Selective Staining of Proteins with Hydrophobic Surface Sites on a

Cell biology, chemogenomics and chemoproteomics ? application to drug discovery. Claude E Gagna , W Clark Lambert. Expert Opinion on Drug Discovery ...
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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 One of the goals of proteomics is to characterize all of the proteins in the proteome with respect to their structure and function. Thousands of new proteins have been predicted from the 30 000-40 000 genes identified in the human genome. A still much larger number of proteins are thought to result from post-transcriptional and post-translational modifications, e.g., glycosylation, phosphorylation, hydroxylation, methylation, acetylation, ubiquitinylation, and so forth. Thus, although the number of unmodified proteins coded by the genome may be between 80 000 and 100 000, the complete human proteome is thought to contain close to 1 000 000 proteins.1 To date, only a fraction of these proteins has been fully characterized. The major technology platforms that have been developed for proteomics include 2-D gel electrophoresis, gel image analysis software, affinity and multidimensional high performance liquid chromatography (HPLC), mass spectrometry (MS), protein shotgun sequencing, isotope-coded affinity tag (ICAT) reagent comparative quantification method, chip based microassays, two-hybrid assays, phage display assays, biomolecular interaction analysis through surface plasmon resonance (SPR) coupled with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS, bioluminescence and fluorescence resonance energy transfer (BRET and FRET), X-ray and NMR analysis of protein three-dimensional structure. Two-dimensional (2-D) electrophoresis has been the method of choice for the separation of the proteins in a complex * To whom the correspondence should be addressed. Telephone: (312) 996-5202. Fax: (312) 996-0431. E-mail: [email protected]. † Current address: Department of Molecular Pharmacology and Biological Chemistry (S215), The Feinberg School of Medicine, Northwestern University. 10.1021/pr025579+ CCC: $25.00

 2003 American Chemical Society

proteomic sample. Developed by O’Farrell in 1975, this technology separates proteins based on differences in molecular charge and mass.2 The protein mixture of interest, which may be a full tissue or body fluid extract or a fraction of the same, is applied to an isoelectric focusing (IEF) gel or an IPG strip with little or no prior sample preparation. In an electric field, a protein will migrate toward the pH region on the IEF gel or the IPG strip in which the pH equals its isoelectric point. After the first dimension separation based on differences in isoelectric points, the IEF gel or the IPG strip is laid atop the second dimension polyacrylamide sodium dodecyl sulfate gel (PAGESDS), and the proteins are run in an orthogonal direction, to achieve separation by size of fully unfolded, SDS coated protein molecules. The gels are stained with chromophores, fluorophores, or silver that bind to all proteins nonspecifically. The stained gels are then photographed utilizing digital imaging systems, with a broad range of image enhancing features. Application of fluorescent staining dyes, which bind noncovalently to proteins, allows for the extraction of selected spots after excision and further investigation via proteolytic fragmentation and MS identification of signature peptides. This technology has provided important information about the expression, molecular weight, isoelectric point, and sequence of new proteins. By examining images of 2-D gels of protein extracts from tissues or cells at different stages of development, under different growth conditions, in the presence of drug molecules or upon infection with a pathogen, researchers have looked for changes in the spotting pattern: (1) movements of individual spots, which may be caused by alterations in the posttranslational modification, e.g., phosphorylation, or (2) the Journal of Proteome Research 2003, 2, 469-475

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research articles disappearance of a spot or the creation of a new one, signaling the shutting off or turning on of the gene expression.3 With significant technical improvements, 2-D gel electrophoresis continues to be the primary high-resolution separation method for complex protein mixtures. Although this platform can lead to the establishment of a more complete library of expressed proteins and thus the identification of new proteins and their primary structural characterization, other approaches are necessary to elucidate the native structure and function of these new proteins. Nondenaturing or native electrophoresis offers a different perspective to the protein-mixture composition. Native threedimensional structure or conformation is maintained through support of stabilizing noncovalent interactions. Disulfide bridges and noncovalent linkages between subunits are preserved in a biologically functional multimeric protein. The metal ions and prosthetic groups are still attached to polypeptide chains in a holoprotein unit. The pattern of separation resulting from this type of electrophoresis emphasizes the native charge-to-mass ratio. The separation may be followed by a selective staining technique, which highlights the presence of proteins that bind certain ligand types, e.g., glycoprotein staining or staining of proteins with hydrophobic surfaces developed in this work. Excision and subsequent structure and function assays of selected spots containing proteins of interest, may also be implemented, if the staining is reversible and the dyes may be dialyzed after excision. We propose the establishment of a subproteomic library of expressed soluble globular proteins whose native structures are typified by the presence of surface hydrophobic sites. Although the majority of hydrophobic amino acid groups are buried in a nonpolar protein interior, in some proteins nonpolar residues are found on the surface, which is expected to be polar due to its aqueous exposure. These nonpolar surface residues may form hydrophobic sites of biologically relevant interaction with small effector molecules, e.g., receptor-ligand, enzymesubstrate, enzyme-inhibitor or enzyme-activator binding, as well as interaction with lipids, peptides and other proteins. Numerous instances of hydrophobic interaction between small molecules and protein targets have been observed, including the binding of two methylepoxyoctadecane enantiomers to two pheromone binding protein (PBP) variants;4 the binding of odorant molecules to the hydrophobic interior of the β-barrel of the porcine odorant binding protein (pOBP);5 an interaction of R1-acid glycoprotein (AAG) with dipyridamole;6 a binding interaction of the protein kinase A catalytic unit and balanol;7 ligand binding to γ-amino butyric acid (GABA)/benzodiazepine receptors;8 the δ-opioid receptor interaction with 4-(N,N-diarylamino) piperidines;9 histamine H3 receptor-antagonist interaction.10 Illustrations of hydrophobic protein-protein interactions include pathological fibrillar aggregations, oligomerizations, enzyme-peptide substrate and toxin binding and structural support. Hydrophobic associations implicated in various pathologies include neurofibrillary tangles of the hyperphosphorylated τ protein (P-τ) that impair synaptic transmission in Alzheimer disease (AD)11 and a strongly hydrophobic aggregation of the eye lens crystallins, associated with the loss of the lens transparency.12 Ligand induced oligomerizations are exemplified by a homooligomerization of an E. coli transcription factor, MalT, induced by maltotriose binding to a ligand site inside a novel superhelix fold13 and the biotin-induced tetramerization of chicken avidin.14 An interaction of an extra470

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cellular Zn-endopeptidase with its peptide substrate is an illustration of a hydrophobic enzyme-protein substrate association and is an important subject of metastasis research.15 The most abundant bee toxin peptide, melittin, is shown to bind bovine brain calmodulin through hydrophobic interactions.16 The surface hydrophobicities of eleven staphylococcal enterotoxins (SE) have been estimated and compared with those of standard proteins on an octyl agarose column by high performance hydrophobic interaction chromatography.17 Fibronectin is a structural protein that promotes cell attachment. Fibronectin activation is ascribed to the nonspecific binding of hydrophobic substrates.18 The glycine rich structural protein (GRP) of French bean is part of a repair mechanism to strengthen the cell walls during elongation growth of the plant. The complete extraction of GRP is only possible by using a detergent, indicating the hydrophobic character of interprotein contacts.19 Among the examples of membrane protein-protein hydrophobic interaction is an oncoprotein binding to an integral membrane protein. A study of a highly hydrophobic bovine papillomavirus protein involved in cell transformation reveals its specific binding to β-type platelet derived growth factor receptor.20,21 The receptor activation seems to be a direct consequence of the complex formation. Integral membrane protein-lipid hydrophobic interactions are exemplified in studies of cytochrome c oxidase solubilization in phosphatidylcholate and membrane reconstitution,22,23 the antimicrobial activity of certain natural peptides due to membrane permeabilization and extensive pore formation24 and a molecular dynamics simulation of an interaction of the enzyme phospholipase A2 with a phospholipid monolayer.25 The hydrophobic sites on protein surfaces may also become exposed as a consequence of conformational changes in response to environmental stress or during protein denaturation.26 The presence of hydrophobic groups on the surface of native protein molecules may thus provide important information about the function of the proteins. Hence, the identification of a protein fraction of complex tissue and body fluid protein extracts with hydrophobic surface sites gains importance as a tool in chemical and structural proteomics.27 It could potentially aid in the discovery of new enzymes, receptors, and regulatory and transporter proteins. In this manuscript, we describe the separation of protein mixtures by nondenaturing gel electrophoresis and their selective staining with polarity sensitive fluorophoric and chromophoric dyes that appear to bind primarily by hydrophobic interactions.

Experimental Section Acrylamide monomers, TRIS base, ammonium persulfate, TMED, chicken egg albumin, carbonic anhydrase from bovine erythrocytes, bovine serum albumin, β-lactoglobulin A from bovine milk, L-lactic dehydrogenase and phosphorylase b from rabbit muscle and soybean trypsin inhibitor, type I-S were obtained from Sigma. The sodium salt of bromophenol blue (BPB) was purchased from Aldrich; 8-anilino-1-naphthalenesulfonic acid (ANSA) was obtained from Molecular Probes. Premixed 30% (29:1) acrylamide/bisacrylamide solution, native TRIS buffer (10X) and BioSafe Coomassie stain were obtained from Bio-Rad. Gradient 4-20% native polyacrylamide gels were purchased from Bio-Rad. Nondenaturing one-dimensional PAGE was carried out with a Bio-Rad Mini-Protean II cell

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Staining Proteins with Hydrophobic Surface Sites

following the protocol provided by Bio-Rad. For the 2-D native electrophoresis, native 7 cm Immobiline Dry Strip, pH range 3-10, and the IPG Native Buffer were obtained from Pharmacia Biotech. The proteins were separated by isoelectric point on the Pharmacia Biotech Multiphor II system. For the second dimension, gradient 4-20% TRIS-glycine gels from Novex and Invitrogen native TRIS-glycine buffer were used. In one-dimensional electrophoresis experiments, selectivity of the staining dyes was probed on gradient 4-20% native polyacrylamide gels. Aliquots of 20 µL of protein solutions in TRIS-glycine native sample buffer were loaded onto the gels. The running buffer was TRIS-glycine at pH 8.3. Electrophoreses were run for 90 min at a constant voltage of 125 V over a 9 cm minigel path. For BPB selectivity experiments, seven gel lanes were loaded with 20 µL aliquots of different proteins, so that the protein concentration in each lane was 12 µM, equivalent to 0.8 µg/µL BSA. For ANSA selectivity experiments, gel lanes were loaded with 20 µL aliquots of 6 µM protein solutions. A duplicate gel, containing all proteins at 0.8 µg/µL, was run simultaneously and stained with Coomassie general stain, to visualize all the proteins. For sensitivity assays, continuous 15% polyacrylamide gels were prepared from the premixed 30% acrylamide/bisacrylamide solution. The gel lanes were loaded with 20 µL aliquots of BSA of decreasing concentration. The running buffer was TRIS-glycine at pH 8.3. Electrophoreses were run for 90 min at a constant voltage of 125 V over a 9 cm minigel path. For the BPB sensitivity test, BSA concentrations ranged from 50 to 0.5 µg per 20 µL well. For ANSA, 20 µL wells contained 66.7 to 0.067 µg BSA. In the two-dimensional native electrophoresis experiments, the first dimension was carried out on a native 7 cm Immobiline Dry Strip with a pH range 3-10. The strip was rehydrated overnight with 130 µL of the protein mixture in Pharmacia Biotech IPG native buffer, pH 3-10 using the sample cups on the sample bar via anodic application, to ensure maximal adsorption of the large proteins onto the strip. For the selectivity assay, the protein mixture contained: 6 µg of ovalbumin, 112 µg glycogen phosphorylase b from rabbit muscle, 4 µg carbonic anhydrase from bovine erythrocytes, 2.5 µg soybean trypsin inhibitor, 8 µg BSA, and 78 µg L-lactate dehydrogenase from rabbit muscle. For the urine protein analysis, about 1 mg of lyophilized urine proteins was dissolved in 130 µL of the native buffer. A sample of urine proteins for 2-D native electrophoresis was prepared as earlier described.28 Briefly, a 40-mL portion of urine was centrifuged at 10 000 rpm for 10 min and 20 mL of the supernatant was transferred to a Pierce Slide-A-Lyzer 10 K Dialysis Cassette and dialyzed at 4 °C against two changes of 2 L of double distilled water over 2 d. The dialyzed urine sample was then lyophilized. After the separation of proteins according to their isoelectric points, the strip was equilibrated in the Novex TRIS-glycine native sample buffer (2×) containing 0.01% SDS for 15 min and dried. SDS was added to facilitate a more complete transfer of the high MW proteins from the strip onto the second dimension gel. Second dimension electrophoreses were run on a pre-cast polyacrylamide mini-gel system, under a constant voltage of 125 V for 90 min. Visualization of the spots containing proteins with hydrophobic surfaces was achieved by staining with the following BPB and ANSA solutions: (1) An 8.67 × 10-5 M BPB solution was prepared in 0.100 M TRIS buffer, pH 8.0. The gel was immersed in approximately

100 mL of this solution and shaken at 100 rpm for 30 min on the Lab Line Orbit Environ-Shaker. The gel was destained by 3 quick rinses with d.d. H2O. Better results were achieved if a small volume of 1 M phosphate buffer, pH 7.0 was used for destaining. Selection of a phosphate buffer as a destaining agent was based on the antichaotropic quality of H2PO4- ion, which strengthens hydrophobic interactions of BSA and BPB in water. The dark blue bands were visualized by a dual UVvisible light Alpha Innotech Corp. IS 1000 transilluminator. (2) A 4.0 × 10-5 M ANSA solution was also prepared in 0.100 M TRIS solution, pH 8.0. The gel was immersed in approximately 100 mL of this solution and shaken at 100 rpm for 20 min. Destaining was achieved by quick rinsing of the gels in d.d. H2O. For the competitive displacement gel experiments (CDGE), continuous 15% native gels were used. Two lanes on each gel were loaded with 20 µL of 15.2 µM BSA (or 20 µg of BSA) and the 1-D native electrophoreses were performed as described above. After the runs, the gels were cut in half and treated as follows: (1) One-half of the first gel was stained with 50 mL of 23 µM BPB. The other half was immersed in 50 mL of 23 µM BPB, also containing 1.67 mM ibuprofen. (2) The first half of the second gel was stained in 50 mL of 25 µM ANSA. The second half was immersed in 50 mL of 25 µM ANSA, also containing 50 µM BPB. All the solutions were prepared in 0.100 M TRIS buffer, pH 8.0. After being shaken at 100 rpm for 20 min, the gels were destained by 3 quick rinses with d.d. H2O. ANSA responsive protein bands were visualized by a UV transilluminator, as the excitation wavelength is 370 nm. The BSA-ANSA complex emission maximum at about 465 nm produces an aqua blue signal. The gels were photographed using a UV/visible Alpha Innotech Corp. IS 1000 digital imaging system. Duplicate control gels were stained with BioSafe Coomassie stain following the protocol provided by Bio-Rad. The protein bands were visualized by a visible light transilluminator. BPB and Coomassie stained gels were scanned using HewlettPackard 5100C Scanjet, to obtain color images. All images were formatted in Paint Shop Pro 4.0. PSP parameters are as follows: for BPB stained gels, hue 175, brightness 25%, contrast 15%.

Results and Discussion The current search for staining dyes was initiated by the observation of an unusual complex between a common electrophoresis tracer bromophenolblue (BPB) and the cytochrome c′ from Chromatium vinosum. The complex formation resulted in a mobility shift on a native gel and could also be observed spectroscopically as a shift and a change in the intensity of the visible absorption band of BPB in solution.29 It was concluded that the mode of binding in this complex was hydrophobic, based on a similar change in absorption maximum of BPB in the presence of nonionic TRITON-100 micelles. Additionally, the binding of BPB to the cytochrome was not affected by the ionic strength of the solution, excluding the possibility that the binding occurs through an ionic interaction.29 Similarly, the visible spectra of BPB solutions exhibit a peak shift and absorption coefficient change upon addition of propanol.30 It has also been demonstrated30 that BPB binds to BSA, a protein that has been shown to have hydrophobic binding sites.31 The absorption maximum of the BSA‚BPB complex exhibits a red shift with respect to that of unbound BPB, characteristic of a change to a more nonpolar environJournal of Proteome Research • Vol. 2, No. 5, 2003 471

research articles 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, phosphorylase 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 IIA and site II in subdomain IIIA. Crystal structures of HSA complexes with warfarin,36 an anticoagulant, and the local anesthetics halothane37 and propofol37 have been reported. Warfarin binds to site I, which is predominantly hydrophobic. The site is formed by the packing of all six helices of subdomain IIA and consists of two chambers in to which a warfarin molecule fits quite snugly.36 The benzyl moiety interacts with residues F211, W214, L219, L238, R218, and H242, whereas the coumarin moiety binds to R222, F223, L238, V241, R257, I260, I264, I290, and A291. We propose that ANSA, a molecule with a similar topology (a naphthalene bicyclic structure and an anilino moiety with a one atom linker) could also bind to site I. Furthermore, it was recently observed that ANSA competitively displaces BPB from BSA, which was monitored by absorption difference spectroscopy.30 Upon titration of the BSA‚ BPB complex by increasing concentrations of ANSA, the absorption difference maximum corresponding to the BSA‚BPB complex decreases, suggesting competitive displacement of BPB by ANSA. The competitive binding of BPB and ANSA suggests that BPB and ANSA interaction occur at a common binding site on BSA. Thus, it can be concluded that ANSA and BPB bind to a common hydrophobic locus on BSA, analogous to the warfarin binding site on HSA. The staining of phosphorylase b was not unexpected, because it has earlier been shown to bind several amphiphilic ligands, including pyridoxal-5′-phosphate and isopropoxycarbonyl-6-methyl-pyridinium (crystal structure with the Brookhaven PDB code 3amv),38 heptulopyranosonamide (1fs4),39 oxoethyl amide (1c50),40 and caffeine (1c8l),41 Likewise, LDH has earlier been reported to bind BPB, although the mode of binding was not proposed,42 By contrast, chicken egg albumin, 472

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Figure 1. Selectivity assays. Lanes 1 through 7 contain 20 µL aliquots of chicken egg albumin (lane 1; 45 000 Da), phosphorylase b from rabbit muscle (lane 2; 97 600 Da) carbonic anhydrase from bovine erythrocytes (lane 3; 31 000 Da), soybean trypsin inhibitor S (lane 4; 21 500 Da), bovine serum albumin or BSA (lane 5; 66 000 Da), β-lactoglobulin A from bovine milk (lane 6, 17 600 Da), and L-lactic dehydrogenase from rabbit muscle (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 separate protein solutions. (A) One-dimensional native gel stained with 8.67 × 10-5 M BPB. Concentrations of proteins in µg/µL are: lane 1, 0.54; lane 2, 1.17; lane 3, 0.37; lane 4, 0.26; lane 5, 0.79; lane 6, 0.21 and lane 7, 2.50, which are equivalent to 12 µM; (B) One-dimensional native gel stained with 4.0 × 10-5 M ANSA. Concentrations of proteins in µg/µL are: lane 1, 0.27; lane 2, 0.58; lane 3, 0.18; lane 4, 0.13; lane 5, 0.40; lane 6, 0.10 and lane 7, 1.25, which are equivalent to 6 µM; (C) Control gel stained with BioSafe Coomassie. Concentrations of all proteins are 0.8 µg/µL.

bovine carbonic anhydrase, trypsin inhibitor, and β-lactoglobulin on the gels do not stain in the presence of BPB or ANSA, but are stained in the presence of a nonspecific dye, e.g., Coomassie. It was not unexpected that the protein from chicken egg did not stain with BPB and ANSA. Although earlier named albumin, it is actually a serpin (serine protease inhibitor) with a completely different structure and function than that of BSA or HSA. The results show that BPB and ANSA are selective with respect to the proteins to which they bind and thus detect those which have solvent accessible hydrophobic sites. A sensitivity test was carried out to show a detectability limit of each staining technique. The sensitivity assay results are presented in Figure 2A-B. The detection limit for our system is determined to be at 0.50 µg protein per 20 µL for a 8.67 ×

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Staining Proteins with Hydrophobic Surface Sites Table 1. Molecular Weights and PI Values for Proteins Used in Gel Assays 1-D gel 2-D gel ANSA/BPB ANSA/BPB stain visiblea stain visiblea

protein

MW (kDa)

pI

BSA carbonic anhydrase LDH ovalbumin phosphorylase b trypsin inhibitor

66 31

5.4, 5.5, 5.6 5.9, 6.0

+ -

+ -

8.5 4.5 6.7 4.7

+ + -

+ + -

209 45 97.6 21.5

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 of a protein spot stained with these polarity sensitive reagents. The sensitivity of both the BPB and ANSA staining solutions is sufficient to enable selective staining of more abundant proteins with hydrophobic surfaces in whole cell lysates, body fluids, and other complex protein mixtures. Even with the current gel bandwidths and without any digital image enhancement, the sensitivity of the ANSA stain (67 ng per band) is comparable to that of the Coomassie general protein stain which averages at 40 ng, or the BioSafe Coomassie by Bio-Rad, whose detection limit is at 28 ng. The detection of hydrophobic surface sites on proteins of lower abundance is also possible upon prefractionation and/or enrichment of a sample. The photographs of two-dimensional gels stained with ANSA and Coomassie are presented in Figure 3A,B. The molecular weights and pI values of the proteins used in this study are listed in Table 1. Native 2-D electrophoretic patterns reveal a greater complexity of the mixture. The isozyme triplets of the BSA monomeric and dimeric structures stain with both ANSA and Coomassie. Further, at least three bands of LDH and at least two phosphorylase b traces stained with ANSA, possibly corresponding to the light and heavy subunit of dimeric LDH and the phosphorylase b monomeric and dimeric structures, respectively. The bovine carbonic anhydrase, chicken ovalbumin and soybean trypsin inhibitor stain only with Coomassie stain. The identification of a subproteomic library that exhibits particular structural features related to protein functions could facilitate functional and structural characterization of new proteins. As noted in the Introduction, a number of proteins have been identified that have hydrophobic surface sites involved in binding of small molecules, lipids, peptides and other proteins. Native 2-D electrophoresis of complex protein samples, followed by staining with a polarity sensitive dye facilitates the discovery of soluble globular proteins whose hydrophobic surface sites may be implicated in their transport, catalytic or regulatory functions. The staining is reversible, such that the dyes may be dialyzed after excision, enabling subsequent structure and function assays of selected spots containing proteins of interest, including proteins unique to certain disease states, or development stages. Furthermore, implementation of modern bioanalytical techniques already in use with 2-D

Figure 2. Sensitivity assays. (A) One-dimensional native gel stained with 8.67 × 10-5 M BPB. Lanes 1 through 7 contain 50, 20, 10, 5.0, 2.0, 1.0, and 0.50 µg BSA per 20 µL, corresponding to BSA concentrations from 38 to 0.38 µM; (B) One-dimensional native gel stained with 4.0 × 10-5 M ANSA. Lanes 1 through 7 contain 66.7, 33.3, 6.7, 3.3, 0.67, 0.33, and 0.067 µg BSA per 20 µL, corresponding to BSA concentrations from 51 µM to 51 nM.

SDS-PAGE, e.g., MALDI-TOF mass spectrometry and sequencing, may establish a library of proteins with hydrophobic surface sites, containing information on their molecular weight and sequence to complement the binding assays and structurefunction correlation, which are unique to nondenaturing approaches. An application of the above approach to the characterization of proteins found in urine is shown in Figure 4. A nondenaturing 2-D gel of urine proteins stained with ANSA is shown in Figure 4.A. Stained bands indicate the presence of proteins with a solvent accessible hydrophobic site. Figure 4B shows a nondenaturing 2D gel of the same sample of urine proteins stained with Coomassie, the general stain for all proteins. It is apparent that only a fraction of the total proteins stain with ANSA. The identification of soluble globular proteins with surface hydrophobic sites by staining with polarity sensitive dyes may also be used to screen pharmacologically active molecules as drug candidates. A drug candidate will be effective in vivo only if it is able to achieve and maintain therapeutic concentration at the site of action.43 Pharmacological characteristics, e.g., solubility, partition coefficient, permeability and protein binding contribute to its in vivo disposition. Many drug candidates are lipophilic, such that their efficacy may depend on their transport by serum proteins, e.g., HSA, to their target, e.g., another protein. Additionally, their approval may be limited by their interactions with other proteins that lead to undesirable side effects. The advances of combinatorial chemistry and drug discovery have increased the interest in rapid and cost-effective solutions for prescreening of novel pharmaceuticals and early elimination of possible problem candidates. Complex protein mixtures, e.g., serum, urine, or cerebrospinal fluid samples or soluble cell extracts may be subjected to the native 2-D electrophoresis protocol, followed by Coomassie, silver, or Ruby staining for total protein and the novel proceJournal of Proteome Research • Vol. 2, No. 5, 2003 473

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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.

Figure 3. Two-dimensional selectivity assays. The protein mixture contained the following proteins: chicken egg albumin (6 µg), phosphorylase b (112 µg), bovine carbonic anhydrase (4 µg), soybean trypsin inhibitor (2.5 µg), BSA (8 µg) and lactic dehydrogenase (78 µg) in 130 µL. (A) Two-dimensional native gel stained with 4.0 × 10-5 M ANSA; (B) Control gel stained with BioSafe Coomassie.

Figure 4. Two-dimensional native gel of urine proteins. The first dimension Immobiline Dry Strip was rehydrated with 130 µL of native buffer, pH 3-10, containing about 1 mg of lyophilized urine proteins. (A) Two-dimensional native gel stained with 4.0 × 10-5 M ANSA; (B) Control gel stained with BioSafe Coomassie.

dure for staining of soluble globular proteins with hydrophobic surface sites. On the Coomassie stained gels, all of the detectable proteins will be visualized. The selectively stained samples should have distinct patterns, since a selective marker, e.g., BPB or ANSA, would stain only the proteins with hydrophobic surface sites. On a gel treated with a mixture of a polarity sensitive dye and a lipophilic (hydrophobic) drug molecule, only the proteins whose hydrophobic surface sites do not bind 474

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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. A simple demonstration of the competitive displacement gel experiment (CDGE) was carried out using the drug ibuprofen, for which earlier studies have shown affinity toward HSA and BSA binding.44,45 Figure 5 shows that BSA exposed to the polarity sensitive chromophore BPB only, produces a normal 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 determined previously by a spectrophotometric competitive titration.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 that the site is indeed hydrophobic. Again, two gel lanes contained the same protein amount. BSA exposed to the environment polarity sensitive fluorophore ANSA only, produces a fluorescent signal due to the interaction of the dye with a hydrophobic region on the surface of the protein. Figure 6 shows that when a competing dye, BPB, is introduced into the ANSA staining solution, the fluorescent signal is reduced to less than one-half of the initial signal. This is explained by the competitive displacement of ANSA by BPB from a common binding locus on the protein. On the basis of the concentration ratio of BPB to ANSA and the extent of displacement, the binding constants of the two dyes are similar. In summary, a rapid and sensitive method for selective staining of soluble globular proteins with hydrophobic surface

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Figure 6. 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 25 µM ANSA. The right half of the gel was stained with a solution containing 25 µM ANSA and 50 µM BPB.

sites has been developed. A fluorescent and visible spectrophotometric approach is offered through ANSA and BPB staining, respectively, for additional versatility of the technique. Staining with both reagents is reversible, enabling excision of stained spots, removal of dyes by dialysis or chromatography and subsequent structure and function assays. As elaborated earlier, the CDGE offers the possibility of evaluating the specificity of drug-target protein interaction for many lipophilic drugs. For a highly specific drug, the staining pattern in the presence of the drug will lack only the band corresponding to the target protein. An analogous assay can aid environmental investigations of protein-toxin interactions. The technique also enables qualitative prescreening of proteomic samples against drugs and drug candidates, whose pharmacological potency is indicated by cell-based assays, but their protein target is unknown.

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