Supramolecular Gel Electrophoresis of Acidic Native Proteins

Aug 22, 2014 - The proteins remain in their native forms during migration, and their activities are .... Development of native protein electrophoresis...
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Supramolecular Gel Electrophoresis of Acidic Native Proteins Kanako Munenobu, Takayuki Hase, Takanori Oyoshi, and Masamichi Yamanaka* Department of Chemistry, Graduate School of Science, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan S Supporting Information *

ABSTRACT: Amphiphilic tris-urea molecules self-assemble into a supramolecular hydrogel in tris(hydroxymethyl)aminomethane−glycine buffer. The supramolecular hydrogel is used as a matrix for the electrophoresis of acidic native proteins, in which proteins are separated based on their isoelectric points rather than their molecular weights. The proteins remain in their native forms during migration, and their activities are retained after electrophoresis. Glucoside substituents on the amphiphilic tris-urea molecule allow for the affinity electrophoresis of a carbohydrate-binding protein to be performed. The proteins can be efficiently recovered from the supramolecular hydrogel using a simple procedure. This is a major advantage of using this noncovalent, self-assembled material.

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which immobilize an aqueous phase, have been developed for use as matrices to manipulate biomaterials. In addition, they have been used in processes such as enzymatic analysis, tissue engineering, and cell culture.22−27 Despite much interest in the biological applications of supramolecular hydrogels, our recent study was the first report of protein electrophoresis using supramolecular hydrogel.28 Supramolecular hydrogels have several advantages when used as protein electrophoresis matrices. Responding to stimuli with a sol phase transition, which is a characteristic that is naturally found in supramolecular gels, allows the efficient recovery of proteins from the gel matrix after electrophoresis. The structural diversity of low-molecular-weight gelators allows affinity electrophoresis to be tailored according to the specific proteins of interest. Here, we describe a supramolecular gel electrophoresis (SUGE) technique that can be used to analyze acidic native proteins. The supramolecular hydrogel that was used as the electrophoretic matrix was constructed via the self-assembly of the amphiphilic tris-urea molecule 1. The separation of acidic protein samples in the supramolecular hydrogel is primarily dependent on the isoelectric points (pI) of the proteins. Furthermore, the separated proteins can be easily recovered from the supramolecular hydrogel retaining their activities.

lectrophoresis is a technique widely used in biological research to analyze nucleic acids and proteins.1,2 Tiselius developed the free-solution boundary electrophoresis of serum globulin, which was the first example of protein electrophoresis.3 Zone electrophoresis using a gel matrix that was originally a starch gel has been widely used for separating and identifying proteins.4 Protein electrophoresis using polyacrylamide gels is an integral method in protein research.5 Sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDSPAGE) is the most frequently used technique for analyzing protein samples. The SDS-PAGE procedure was established in the early 1970s and has remained almost unchanged.6 Alternative protein electrophoresis techniques have been developed such as affinity electrophoresis, which is an efficient method for detecting specific classes of proteins.7 For instance, Phos-tag SDS-PAGE can be used to identify phosphorylated proteins.8 In this technique, a well-designed phosphorylated protein receptor (Phos-tag)9 is co-polymerized with polyacrylamide gel. This matrix results in reduced mobility that is dependent on the phosphorylation status of the protein. Twodimensional electrophoresis and subsequent mass spectrometry have become indispensable techniques in proteomics research.10,11 Despite methodological advancements, polyacrylamide gel has been used as the matrix for protein electrophoresis for over 50 years. Polyacrylamide gels exhibit a strong affinity for proteins; thus, the recovery of proteins after PAGE is laborious. Separation manner in polyacrylamide gel is generally monotonous. The discovery of a novel gel matrix would be a major breakthrough in the field of protein electrophoresis. Gel materials can be constructed not only by polymeric compounds, but also by the self-assembly of small molecules called low-molecular-weight gelators.12−17 The potential applications of these self-assembled supramolecular gels in material science have attracted the interest of researchers in recent decades.18−21 In particular, supramolecular hydrogels, © 2014 American Chemical Society

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EXPERIMENTAL SECTION Gelation Experiments. A mixture of 129 and tris(hydroxymethyl)aminomethane (Tris)−glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3) in a test tube was heated at 100 °C. The solution obtained was gradually cooled to ambient temperature. The formation of a gel was evaluated by Received: July 17, 2014 Accepted: August 22, 2014 Published: August 22, 2014 9924

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Figure 1. Outline of the native-SUGE procedure.

procedure contains proteins in their native states. The separation pattern was performed by analyzing the supernatants using a standard SDS-PAGE system, followed by CBB staining. Preparation of the Supramolecular Hydrogel of the Tris-Glycine Buffer. We have recently developed the amphiphilic tris-urea low-molecular-weight hydrogelator 1 that efficiently gelatinizes a wide range of aqueous solutions (Figure 1).29 Amphiphilic tris-urea 1 formed a supramolecular hydrogel with Tris−glycine buffer or with other buffers that are typically used in native-PAGE experiments.30 The minimum concentration of 1 required to achieve gelation of the Tris− glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3) was estimated to be 0.25 wt % (1.9 mM) (Figure 2A). Hydrogels at concentrations of 1 below 0.5 wt % were semitransparent, whereas hydrogels at concentrations of 1 above 0.5 wt % were opaque. Once formed, the hydrogels of 1 were stable for at least one year at ambient temperatures, regardless of the concentration used, with no crystallization or melting. The

inverted tube test. The mixture was defined as a gel if it remained at the top of the inverted test tube. General Procedure for Native-SUGE. A mixture of 1 (2.0 wt %) and Tris−glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3) in a glass tube (with ϕ 5 mm) was heated to 100 °C. The solution obtained was drawn into a glass capillary (with ϕ 2 mm, length 120 mm). The solution in the glass capillary was left at the ambient temperature for 1 d, then the gelatinated matrix was used for electrophoresis as described below. The length of the supramolecular hydrogel was adjusted to 80 mm. A solution of protein(s) (typically 6.0 μg of each) was placed at one end of the hydrogel and allowed to become absorbed by the gel by standing the glass capillary vertically. Both ends of the glass capillary were then filled with an agarose gel (2.0 wt %). The glass capillary was immersed in Tris−glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3) in a submarine electrophoresis system, and electrophoresis was performed at 100 V for 100 min. The electrophoresed gel was removed from the glass capillary and divided into eight equal parts (numbered 1 to 8, from the anode end). Each hydrogel part was placed in a microtube and frozen at −20 °C for 3 h. Deionized (DI) water (15 μL) was added to the hydrogel, then the mixture was centrifuged (at 14 500 g) for 2 min. The supernatant obtained was freeze-dried and then Tris−glycine buffer (15 μL) was added. The solution was analyzed using typical SDS-PAGE procedure followed staining with Coomassie brilliant blue (CBB).

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RESULTS AND DISCUSSION SUGE Strategy for Acidic Native Proteins. SUGE for acidic native proteins (native-SUGE) is based on the use of a supramolecular hydrogel as an alternative matrix to the polyacrylamide gel that is used in native-PAGE. A typical experimental procedure of native-SUGE is outlined in Figure 1. A supramolecular hydrogel was prepared from amphiphilic trisurea 129 and a Tris−glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3). A glass capillary (with ϕ 2 mm, length 120 mm) was filled to 80 mm with the supramolecular hydrogel (2.0 wt %). A sample protein solution was adsorbed onto one end of the hydrogel. The glass capillary was immersed in Tris− glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3) in a submarine electrophoresis system. Next, electrophoresis was performed at 100 V for 100 min, allowing the migration of proteins from the cathode to the anode. The electrophoresed gel was then removed from the glass capillary and divided into eight equal parts (numbered 1−8 from the anode), and proteins were isolated from the supramolecular hydrogel by freezing and centrifugation. The supernatant derived from this

Figure 2. Formation of the supramolecular hydrogel: (A) photographs of mixtures of 1 and Tris−glycine buffer, using concentrations of 1, from left to right, of 0.10, 0.25, and 2.0 wt %; (B) SEM image of a xerogel of 1. 9925

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Figure 3. Native-SUGE of acidic native proteins. SDS-PAGE analyses after native-SUGE (hydrogel of 1) separation of (A) D-lactate dehydrogenase (LDH, 146 kDa, pI = 4.0) and β-galactosidase (β-Gal, 540 kDa, pI = 4.6); (B) LDH and ovalbumin (OVA, 45 kDa, pI = 4.7); and (C) β-Gal and OVA.

A mixture of β-Gal and OVA was next used to perform native-SUGE (Figure 3C). SDS-PAGE analysis showed β-Gal bands of similar densities in lanes 4 and 5. OVA bands were found in lanes 5 and 6, with lane 5 containing the strongest band. In this experiment, the larger and more acidic β-Gal was more mobile than the smaller and less-acidic OVA. These results indicate that native-SUGE performed using the supramolecular hydrogel of 1 caused acidic native proteins to be separated depending on their pI values rather than their molecular weights. The typical native-PAGE experiments of these proteins showed considerably different results (see Figure S2 in the Supporting Information). LDH and β-Gal, and β-Gal and OVA were observed as distinct, separated bands. Conversely, LDH and OVA exhibited the same mobility, because of their comparable combination of molecular weights and isoelectric points. The separation of LDH and OVA illustrates one advantage of native-SUGE over native-PAGE. The meshes present in the supramolecular hydrogel of 1 (2.0 wt %) may be coarser than the meshes in polyacrylamide gel (>10%), meaning that the molecular sieve effect should have little effect in native-SUGE, and that the net negative charge of the proteins is the predominant factor affecting their separation. SUGE of Fluorescent Proteins. Native-SUGE was performed using green fluorescent protein (GFP, 27 kDa, pI = 5.57), and the green fluorescent band was observed in the supramolecular hydrogel of 1 during and after electrophoresis via ultraviolet (UV, 365 nm) irradiation of the gel (see Figure S3 in the Supporting Information). This indicates that the GFP remained in its native form throughout the electrophoretic process. Native-SUGE was also performed using a mixture of OVA and GFP (Figure 4A). A green fluorescent band of GFP was observed by irradiating the gel with UV light. After SDSPAGE analysis, OVA was found in lanes 4 and 5 (with lane 4 containing the strongest band) and GFP was found in lanes 5 and 6 (with lane 5 containing the strongest band). The green fluorescent band observed in the supramolecular hydrogel of 1 corresponded with the GFP band detected following SDSPAGE analysis. The more-acidic OVA was more mobile than the less-acidic GFP. Native-SUGE was performed using a mixture of GFP and red fluorescent protein (RFP, 27 kDa, pI = 5.65); green and red fluorescent bands were observed in the supramolecular hydrogel of 1 during and after electrophoresis via irradiating the gel with UV light (Figure 4B). After SDSPAGE, the GFP was found in lanes 5 and 6 (with lane 5 containing the strongest band) and the RFP was found in lanes 6 and 7 (with lane 7 containing the strongest band). The more-

thermal stability (Tgel) of the hydrogel increased as the concentration of 1 was increased, which is a typical characteristic of supramolecular gels (Tgel = 31 °C for 0.25 wt %, 106 °C for 2.0 wt %; see Table S1 in the Supporting Information).31 A scanning electron microscopy (SEM) image of a xerogel prepared from Tris−glycine buffer with 2.0 wt % of 1 showed that there were nanofibers present with diameters ranging from 90 nm to 240 nm (Figure 2B). SUGE of Acidic Native Proteins. In typical native-PAGE, proteins are separated according to their molecular weights, isoelectric points, three-dimensional (3D) structures, etc.32,33 The supramolecular hydrogel of 1 has a fibrous network that differs from that of a polyacrylamide gel, leading to the separation of proteins in a different manner. Three acidic native proteins, D-lactate dehydrogenase (LDH, tetramer = 146 kDa, pI = 4.0), β-galactosidase (β-Gal, tetramer = 540 kDa, pI = 4.6), and ovalbumin (OVA, monomer = 45 kDa, pI = 4.7), were used in the initial experiments investigating the use of nativeSUGE using the supramolecular hydrogel of 1. Two of the three proteins were applied to the native-SUGE system and their separation patterns were analyzed by SDS-PAGE (see Figure 3 and Figure S1 in the Supporting Information). First, a mixture of LDH and β-Gal was used, and the results from SDSPAGE analysis are shown in Figure 3A. Note that LDH was found in lanes 4 and 5, with lane 4 containing the strongest band. β-Gal was found in lanes 5 and 6, with lane 5 containing the strongest band. Therefore, the smaller (146 kDa) and more acidic (pI = 4.0) LDH was more mobile than the larger (540 kDa) and less-acidic (pI = 4.6) β-Gal in this system. LDH and β-Gal retained their enzymatic activities after electrophoresis in the supramolecular hydrogel of 1 (vide infra), indicating that these proteins remained in their native tetrameric forms. LDH and β-Gal were denatured in the process of performing SDSPAGE after electrophoresis in the supramolecular hydrogel of 1 had been performed; thus, LDH and β-Gal were found to have molecular weights of 36.5 kDa and 135 kDa, respectively, by SDS-PAGE analysis. Next, we used a mixture of LDH and OVA to perform native-SUGE. In this experiment, LDH was found in lanes 3 and 4 (with lane 4 containing the strongest band), and OVA was found in lanes 5 and 6 (with lane 5 containing the strongest band) in the SDS-PAGE (Figure 3B). LDH retained its native tetrameric form during electrophoresis through the supramolecular hydrogel of 1. In this test, the larger and more-acidic LDH was more mobile than the smaller and less-acidic OVA. 9926

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the supramolecular hydrogel of 1, the GFP retained its native structure, but the denaturation of GFP can easily be reversed.34 The activity of the LDH after the native-SUGE was measured to confirm that it had remained in its native form. LDH catalyzes the oxidation of lactate to pyruvate in the presence of nicotinamide adenine dinucleotide (NAD+). The enzymatic activity of LDH can be determined by the absorption of UV light at 340 nm by the reduced form, NADH. The LDH activity after electrophoresis through the supramolecular hydrogel of 1 was determined using a D-/L-lactic acid assay kit (Biocon Japan, Ltd.), according to the manufacturer’s instruction. LDH (6.0 μg) was electrophoresed through the supramolecular hydrogel of 1 (2.0 wt %, ϕ = 2 mm, length 20 mm) at 100 V for 30 min, and the entire gel was used for the assay. The LDH activity after electrophoresis was ∼97% of the original LDH activity. The supramolecular hydrogel of 1 showed no catalytic activity in the D-/L-lactic acid assay. The catalytic activity of β-Gal after electrophoresis through the supramolecular hydrogel of 1 was confirmed using the supernatant to hydrolyze ο-nitrophenyl-β-D-galactopyranoside (ONPG) to give ο-nitrophenol (ONP) (see Figure S4 in the Supporting Information). Two aliquots of β-Gal were subjected to electrophoresis through the supramolecular hydrogel of 1 (2.0 wt %, ϕ = 2 mm, length 80 mm) at 100 V for 100 min under the same conditions. One sample was then subjected to SDS-PAGE, after which the strongest β-Gal band was found in lane 3. The other sample was extracted and used to hydrolyze ONPG, and the extract from lane 3 showed the strongest ONP production. The intensities of catalytic activity were comparable with the result of SDS-PAGE analysis. Affinity Electrophoresis of Lectin Using the NativeSUGE Technique. Carbohydrate-binding proteins (lectins) have many important roles in living organisms, including information transmission.35 Affinity electrophoretic systems for lectins have been developed and are effective for identifying protein functions.7 The surfaces of the nanofibers formed through the self-assembly of the amphiphilic tris-urea 1 were densely coated with glucosides that were introduced as

Figure 4. Native-SUGE of fluorescent proteins. Photographs of the UV-irradiated glass capillaries after electrophoresis and SDS-PAGE analyses of (A) green fluorescent protein (GFP, 27 kDa, pI = 5.57) and OVA; (B) GFP and red fluorescent protein (RFP, 27 kDa, pI = 5.65).

acidic GFP was more mobile than the less-acidic RFP, despite them having similar isoelectric points. Protein Activity Assay. It is preferable for proteins to retain their native three-dimensional (3D) structures and activities during electrophoresis. During native-SUGE using

Figure 5. Native-SUGE of a carbohydrate-binding protein. SDS-PAGE of the native-SUGE (hydrogel of 1) and the mechanisms that were assumed to lead to the results, for (A) concanavalin A (ConA, 112 kDa (as tetramer), pI = 4.4−5.5) in Tris−glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3); (B) denatured ConA in Tris−glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3); (C) ConA in Tris−glycine−MeαMan buffer (25 mM Tris, 192 mM glycine, 51 mM MeαMan, pH 8.3). 9927

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containing electrophoresed OVA was centrifuged (at 14 500 g) for 2 min. The supernatant was subjected to SDS-PAGE together with weighted references, and a quantitative evaluation was accomplished by analyzing the CBB-stained polyacrylamide gel plate using ImageJ software. The recovery of OVA, estimated from the integrated optical density, was 24%. Only 4% of the OVA was recovered from polyacrylamide gel using the same procedure. Unfortunately, this procedure was ineffective for recovering ConA, the recovery being only 8%. The recovery of proteins was improved dramatically when the electrophoresed hydrogel of 1 was frozen before the extraction process. Therefore, the recovery procedure was as follows. A sample of a supramolecular hydrogel of 1, containing an electrophoresed protein, was frozen at −20 °C for 3 h. Deionized water was then added to the hydrogel and the mixture was centrifuged (at 14 500 g) for 2 min. The supernatant was freeze-dried, and the sample obtained was dissolved in Tris−glycine buffer. The solution was analyzed by SDS-PAGE, as described above. The recoveries of OVA and ConA were improved, reaching 58% and 43%, respectively, following this procedure. By freezing the hydrogel, the ordered aggregates may be broken up, thereby weakening the interactions between the proteins and the hydrogelator.

hydrophilic groups. The glucoside-coated nanofibers can interact with appropriate lectins during electrophoresis, and the lectins involved in these interactions do not migrate as far as noncarbohydrate-binding proteins in the gel. A wellcharacterized lectin, concanavalin A (ConA, tetramer = 112 kDa, pI = 4.4−5.5), was subjected to affinity electrophoresis using the supramolecular hydrogel of 1. A moderate association between 1 and ConA was expected, from the known affinity between α-methyl-D-glucopyranoside and ConA (Ka = 1.96 × 103 M−1).36,37 The electrophoresis of ConA was performed under the typical native-SUGE conditions (2.0 wt % of 1, 100 V, 100 min) (Figure 5A). SDS-PAGE showed that most of the ConA remained at the cathode at the starting point (lane 8), and the ConA remained in lane 8 even when the electrophoretic time was increased to 300 min (see Figure S5 in the Supporting Information). In contrast, the electrophoresis of denaturedConA under the same conditions showed considerably different results (Figure 5B), with the denatured-ConA migrating toward the anode during electrophoresis using the supramolecular hydrogel of 1, and was found in lanes 4 and 5 after SDS-PAGE. These results indicate that native ConA interacted with the glucosides on the nanofiber surfaces, which inhibited its electrophoretic mobility. The addition of a saccharide with a strong affinity for ConA to the electrophoresis buffer might improve the electrophoretic migration of native ConA in the native-SUGE. The ConA and saccharide complex in the buffer prevents ConA interacting with the glucosides on the nanofiber surfaces and allows ConA to migrate toward the anode during electrophoresis. The saccharide α-methyl-D-mannopyranoside (MeαMan) was selected for this task because it has strong affinity for ConA (Ka = 0.82 × 104 M−1).36,37 A Tris−glycine− MeαMan buffer (25 mM Tris, 192 mM glycine, 51 mM MeαMan, pH 8.3) was used in native-SUGE for native ConA. After SDS-PAGE analysis, ConA was found in lanes 5 and 6 (Figure 5C). The native ConA migrated much further toward the anode in the presence of MeαMan than it did under the typical native-SUGE conditions used to produce the results shown in Figure 5A. Recovery of Protein Samples from Supramolecular Hydrogel. Recovering proteins from the gel matrix after electrophoresis allows the proteins to be used in further investigations. The amide groups in polyacrylamide interact strongly with the polyamide framework of proteins. Therefore, separating proteins from polyacrylamide gel after electrophoresis is a difficult task. Several methods have been developed to recover proteins from a polyacrylamide gel.38 The passive elution method is the most straightforward but usually results in poor extraction efficiencies. Dissolving the gel and degrading the polyacrylamide, with H2O2, for example, gives better extraction efficiencies, but the proteins are often damaged during the process. Electroelution is a practical method for recovering proteins from a polyacrylamide gel, although it requires specialized equipment and is a long and complicated procedure. Supramolecular hydrogels, which are formed through weak noncovalent interactions between small molecules, offer potential advantages when used as electrophoretic matrices as they allow the efficient recovery of proteins. The electrophoresed proteins can be extracted from the supramolecular hydrogel of 1 using a very simple procedure (see Figure S6 in the Supporting Information). A mixture of Tris−glycine buffer and the supramolecular hydrogel of 1

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CONCLUSIONS In conclusion, we have developed an electrophoretic procedure for separating native proteins using supramolecular hydrogel of 1 as the matrix. We have showed that acidic proteins can be separated according to their isoelectric points rather than their molecular weights. Furthermore, the activities of the proteins were retained, even after electrophoresis in the supramolecular hydrogel of 1. Affinity electrophoresis of ConA was achieved using the interactions between the glucosides of 1 and ConA. The mobility of ConA during electrophoresis was controlled by the addition of a saccharide (which bound strongly to the ConA) to the buffer. The structural alteration of the lowmolecular-weight hydrogelator would make further affinity electrophoresis possible. Proteins were efficiently recovered from the supramolecular hydrogel of 1 after electrophoresis using a very simple procedure. Therefore, these data indicate that supramolecular hydrogels are valuable matrices for use in protein electrophoresis.

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ASSOCIATED CONTENT

S Supporting Information *

Table S1 and Figures S1−S6. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Fax: +81-54-237-3384. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for the Scientific Research on the Innovative Areas: “Fusion Materials” (Area No. 2206, No. 23107514) and a Grant-in-Aid for the Scientific Research (B) (No. 24310089). 9928

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