Methods of Isoelectr ic Focusing R. H. BYLES, WILLIAM MCGUIRE AND MARK F. SANDERS Laboratory of Biological Anthropology, Department of Anthropology, liniversity of California, Los Angeles, Los Angeles, California 90024
K E Y WORDS Isoelectric focusing variation
ABSTRACT The method of isoelectric focusing has been avoided by many workers because of expense, technical difficulty, and problems of interpretation. Inexpensive, easy, and interpretable results are possible using equipment and reagents commonly available. Methods which allow these results are presented and explained. Within the last few years a number of techniques have been developed which represent major advances in the ability of investigators to discriminate variation in the proteins of many species of organisms. These are of potential benefit t o physical anthropologists interested in the biochemical variation in human and non-human primates. Most noteworthy among these new methods are double internal standard disc electrophoresis pioneered by Johnson (N.D.), and isoelectric focusing. The latter technique, however, has not been exploited as a research tool for survey studies. The ability of this method to distinguish differences in free electric mobility among otherwise indistinguishable protein molecules would ordinarily have been adopted by many laboratories, except for practical and fiscal considerations which put this powerful tool out of the reach of modestly funded research projects. The objections to this technique which have been brought to our attention include: 1. Expense. 2. Technical requirements in terms of trained personnel and equipment. 3. The lack of storable “hard-copy” for permanent records. 4. The general attitude that these high resolution techniques produce “uninterpretable” data. Several avenues now open obviate or eliminate all of these objections and this research tool can be made accessible t o even small laboratory facilities. IEF PRINCIPLES
Traditional electrophoretic techniques are AMvr. J. PHYS. ANTHROP. (1979) 50: 27-34
dependent upon, and limited by. the migration of the protein under the influence of an electric field through a supporting matrix buffered at a constant pH. The rate of protein migration is jointly determined by the free electric mobility of the protein and a configurational retardation from the interaction of the protein molecule with the supporting matrix. The dependence of the observed migration rate upon these two variables increases the likelihood that two different proteins will exhibit the same mobility in a particular electrophoretic system; the decreased sensitivity from such co-migration is particularly severe in the case of allozymes of the same protein in which differences in molecular size and charge are expected to be minimal. In addition, the dynamic nature of this separation technique places heavy emphasis upon rigid control of external variables such as field strength, migration time, and temperature. Thin layer isoelectric focusing is essentially free of these problems as it is based upon the migration of a given protein t o a fixed point within a stable pH gradient under the influence of an electric field. The fixed point where protein migration ceases, the isoelectric point (PI), corresponds t o the pH at which the protein has neutral charge. Since the pH gradient is stabilized within the gel by carrier ampholytes, once a protein molecule reaches its PI, it is no longer influenced by the electric field and will remain in a fixed position almost indefinitely. Thus, isoelectric focusing is a static technique; the final position of a given protein species is not dependent upon position of application or migration time, but rather
R. H. BYLES, W. MCGUIRE AND M. F. SANDERS
upon the net surface charge of the protein. In addition, since the rate of migration of a molecule to the isoelectric point is not a consideration, molecular size is not a contributing factor in this type of analysis. Finally, isoelectric focusing tremendously reduces the likelihood that even very closely related allozymes will share an identical position since, a t low values of ApH/distance, a difference in isoelectric point of only 0.01 pH units is sufficient to achieve separation. COST
There are two traditional sources of expense for the routine user of isoelectric focusing technique: the high cost of the carrier ampholytes required t o produce the pH gradient and the expensive apparati commercially marketed. Even a modestly equipped and funded laboratory can make both of these potentially expensive components at a small fraction of the cost of the commercial products.
Ampholytes Isoelectric focusing replaces the normal fixed pH buffer with a dilute solution of a complex buffer known as a carrier ampholyte. Older column isoelectric focusing apparati required the use of considerable quantities of this reagent, and therefore made this a far from frugal methodology. While recent developments, particularly those of Radola ('73) using readily available Sephadex G-75-40 as the supporting medium have provided resolution better than earlier systems while using far less carrier ampholyte, the cost of this reagent has remained an important, and often limiting, consideration. We describe below a method for the production of carrier ampholyte using readily available reagents and inexpensive equipment, producing a high-quality product a t minimum cost. Ampholytes are produced in a polymerization reaction between acrylic acid and any of a number of polyamino compounds, as described in an excellent article by Vesterberg ('69). We have chosen to use pentaethylene hexamine as the polyamino compound since the resulting ampholyte product has good buffering capacity over a wide pH range, making it especially suitable for routine use. Pentaethylene hexamine is a very caustic liquid and, as commercially available, possesses a light yellow color. This color will appear in the final product, but is not objectiona-
ble unless direct ultraviolet scanning of the gel is contemplated. In such cases, the yellow color may be removed from the hexamine by distillation a t 180-185°C. The acrylic acid reagent contains a polymerization inhibitor which must be removed prior to use in the synthetic reaction by distillation under vacuum, provided by a water respirator. Temperature is controlled by a monitored water bath. We strongly emphasize the need to perform this distillation under vacuum to prevent explosive polymerization of this highly irritating acid. In addition, proper safety equipment, including goggles and a heavy lab coat, are considered necessities. The reaction mixture used in the synthesis consists crylic acid and pentaethylene hexamine in a 3: 1molar ratio, with enough water added t o keep the acrylic acid concentration at about 3 M. In the usual case, 0.18 moles of acrylic acid are mixed with 0.06 moles of pentaethylene hexamine and 60 ml of water. Only the precautions normally taken with the mixing of strong acid and strong base are required. The mixture is refluxed for five hours at 7 0 T , during which time the color of the mixture will normally turn from yellow to orange-brown. After refluxing, the mixture is lyophilized (or flash evaporated) and dissolved in a minimal volume of water. This solution is weighed and the concentration of ampholyte determined; usual stock concentrations are 3 5 -40%. The material yielded by this procedure compares favorably in buffering capability to that available commercially. The workable pH range of such synthetic preparations is roughly 3.5-10.As we find that both the commercial ampholyte and that prepared as described above have a rather low buffering ability in the acid range, we also use an ampholyte mixture prepared from a reaction mixture with acrylic acid and pentaethylene hexamine in a 4 : l molar ratio. This preparation has considerably more buffer capacity in the acid range and is used 1:l with the previous ampholyte mixture in gel preparation.
Electro-focusing apparatus Reduced t o simplest terms, the equipment necessary to perform isoelectric focusing analyses is (1) a chamber enclosing a metal plate with the capacity to he maintained a t about 4°C and to support both a gel plate and the electrodes used to generate the electric k, (2) a power supply capable of supplying 500
Fig. 1 Isoelectric focusing apparatus. The apparatus is a double walled plexiglass box fitted with a cooling hlock, electrode sockets, electrical input socket, and inflow and outflow connections to the cooling hlock.
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Fig. 2 Standard protein sample. Isoelectric points (PI) for several protein bands are given at the right. The numbered brackets a t t h e left indicate the bands and pI ranges of the seven standard constituents; (1) cytochrome c (2) ribonuclease (3) equine myoglobin (4) beta lactoglobulin (5) ferritin (6) whale myoglobin (7) conalbumin.
R. H. BYLES, W. MCGUIRE AND M. F. SANDERS
regulated volts and with a current range of 0100 mA, and (3) a source of circulating coolant capable of holding 4 +- 1°C. The isoelectric focusing chamber (fig. 1) consists of a Plexiglas outer shell within which is mounted a hollowed aluminum cooling block; the top surface of this block is blanchard ground to insure maximum contact with the gel plates and teflon coated to minimize electrical hazard. The outer shell is provided with electrical connections for the electrodes, and the inlet and outlet connections for the cooling block. The cover of the chamber should be provided with safety switches to prevent the chamber from being opened while current is flowing. Although platinum electrodes for isoelectric focusing are commercially available. we find that flattened carbon rods mounted in plastic holders provide equal performance a t a fraction of the cost. Another expensive item associated with focusing methods is t h e microelectrode used to ascertain the pH a t which the samples reach neutrality. Not only is this expensive, but it also requires that the paper print and the gel be accurately aligned so that pH readings on t h e gel can be given correspondence on the paper print. This process and this tool are unnecessary as other more convenient and accurate means are available to serve the same purpose. Radola ('73) gives a list of proteins with established PI and a mixture of these proteins will produce a standard sample which covers the full pH range. If this standard is run alongside of the sample protein, this system is most accurate and inexpensive. We have had superior results with this technique. Figure 2 shows a run using this standard and, as can be seen, the resolution possible in the standard allows close approximation of the p1 of the sample. The central piece of apparatus described above, as well as many alternate means to t h e same end, can be constructed a t minimum cost. Dimensions can be adjusted to suit t h e needs of each laboratory. We are confident that the objection of expense using this technique can be met easily. Our investment in reagents is very small, and the total cost of construction of the apparatus was less than three hundred dollars. TECHNICAL REQUIREMENTS AND PROCEDURES
The methodology for isoelectric focusing in
granulated gels used in this laboratory is the technique described by Radola ('731, modified slightly to enhance resolution and reduce costs. Composition of the isoelectric focusing gel is as follows: Sephadex G-75-40 (superfine), 8%; combined carrier ampholytes (described above), 2%; arginine and lysine (0.1% each) are routinely added to stabilize the basic region of the gel. Approximately 35 ml of this gel is evenly applied by pipet to a cleaned glass plate, 20 x 20 cm. An extremely smooth gel surface is obtained by tapping the bottom of the glass plate on the edge of a lab bench. The smooth gel is allowed to dry at room temperature for one and one-half to two hours or until small fissures 1-2 mm long appear around the edges; the finished gel has a very smooth surface and is approximately 0.6 mm thick. Unused portions of the gel mixture may be stored a t 0-3°C:for extended periods; it is recommended that refrigerated gel be allowed to come to room temperature before pouring. An incidental but appealing feature of the granulated gels is that if the gel is too thick (does not smooth out) or too thin (runs from the edge of the plate), it may be scraped from the plate and reused. The poured gel is placed on the cooling block of the unit and connected to the electrodes via Whatman 3MM paper strips soaked in 0.1 M H,SO, (anode) and 0.4 M NaOH (cathode); almost any strong, nonvolatile acid and base of similar concentration may be substituted for those given. The electrodes are applied directly onto the paper strips - both electrodes and connecting paper should completely cover ) apthe ends of the gel. Samples (20-40 ~ 1are plied in bands using No. 2 coverslips as applicators; care must be exercised to avoid damaging the gel during sample application. Focusing is accomplished by application of 200-400 regulated volts for a period of six to eight hours. Starting voltage should be adjusted to keep current flow a t or below 2.5 mAicm of electrode. During the focusing process, the current flow will drop to near zero as the pH gradient is formed. Hardcopy procedures We follow the procedure of Radola ('73) in printing the results of isoelectric focusing onto filter paper and then staining. The only modification made is the observation that the smooth finish and extreme durability of
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Fig. 3 Native human albumin. The pl of native human albumin is approximately 4.3 as determined by comparison to the standard.
Photo 1 This print shows the position of a series of primate albumins with reference to the standard proteins. T w o grey mangabey individuals and two black mangabey individuals have provided albumin samples for this run.
R. H. BYLES, W.MCGIJIRE AND M. F. SANDERS
Fig. 4 Denatured human albumin. Denaturation fragments of human seruni albumin have PI’S ranging between 4.3(native albumin) and 6.1 (a) 9 denaturation fragments (b) denatured, nun fractioned albumin (c) native, non -fructioned albumin.
Photo 2 Denatured human serum albumin is focused alongside the protein standard. Eleven bands including native albumin, denatured albumin, and nine denaturation fragments are visible.
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4.2 Fig 5 Human IrmRferrin Samples from a human tranuferrtn homozvgute and hcterozvgote are shown companson to the standard
Whatman No. 50 paper is particularly useful in the printing process. The filter paper is cut to a size slightly larger than t h e gel, rolled smoothly onto t h e gel surface to avoid air bubbles, allowed to stand for one minute, carefully removed and washed. Two sequential 15-minute washes of 10% trichloroacetic acid are used to remove ampholytes, followed by a 3-minute wash in destaining solution (methano1:water:acetic acid, 33:66:10) to remove TCA. Staining is performed in 0.1-0.2% Coomassie Blue G-250 in methano1:water:acetic acid, 5:5: 1 (Coomassie Blue R-250 is also suitable) for 30 minutes. The prints are destained until clear in the solution mentioned above. The print technique is especially convenient as i t allows a permanent record of each run to be stored in a compact set of laboratory notes. INTERPRETATION OF MICROHETEROGENEITY
Many conclude t h a t the variation using
these methods, in addition to the variation discovered using less efficient systems, is somehow suspicious, uninterpretable, or artifactual. Focusing in granulated gels has produced cases of real variation which were “cryptic” using other means, and the ability to define the nature of the variants found has improved with the use of the technique. For example, we are investigating several proteins from human plasma using this method and all of the microheterogeneity found has been intelligible. A survey of human serum albumin has produced little evidence of allelic heterogeneity and figure 3 shows the PI of ordinary albumin. Figure 4,however, shows the results of subjecting albumin samples to denaturing heat (90°C) for 30 minutes. Eleven bands can be distinguished in the print. Sponar et al. (‘631, using fractionization analysis on the same molecule, likewise reported eleven comDonents. The two lower bands are the two entire albumin molecules. The bottom band is
R. H. BYLES, W. MCGTJIRE AND M. F. SANDERS
native albumin not yet denatured; the one above (2) is intact, but denatured albumin. The other nine bands are the denaturation fractions eluted off of the fractionization column. Disc electrophoresis of this same material is able to resolve only five electromorphu. Isoelectric focusing distinguished all eleven of the fractions clearly. Transferrins are also being subjected to isoelectric focusing analysis, as well ax double standard disc techniques, in this laboratory. Figure 5 shows the results of isoelectric focusing of transferrins isolated by rivanol from whole serum. Several components are visible as a result of isoelectric focusing. In the past we have found that the increased resolution of this focusing system produces useful data with great precision. CONCLUSION
Isoelectric focusing is a method for distin-
guishing molecules with different isoelectric points. The disadvantages to this method which have prevented many from adopting it in the past are now no longer in existence. For this reason, this method of high resolution protein analysis is within the capability of most laboratories, and within the budgets of virtually all. LITERATURE CITED Johnson, G. M. N.D. Hidden heterogeneity among electrophoretic alleles. Carnegie Institution Dept. Plant Biol, Publication No. 572. Radola, B. J. 1973 Isoelectric focusing in layers of granulated gels. I. Thin-layer isoelectric focusing of proteins. Biochim. Biophys. Acta., 295: 412-428. Sponar, J., I. Fric, S. Stokrova and J. Kovarikova 1963 On heterogeneity of human serum albumin. Coll. Czech. Chem. Comm., 28: 1831-1837. Vesterberg, 0. 1969 Synthesis and isoelectric fractionation of carrier ampholytes. Acta Chem. Scand., 23: 2653-2666.