing conditions. The application of these staining techniques may help elucidate new
catalytic, transport, and regulatory functionalities in complex proteomic screenings.
Keywords: chemical proteomics • subproteomic library • hydrophobic surface sites • polarity sensitive staining •
nondenaturing gel electrophoresis
Introdution proteomic sample. Developed by O’Farrell in 1975, this tech-
nology separates proteins based on differences in molecular
One of the goals of proteomics is to characterize all of the charge and mass.2 The protein mixture of interest, which may
proteins in the proteome with respect to their structure and be a full tissue or body fluid extract or a fraction of the same,
function. Thousands of new proteins have been predicted from is applied to an isoelectric focusing (IEF) gel or an IPG strip
the 30 000-40 000 genes identified in the human genome. A
with little or no prior sample preparation. In an electric field,
still much larger number of proteins are thought to result from
a protein will migrate toward the pH region on the IEF gel or
post-transcriptional and post-translational modifications, e.g.,
the IPG strip in which the pH equals its isoelectric point. After
glycosylation, phosphorylation, hydroxylation, methylation,
the first dimension separation based on differences in isoelec-
acetylation, ubiquitinylation, and so forth. Thus, although the
tric points, the IEF gel or the IPG strip is laid atop the second
number of unmodified proteins coded by the genome may be
between 80 000 and 100 000, the complete human proteome dimension polyacrylamide sodium dodecyl sulfate gel (PAGE-
is thought to contain close to 1 000 000 proteins.1 To date, only SDS), and the proteins are run in an orthogonal direction, to
a fraction of these proteins has been fully characterized. achieve separation by size of fully unfolded, SDS coated protein
molecules. The gels are stained with chromophores, fluoro-
The major technology platforms that have been developed
for proteomics include 2-D gel electrophoresis, gel image phores, or silver that bind to all proteins nonspecifically. The
analysis software, affinity and multidimensional high perfor- stained gels are then photographed utilizing digital imaging
mance liquid chromatography (HPLC), mass spectrometry systems, with a broad range of image enhancing features. Appli-
(MS), protein shotgun sequencing, isotope-coded affinity tag cation of fluorescent staining dyes, which bind noncovalently
(ICAT) reagent comparative quantification method, chip based to proteins, allows for the extraction of selected spots after
microassays, two-hybrid assays, phage display assays, biomo- excision and further investigation via proteolytic fragmentation
lecular interaction analysis through surface plasmon resonance and MS identification of signature peptides. This technology
(SPR) coupled with matrix-assisted laser desorption ionization has provided important information about the expression,
time-of-flight (MALDI-TOF) MS, bioluminescence and fluores- molecular weight, isoelectric point, and sequence of new
cence resonance energy transfer (BRET and FRET), X-ray and proteins.
NMR analysis of protein three-dimensional structure. By examining images of 2-D gels of protein extracts from
Two-dimensional (2-D) electrophoresis has been the method tissues or cells at different stages of development, under
of choice for the separation of the proteins in a complex different growth conditions, in the presence of drug molecules
or upon infection with a pathogen, researchers have looked
* To whom the correspondence should be addressed. Telephone: (312) for changes in the spotting pattern: (1) movements of indi-
996-5202. Fax: (312) 996-0431. E-mail: RKassner@uic.edu.
† Current address: Department of Molecular Pharmacology and Biological vidual spots, which may be caused by alterations in the post-
Chemistry (S215), The Feinberg School of Medicine, Northwestern University. translational modification, e.g., phosphorylation, or (2) the
10.1021/pr025579+ CCC: $25.00 © 2003 American Chemical Society Journal of Proteome Research 2003, 2, 469-475 469
Published on Web 08/16/2003

3. research articles Bertsch and Kassner
disappearance of a spot or the creation of a new one, signaling cellular Zn-endopeptidase with its peptide substrate is an
the shutting off or turning on of the gene expression.3 illustration of a hydrophobic enzyme-protein substrate as-
With significant technical improvements, 2-D gel electro- sociation and is an important subject of metastasis research.15
phoresis continues to be the primary high-resolution separation The most abundant bee toxin peptide, melittin, is shown to
method for complex protein mixtures. Although this platform bind bovine brain calmodulin through hydrophobic interac-
can lead to the establishment of a more complete library of tions.16 The surface hydrophobicities of eleven staphylococcal
expressed proteins and thus the identification of new proteins enterotoxins (SE) have been estimated and compared with
and their primary structural characterization, other approaches those of standard proteins on an octyl agarose column by high
are necessary to elucidate the native structure and function of performance hydrophobic interaction chromatography.17
these new proteins. Fibronectin is a structural protein that promotes cell attach-
Nondenaturing or native electrophoresis offers a different ment. Fibronectin activation is ascribed to the nonspecific
perspective to the protein-mixture composition. Native three- binding of hydrophobic substrates.18 The glycine rich structural
dimensional structure or conformation is maintained through protein (GRP) of French bean is part of a repair mechanism to
support of stabilizing noncovalent interactions. Disulfide bridges strengthen the cell walls during elongation growth of the plant.
and noncovalent linkages between subunits are preserved in a The complete extraction of GRP is only possible by using a
biologically functional multimeric protein. The metal ions and detergent, indicating the hydrophobic character of interprotein
prosthetic groups are still attached to polypeptide chains in a contacts.19
holoprotein unit. The pattern of separation resulting from this Among the examples of membrane protein-protein hydro-
type of electrophoresis emphasizes the native charge-to-mass phobic interaction is an oncoprotein binding to an integral
ratio. The separation may be followed by a selective staining membrane protein. A study of a highly hydrophobic bovine
technique, which highlights the presence of proteins that bind papillomavirus protein involved in cell transformation reveals
certain ligand types, e.g., glycoprotein staining or staining of its specific binding to β-type platelet derived growth factor
proteins with hydrophobic surfaces developed in this work. receptor.20,21 The receptor activation seems to be a direct
Excision and subsequent structure and function assays of consequence of the complex formation.
selected spots containing proteins of interest, may also be Integral membrane protein-lipid hydrophobic interactions
implemented, if the staining is reversible and the dyes may be are exemplified in studies of cytochrome c oxidase solubiliza-
dialyzed after excision. tion in phosphatidylcholate and membrane reconstitution,22,23
We propose the establishment of a subproteomic library of the antimicrobial activity of certain natural peptides due to
expressed soluble globular proteins whose native structures are membrane permeabilization and extensive pore formation24
typified by the presence of surface hydrophobic sites. Although and a molecular dynamics simulation of an interaction of the
the majority of hydrophobic amino acid groups are buried in enzyme phospholipase A2 with a phospholipid monolayer.25
a nonpolar protein interior, in some proteins nonpolar residues The hydrophobic sites on protein surfaces may also become
are found on the surface, which is expected to be polar due to exposed as a consequence of conformational changes in re-
its aqueous exposure. These nonpolar surface residues may sponse to environmental stress or during protein denatur-
form hydrophobic sites of biologically relevant interaction with ation.26
small effector molecules, e.g., receptor-ligand, enzyme- The presence of hydrophobic groups on the surface of native
substrate, enzyme-inhibitor or enzyme-activator binding, as protein molecules may thus provide important information
well as interaction with lipids, peptides and other proteins. about the function of the proteins. Hence, the identification
Numerous instances of hydrophobic interaction between of a protein fraction of complex tissue and body fluid protein
small molecules and protein targets have been observed, extracts with hydrophobic surface sites gains importance as a
including the binding of two methylepoxyoctadecane enanti- tool in chemical and structural proteomics.27 It could poten-
omers to two pheromone binding protein (PBP) variants;4 the tially aid in the discovery of new enzymes, receptors, and
binding of odorant molecules to the hydrophobic interior of regulatory and transporter proteins.
the β-barrel of the porcine odorant binding protein (pOBP);5 In this manuscript, we describe the separation of protein
an interaction of R1-acid glycoprotein (AAG) with dipy- mixtures by nondenaturing gel electrophoresis and their selec-
ridamole;6 a binding interaction of the protein kinase A catalytic tive staining with polarity sensitive fluorophoric and chro-
unit and balanol;7 ligand binding to γ-amino butyric acid mophoric dyes that appear to bind primarily by hydrophobic
(GABA)/benzodiazepine receptors;8 the δ-opioid receptor in- interactions.
teraction with 4-(N,N-diarylamino) piperidines;9 histamine H3
receptor-antagonist interaction.10 Experimental Section
Illustrations of hydrophobic protein-protein interactions Acrylamide monomers, TRIS base, ammonium persulfate,
include pathological fibrillar aggregations, oligomerizations, TMED, chicken egg albumin, carbonic anhydrase from bovine
enzyme-peptide substrate and toxin binding and structural erythrocytes, bovine serum albumin, β-lactoglobulin A from
support. Hydrophobic associations implicated in various pa- bovine milk, L-lactic dehydrogenase and phosphorylase b from
thologies include neurofibrillary tangles of the hyperphospho- rabbit muscle and soybean trypsin inhibitor, type I-S were
rylated τ protein (P-τ) that impair synaptic transmission in obtained from Sigma. The sodium salt of bromophenol blue
Alzheimer disease (AD)11 and a strongly hydrophobic aggrega- (BPB) was purchased from Aldrich; 8-anilino-1-naphthalene-
tion of the eye lens crystallins, associated with the loss of the sulfonic acid (ANSA) was obtained from Molecular Probes.
lens transparency.12 Ligand induced oligomerizations are ex- Premixed 30% (29:1) acrylamide/bisacrylamide solution, native
emplified by a homooligomerization of an E. coli transcription TRIS buffer (10X) and BioSafe Coomassie stain were obtained
factor, MalT, induced by maltotriose binding to a ligand site from Bio-Rad. Gradient 4-20% native polyacrylamide gels were
inside a novel superhelix fold13 and the biotin-induced tet- purchased from Bio-Rad. Nondenaturing one-dimensional
ramerization of chicken avidin.14 An interaction of an extra- PAGE was carried out with a Bio-Rad Mini-Protean II cell
470 Journal of Proteome Research • Vol. 2, No. 5, 2003

4. Staining Proteins with Hydrophobic Surface Sites research articles
following the protocol provided by Bio-Rad. For the 2-D native 100 mL of this solution and shaken at 100 rpm for 30 min on
electrophoresis, native 7 cm Immobiline Dry Strip, pH range the Lab Line Orbit Environ-Shaker. The gel was destained by 3
3-10, and the IPG Native Buffer were obtained from Pharmacia quick rinses with d.d. H2O. Better results were achieved if a
Biotech. The proteins were separated by isoelectric point on small volume of 1 M phosphate buffer, pH 7.0 was used for
the Pharmacia Biotech Multiphor II system. For the second destaining. Selection of a phosphate buffer as a destaining
dimension, gradient 4-20% TRIS-glycine gels from Novex and agent was based on the antichaotropic quality of H2PO4- ion,
Invitrogen native TRIS-glycine buffer were used. which strengthens hydrophobic interactions of BSA and BPB
In one-dimensional electrophoresis experiments, selectivity in water. The dark blue bands were visualized by a dual UV-
of the staining dyes was probed on gradient 4-20% native visible light Alpha Innotech Corp. IS 1000 transilluminator.
polyacrylamide gels. Aliquots of 20 µL of protein solutions in (2) A 4.0 × 10-5 M ANSA solution was also prepared in 0.100
TRIS-glycine native sample buffer were loaded onto the gels. M TRIS solution, pH 8.0. The gel was immersed in ap-
The running buffer was TRIS-glycine at pH 8.3. Electrophoreses proximately 100 mL of this solution and shaken at 100 rpm for
were run for 90 min at a constant voltage of 125 V over a 9 cm 20 min. Destaining was achieved by quick rinsing of the gels
minigel path. in d.d. H2O.
For BPB selectivity experiments, seven gel lanes were loaded For the competitive displacement gel experiments (CDGE),
with 20 µL aliquots of different proteins, so that the protein continuous 15% native gels were used. Two lanes on each gel
concentration in each lane was 12 µM, equivalent to 0.8 µg/µL were loaded with 20 µL of 15.2 µM BSA (or 20 µg of BSA) and
BSA. For ANSA selectivity experiments, gel lanes were loaded the 1-D native electrophoreses were performed as described
with 20 µL aliquots of 6 µM protein solutions. A duplicate gel, above. After the runs, the gels were cut in half and treated as
containing all proteins at 0.8 µg/µL, was run simultaneously follows: (1) One-half of the first gel was stained with 50 mL of
and stained with Coomassie general stain, to visualize all the 23 µM BPB. The other half was immersed in 50 mL of 23 µM
proteins. BPB, also containing 1.67 mM ibuprofen. (2) The first half of
For sensitivity assays, continuous 15% polyacrylamide gels the second gel was stained in 50 mL of 25 µM ANSA. The
were prepared from the premixed 30% acrylamide/bisacryla- second half was immersed in 50 mL of 25 µM ANSA, also
mide solution. The gel lanes were loaded with 20 µL aliquots containing 50 µM BPB. All the solutions were prepared in 0.100
of BSA of decreasing concentration. The running buffer was M TRIS buffer, pH 8.0. After being shaken at 100 rpm for 20
TRIS-glycine at pH 8.3. Electrophoreses were run for 90 min min, the gels were destained by 3 quick rinses with d.d. H2O.
at a constant voltage of 125 V over a 9 cm minigel path. For ANSA responsive protein bands were visualized by a UV
the BPB sensitivity test, BSA concentrations ranged from 50 to transilluminator, as the excitation wavelength is 370 nm. The
0.5 µg per 20 µL well. For ANSA, 20 µL wells contained 66.7 to BSA-ANSA complex emission maximum at about 465 nm
0.067 µg BSA. produces an aqua blue signal. The gels were photographed
In the two-dimensional native electrophoresis experiments, using a UV/visible Alpha Innotech Corp. IS 1000 digital imaging
the first dimension was carried out on a native 7 cm Immo- system.
biline Dry Strip with a pH range 3-10. The strip was rehydrated Duplicate control gels were stained with BioSafe Coomassie
overnight with 130 µL of the protein mixture in Pharmacia stain following the protocol provided by Bio-Rad. The protein
Biotech IPG native buffer, pH 3-10 using the sample cups on bands were visualized by a visible light transilluminator.
the sample bar via anodic application, to ensure maximal BPB and Coomassie stained gels were scanned using Hewlett-
adsorption of the large proteins onto the strip. For the selectiv-
Packard 5100C Scanjet, to obtain color images. All images were
ity assay, the protein mixture contained: 6 µg of ovalbumin,
formatted in Paint Shop Pro 4.0. PSP parameters are as
112 µg glycogen phosphorylase b from rabbit muscle, 4 µg
follows: for BPB stained gels, hue 175, brightness 25%, contrast
carbonic anhydrase from bovine erythrocytes, 2.5 µg soybean
15%.
trypsin inhibitor, 8 µg BSA, and 78 µg L-lactate dehydrogenase
from rabbit muscle. For the urine protein analysis, about 1 mg Results and Discussion
of lyophilized urine proteins was dissolved in 130 µL of the
native buffer. A sample of urine proteins for 2-D native The current search for staining dyes was initiated by the
electrophoresis was prepared as earlier described.28 Briefly, a observation of an unusual complex between a common elec-
40-mL portion of urine was centrifuged at 10 000 rpm for 10 trophoresis tracer bromophenolblue (BPB) and the cytochrome
min and 20 mL of the supernatant was transferred to a Pierce c′ from Chromatium vinosum. The complex formation resulted
Slide-A-Lyzer 10 K Dialysis Cassette and dialyzed at 4 °C against in a mobility shift on a native gel and could also be observed
two changes of 2 L of double distilled water over 2 d. The spectroscopically as a shift and a change in the intensity of
dialyzed urine sample was then lyophilized. After the separation the visible absorption band of BPB in solution.29 It was
of proteins according to their isoelectric points, the strip was concluded that the mode of binding in this complex was
equilibrated in the Novex TRIS-glycine native sample buffer hydrophobic, based on a similar change in absorption maxi-
(2×) containing 0.01% SDS for 15 min and dried. SDS was mum of BPB in the presence of nonionic TRITON-100 micelles.
added to facilitate a more complete transfer of the high MW Additionally, the binding of BPB to the cytochrome was not
proteins from the strip onto the second dimension gel. Second affected by the ionic strength of the solution, excluding the
dimension electrophoreses were run on a pre-cast polyacry- possibility that the binding occurs through an ionic interac-
lamide mini-gel system, under a constant voltage of 125 V for tion.29 Similarly, the visible spectra of BPB solutions exhibit a
90 min. peak shift and absorption coefficient change upon addition of
Visualization of the spots containing proteins with hydro- propanol.30 It has also been demonstrated30 that BPB binds to
phobic surfaces was achieved by staining with the following BSA, a protein that has been shown to have hydrophobic
BPB and ANSA solutions: binding sites.31 The absorption maximum of the BSA‚BPB
(1) An 8.67 × 10-5 M BPB solution was prepared in 0.100 M complex exhibits a red shift with respect to that of unbound
TRIS buffer, pH 8.0. The gel was immersed in approximately BPB, characteristic of a change to a more nonpolar environ-
Journal of Proteome Research • Vol. 2, No. 5, 2003 471

5. research articles Bertsch and Kassner
ment.30 Thus, a stain on a gel immersed in a BPB solution
indicates a hydrophobic type interaction between a protein
from the mixture and the dye.
A literature search26,32-34 for other suitable dyes yielded
1-anilinonaphthalene-8-sulfonic acid (1,8-ANSA), a lipophilic
fluorophore. ANSA is virtually nonfluorescent in an aqueous
medium, but becomes strongly fluorescent as the polarity of a
medium decreases. ANSA has also been shown to bind to
hydrophobic sites on HSA.35 The fluorescent response on an
ANSA stained gel may then be attributed to a hydrophobic
interaction between a protein from the mixture and the dye.
To demonstrate the feasibility of these dyes as staining
agents, selectivity and sensitivity tests were performed.
A selectivity test was designed to show that under the
staining conditions, only a select subset of components of a
complex protein mixture would yield a detectable signal,
indicative of binding of the staining dye to the molecules of
the subset. The images of one-dimensional gel selectivity assays
stained with BPB and ANSA, including the Coomassie stained
control gel, are presented in Figures 1A-C.
As shown in Figure 1A, BSA monomer and dimer, phospho-
rylase b and L-lactic dehydrogenase produced a visible spot
upon staining with BPB. As shown in Figure 1B, the same
proteins produced a fluorescent signal upon staining with
ANSA, indicating the binding of both polarity sensitive dyes to
these proteins.
The results shown in Figure 1 suggest the presence of solvent
accessible hydrophobic sites of interaction of these proteins
with the polarity sensitive dyes. The binding of ANSA to BSA is
consistent with the binding of ANSA to HSA, which has been
shown to have at least two binding sites to which a variety of
hydrophobic drug molecules may bind: site I in subdomain Figure 1. Selectivity assays. Lanes 1 through 7 contain 20 µL
IIA and site II in subdomain IIIA. Crystal structures of HSA aliquots of chicken egg albumin (lane 1; 45 000 Da), phospho-
complexes with warfarin,36 an anticoagulant, and the local rylase b from rabbit muscle (lane 2; 97 600 Da) carbonic anhy-
anesthetics halothane37 and propofol37 have been reported. drase from bovine erythrocytes (lane 3; 31 000 Da), soybean
Warfarin binds to site I, which is predominantly hydrophobic. trypsin inhibitor S (lane 4; 21 500 Da), bovine serum albumin or
The site is formed by the packing of all six helices of subdomain BSA (lane 5; 66 000 Da), β-lactoglobulin A from bovine milk (lane
IIA and consists of two chambers in to which a warfarin 6, 17 600 Da), and L-lactic dehydrogenase from rabbit muscle
molecule fits quite snugly.36 The benzyl moiety interacts with (lane 7, 209 000 Da). Lane 8 contained 20 µL of the mixture of all
the above proteins, at a concentration equivalent to those of the
residues F211, W214, L219, L238, R218, and H242, whereas the
separate protein solutions. (A) One-dimensional native gel
coumarin moiety binds to R222, F223, L238, V241, R257, I260, stained with 8.67 × 10-5 M BPB. Concentrations of proteins in
I264, I290, and A291. We propose that ANSA, a molecule with µg/µL are: lane 1, 0.54; lane 2, 1.17; lane 3, 0.37; lane 4, 0.26;
a similar topology (a naphthalene bicyclic structure and an lane 5, 0.79; lane 6, 0.21 and lane 7, 2.50, which are equivalent
anilino moiety with a one atom linker) could also bind to site to 12 µM; (B) One-dimensional native gel stained with 4.0 × 10-5
I. Furthermore, it was recently observed that ANSA com- M ANSA. Concentrations of proteins in µg/µL are: lane 1, 0.27;
petitively displaces BPB from BSA, which was monitored by lane 2, 0.58; lane 3, 0.18; lane 4, 0.13; lane 5, 0.40; lane 6, 0.10
absorption difference spectroscopy.30 Upon titration of the BSA‚ and lane 7, 1.25, which are equivalent to 6 µM; (C) Control gel
BPB complex by increasing concentrations of ANSA, the stained with BioSafe Coomassie. Concentrations of all proteins
absorption difference maximum corresponding to the BSA‚BPB are 0.8 µg/µL.
complex decreases, suggesting competitive displacement of bovine carbonic anhydrase, trypsin inhibitor, and β-lactoglo-
BPB by ANSA. The competitive binding of BPB and ANSA bulin on the gels do not stain in the presence of BPB or ANSA,
suggests that BPB and ANSA interaction occur at a common but are stained in the presence of a nonspecific dye, e.g.,
binding site on BSA. Thus, it can be concluded that ANSA and Coomassie. It was not unexpected that the protein from chicken
BPB bind to a common hydrophobic locus on BSA, analogous egg did not stain with BPB and ANSA. Although earlier named
to the warfarin binding site on HSA. albumin, it is actually a serpin (serine protease inhibitor) with
The staining of phosphorylase b was not unexpected, a completely different structure and function than that of BSA
because it has earlier been shown to bind several amphiphilic or HSA. The results show that BPB and ANSA are selective with
ligands, including pyridoxal-5′-phosphate and isopropoxy- respect to the proteins to which they bind and thus detect those
carbonyl-6-methyl-pyridinium (crystal structure with the which have solvent accessible hydrophobic sites.
Brookhaven PDB code 3amv),38 heptulopyranosonamide (1fs4),39 A sensitivity test was carried out to show a detectability limit
oxoethyl amide (1c50),40 and caffeine (1c8l),41 Likewise, LDH of each staining technique. The sensitivity assay results are
has earlier been reported to bind BPB, although the mode of presented in Figure 2A-B. The detection limit for our system
binding was not proposed,42 By contrast, chicken egg albumin, is determined to be at 0.50 µg protein per 20 µL for a 8.67 ×
472 Journal of Proteome Research • Vol. 2, No. 5, 2003

6. Staining Proteins with Hydrophobic Surface Sites research articles
Table 1. Molecular Weights and PI Values for Proteins Used
in Gel Assays
1-D gel 2-D gel
ANSA/BPB ANSA/BPB
protein MW (kDa) pI stain visiblea stain visiblea
BSA 66 5.4, 5.5, 5.6 + +
carbonic 31 5.9, 6.0 - -
anhydrase
LDH 209 8.5 + +
ovalbumin 45 4.5 - -
phosphorylase b 97.6 6.7 + +
trypsin inhibitor 21.5 4.7 - -
a
A “+” sign indicates that a protein stains with a polarity sensitive probe,
whereas a “-” sign indicates a negative result. The l-lactic dehydrogenase
and phosphorylase b dissociate into subunits in the presence of 0.01% SDS
during the separation in the second dimension on the 2-D gel.
10-5 M BPB staining solution, which corresponds to a 380 nM
BSA solution. The detection limit is at 67 ng BSA per 20 µL
sample for a 4.0 × 10-5 M ANSA solution, corresponding to a
BSA concentration of 51 nM. If gels with narrower lanes were
used, then the detection limit would presumably be even lower.
Advanced digital imaging may further enhance the detectability Figure 2. Sensitivity assays. (A) One-dimensional native gel
stained with 8.67 × 10-5 M BPB. Lanes 1 through 7 contain 50,
of a protein spot stained with these polarity sensitive reagents.
20, 10, 5.0, 2.0, 1.0, and 0.50 µg BSA per 20 µL, corresponding to
The sensitivity of both the BPB and ANSA staining solutions
BSA concentrations from 38 to 0.38 µM; (B) One-dimensional
is sufficient to enable selective staining of more abundant native gel stained with 4.0 × 10-5 M ANSA. Lanes 1 through 7
proteins with hydrophobic surfaces in whole cell lysates, body contain 66.7, 33.3, 6.7, 3.3, 0.67, 0.33, and 0.067 µg BSA per 20
fluids, and other complex protein mixtures. Even with the µL, corresponding to BSA concentrations from 51 µM to 51 nM.
current gel bandwidths and without any digital image enhance-
ment, the sensitivity of the ANSA stain (67 ng per band) is SDS-PAGE, e.g., MALDI-TOF mass spectrometry and sequenc-
comparable to that of the Coomassie general protein stain ing, may establish a library of proteins with hydrophobic
which averages at 40 ng, or the BioSafe Coomassie by Bio-Rad, surface sites, containing information on their molecular weight
whose detection limit is at 28 ng. The detection of hydrophobic and sequence to complement the binding assays and structure-
surface sites on proteins of lower abundance is also possible function correlation, which are unique to nondenaturing
upon prefractionation and/or enrichment of a sample. approaches.
The photographs of two-dimensional gels stained with ANSA An application of the above approach to the characterization
and Coomassie are presented in Figure 3A,B. The molecular of proteins found in urine is shown in Figure 4. A nondena-
weights and pI values of the proteins used in this study are turing 2-D gel of urine proteins stained with ANSA is shown in
listed in Table 1. Native 2-D electrophoretic patterns reveal a Figure 4.A. Stained bands indicate the presence of proteins with
greater complexity of the mixture. The isozyme triplets of the a solvent accessible hydrophobic site. Figure 4B shows a
BSA monomeric and dimeric structures stain with both ANSA nondenaturing 2D gel of the same sample of urine proteins
and Coomassie. Further, at least three bands of LDH and at stained with Coomassie, the general stain for all proteins. It is
least two phosphorylase b traces stained with ANSA, possibly apparent that only a fraction of the total proteins stain with
corresponding to the light and heavy subunit of dimeric LDH ANSA.
and the phosphorylase b monomeric and dimeric structures, The identification of soluble globular proteins with surface
respectively. The bovine carbonic anhydrase, chicken ovalbu- hydrophobic sites by staining with polarity sensitive dyes may
min and soybean trypsin inhibitor stain only with Coomassie also be used to screen pharmacologically active molecules as
stain. drug candidates. A drug candidate will be effective in vivo only
The identification of a subproteomic library that exhibits if it is able to achieve and maintain therapeutic concentration
particular structural features related to protein functions could at the site of action.43 Pharmacological characteristics, e.g.,
facilitate functional and structural characterization of new solubility, partition coefficient, permeability and protein bind-
proteins. As noted in the Introduction, a number of proteins ing contribute to its in vivo disposition. Many drug candidates
have been identified that have hydrophobic surface sites are lipophilic, such that their efficacy may depend on their
involved in binding of small molecules, lipids, peptides and transport by serum proteins, e.g., HSA, to their target, e.g.,
other proteins. Native 2-D electrophoresis of complex protein another protein. Additionally, their approval may be limited
samples, followed by staining with a polarity sensitive dye by their interactions with other proteins that lead to undesirable
facilitates the discovery of soluble globular proteins whose side effects. The advances of combinatorial chemistry and drug
hydrophobic surface sites may be implicated in their transport, discovery have increased the interest in rapid and cost-effective
catalytic or regulatory functions. The staining is reversible, such solutions for prescreening of novel pharmaceuticals and early
that the dyes may be dialyzed after excision, enabling subse- elimination of possible problem candidates.
quent structure and function assays of selected spots containing Complex protein mixtures, e.g., serum, urine, or cerebrospi-
proteins of interest, including proteins unique to certain disease nal fluid samples or soluble cell extracts may be subjected to
states, or development stages. Furthermore, implementation the native 2-D electrophoresis protocol, followed by Coomassie,
of modern bioanalytical techniques already in use with 2-D silver, or Ruby staining for total protein and the novel proce-
Journal of Proteome Research • Vol. 2, No. 5, 2003 473

7. research articles Bertsch and Kassner
Figure 5. Competitive displacement gel experiment. Two lanes
were loaded with equivalent amounts of BSA, e.g., 20 µL of 1.0
µg/µL solution (15 µM). After native electrophoresis, the gel was
cut in half and treated as described in the Material and methods
section. The left half was stained with 23 µM BPB. The right half
of the gel was stained with a solution containing 23 µM BPB and
1.67 mM ibuprofen.
the drug would bind the dye. Ideally, only the expected binding
of the drug to a target carrier protein would be observed. Loss
of additional signals upon binding of the drug with respect to
the selectively stained gel in the absence of the drug may
indicate possible side effects due to nonspecific binding of the
drug.
Figure 3. Two-dimensional selectivity assays. The protein mix-
A simple demonstration of the competitive displacement gel
ture contained the following proteins: chicken egg albumin (6
µg), phosphorylase b (112 µg), bovine carbonic anhydrase (4 µg),
experiment (CDGE) was carried out using the drug ibuprofen,
soybean trypsin inhibitor (2.5 µg), BSA (8 µg) and lactic dehy- for which earlier studies have shown affinity toward HSA and
drogenase (78 µg) in 130 µL. (A) Two-dimensional native gel BSA binding.44,45 Figure 5 shows that BSA exposed to the
stained with 4.0 × 10-5 M ANSA; (B) Control gel stained with polarity sensitive chromophore BPB only, produces a normal
BioSafe Coomassie. signal due to the interaction of the dye with a hydrophobic
region on the surface of the protein. When the drug ibuprofen
is introduced into the staining solution, the signal is greatly
diminished. The loss of the stained spot in the presence of
ibuprofen can be explained by the competitive displacement
of the dye by the drug.
The displacement efficacy at a certain pH, ionic strength,
and temperature is dependent upon two factors: (1) the
strength of binding, characterized by the free energy of binding
or the binding constant; and (2) the drug vs dye concentration
ratio. The higher the binding constant of the drug, the lower
the concentration limit at which the displacement is detectable
as a loss of the signal. The particular concentration ratio for
this assay was selected based on the binding constant deter-
mined previously by a spectrophotometric competitive titra-
tion.30 For a novel protein-drug interaction, for which an initial
assessment of protein-drug binding affinity is not available, a
millimolar to micromolar drug concentration range seems most
appropriate. Alternatively, the drug concentration may be
chosen based on blood concentrations during treatment with
analogous agents.
A similar experiment served as qualitative evidence that BPB
and ANSA bind to the same site on the protein surface and
Figure 4. Two-dimensional native gel of urine proteins. The first
that the site is indeed hydrophobic. Again, two gel lanes
dimension Immobiline Dry Strip was rehydrated with 130 µL of contained the same protein amount. BSA exposed to the
native buffer, pH 3-10, containing about 1 mg of lyophilized urine environment polarity sensitive fluorophore ANSA only, pro-
proteins. (A) Two-dimensional native gel stained with 4.0 × 10-5 duces a fluorescent signal due to the interaction of the dye with
M ANSA; (B) Control gel stained with BioSafe Coomassie. a hydrophobic region on the surface of the protein.
Figure 6 shows that when a competing dye, BPB, is intro-
dure for staining of soluble globular proteins with hydrophobic duced into the ANSA staining solution, the fluorescent signal
surface sites. On the Coomassie stained gels, all of the detect- is reduced to less than one-half of the initial signal. This is
able proteins will be visualized. The selectively stained samples explained by the competitive displacement of ANSA by BPB
should have distinct patterns, since a selective marker, e.g., BPB from a common binding locus on the protein. On the basis of
or ANSA, would stain only the proteins with hydrophobic the concentration ratio of BPB to ANSA and the extent of
surface sites. On a gel treated with a mixture of a polarity displacement, the binding constants of the two dyes are similar.
sensitive dye and a lipophilic (hydrophobic) drug molecule, In summary, a rapid and sensitive method for selective
only the proteins whose hydrophobic surface sites do not bind staining of soluble globular proteins with hydrophobic surface
474 Journal of Proteome Research • Vol. 2, No. 5, 2003

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