isoelectric focusing in polyacrylamide gels

ISOELECTRIC FOCUSING IN POLYACRYLAMIDE GELS#

Daniel Wellner and Melvin B. Hayest Department of Biochemistry

Cornell University Medical College New Ywk , New York 10021

A method for the separation of proteins by isoelectric focusing in polyacrylamide gels was developed in our laboratory as an analytical tool for the study of L-amino acid oxidase isozymes.l.2 Although we also used the sucrose gradient method of Vesterberg and Svenssons successfully for separating the isoenzymes, that method had a number of drawbacks for our purpose. In the first place, our samples were often too small to apply to the available large-scale sucrose gradient columns. An- other problem was the limitation in the number of experiments that could be carried out in any given time because of the lengthy procedure, involved in pre- paring the sucrose gradient, allowing the separation to take place, collecting the fractions, and analyzing them first for pH, then for protein, and then for enzymatic activity.

We thought that for many applications, polyacrylamide gel would provideo a suitable substitute for a sucrose gradient as an anticonvection medium in isoelectric focusing. It appears that the time was right for such an idea because it was conceived independently in several other laboratories around the world.4-10 In this paper, we would like to indicate the advantages of this method and to illus- trate some of its applications, particularly to the L-amino acid oxidases of snake venom.

For analytical purposes, the use of polyacrylamide gel has several distinct advantages over the sucrose gradient method. (1) One of these is that the separation takes less time. We found in our experiments that about 9 hr at 120 V was opti- mal for a 10-cm gel. By using a higher voltage and appropriate cooling, it is possible to reduce this time even further. (2) Only very simple equipment is needed. An apparatus similar to that used by Davis11 for disc dectrophoresis is perfectly suitable. Such equipment is inexpensive and is now available in most biochemical laboratories. (3) In addition, the small amounts of carrier ampholytes needed places the method within reach of laboratories with very limited budgets. (4) The gels can be analyzed for protein, enzymatic activity, or radioactivity, just as in disc electrophoresis. For protein staining, however, it should be noted that some of the dyes commonly employed, such as amido black, may also stain the carrier ampholytes. Thus, unless the ampholytes are thoroughly washed out, such dyes sometimes given rise to a dense background, making observation of the protein bands difficult. This problem may be avoided by using Coomassie blue, which we found to be suitable for staining proteins in focused gels without the necessity of removing the carrier ampholytes. The protein bands may be fixed and stained simultaneously by immersing the gel for a few hours in a solution containing 5% trichloroacetic acid, 5% sulfosalicylic acid, 18% methanol, and 0.02% Coomasie blue.12 Destaining may be accomplished by washing the gels in water or in dilute acetic acid, but this is not necessary for the bands to become visible. (5) Very small samples, of the order of a few micrograms of protein. can be easily detected, either by protein or enzymatic staining. (6) Many samples can be run simultaneously in the same apparatus for comparative purposes. (7) The technique may be com-

Supported by the National Institutes of Health, United States Public Health Service. t Present address: National Institutes of Health, Bethesda, Md. 20014.

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Wellner & Hayes: Polyacrylamide Gels 35

bined with gel electrophoresis to obtain a two-dimensional separation. Thus, the focused gel cylinder may be embedded in a rectangular gel slab and electrophoresed in the second dimension. It is zometimes possible to separate more components in such a way than with either electrofocusing or electrophoresis alone. (8) Last, but perhaps not least, the method involves considerably less work than the sucrose gradient technique.

One disadvantage of the gel method compared with the sucrose gradient technique is that it is more difficult to obtain accurate measurements of the isoelectric points of the separated components. One way in which one can obtain an approxi- mate value is to slice the gel, elute the slices in distilled water or in dilute salt solutions, and measure the pH of the solutions obtained. However, this method is

FIGURE 1. L-Amino acid. oxidase crystals. (From Wellner & Meister.13 Reprinted by permission of the Journal of Biological Chemistry).

time consuming and subject to error. We found that a much simpler and reproduci- ble method consists of touching the surface of the gel at different positions along its length with a pH microelectrode immediately after taking the gel out of its tube. This takes less than five min per gel, and the same gel can then be analyzed for protein or enzymatic activity.

The application of the gel technique to the characterization of enzymes may be illustrated by some of the results we obtained with L-amino acid oxidase. These results emphasize the extent to which the apparent homogeneity of a protein is a function of the resolving power of the method employed to measure it. FIGURE 1 shows a picture of L-amino acid oxidase crystals. These are obtained by

dialyzing the purified enzyme against distilled water. I t is of interest that, although the enzyme is quite insoluble in the absence of salts, it dissolves readily in a 1%

36 Annals New York Academy of Sciences

FIGURE 2. Sedimentation velocity pattern of &-amino acid oxidase. Rotor speed: 59,7780 ' rpm. Sedimentation is from right to left.

solution of carrier ampholytes. Our initial thoughts that we had a pure protein were reinforced by the results of ultracentrifugal analysis as shown in FIGURE 2. In sedimentation velocity studies, the enzyme exhibited a single symmetrical peak with a sedimentation coefficient of 6.7s. The molecular weight was found to be 130,000 by the approach-to-equilibrium method. The enzyme is a flavoprotein with two moles of FAD per mole of protein.13

Electrophoresis in the Tiselius apparatus (a technique no longer widely u d ) revealed that the enzyme consisted of three components.13 Sampling of the electrophoresis cell was used to show that the three components, named A, B, and C in order of decreasing negative charge, had approximately equal specific activities. This was one of the early demonstrations of the existence of isoenzymes. It could be shown that the three species did not arise as a result of limited proteolysis in the venom because prolonged incubation of the venom at 37°C did not result in changes in the electrophoretic pattern.13 In addition, the distribution of isoenzymes differed among individual snakes.

As shown in FIGURE 3, the three isoenzyme bands could also be separated by disc electrophoresis. It soon became apparent, however, that the bands were broader than one would expect for single proteins. When the gels were stained either for protein by using amido black, or for enzymatic activity by using a system containing L-leucine, phenazine methosulfate, and triphenyltetrazolium, the same three bands were seen?

Only when we used isoelectric focusing, however, did we realize how heterogeneous the crystalline enzyme was. FIGURE 4 shows the results obtained when L-amino acid oxidase is focused in a 10-cm gel using a pH gradient of 3-10. The gel was stained with Coomassie blue. At least 18 components, with isoelectric points rang-


ing from 5.2 to 8.4, can be seen. This is the same preparation, crystallized from pooled Crotalus adamanteus venom, that gave three bands on gel electrophoresis. When similar gels were assayed for enzymatic activity, the same pattern was obtained, showing that all the components are active forms of the enzyme.

FIGURE 5 shows the isoelectric focusing patterns of venom preparations obtained from three different snakes. Electrophoresis of their venoms revealed that the first snake possessed all three isoenzymic bands (A, B, and C), where the second possessed only C, and the third only A. These findings suggest the possibility that the distribution of isoenzymes may be, at least in part, genetically determined. Additional evidence that the multiplicity of enzyme forms does not arise from the proteolytic activity of the venom is the finding of an identical pattern of isoenzymes in the erythrocytes, in the venom, and in the homogenized venom gland of the same snake.2

The possibility of artifacts was also considered. For example, irreversible binding of some of the ampholytes to the enzyme might be expected to cause additional components to appear. Binding of ampholytes labeled by the Wilzbach14 technique was therefore studied by gel filtration on Sephadex columns. The results of

FIGURE 3. Separation of L-amino acid oxidase hnzymes by disc electrophoresis. Gel 1 shows the three isoenzymes of a crystalline reparation from pooled venom. Gels 2,3, and 4 show isoenzymes C, B, and A, respectivery, obtained by ion-exchange chromatography on a DEAE-cellulose column. The gels were atained for enzymatic activity3 Top: cathode.


FIGURE 4. Separatisn of L-amino acid midase isoenzymes by isoelectric bas ing in gel. Crystalline enzyme from pooled venom was focused in 10-cm gels containing 2% pH 3-10 carrier ampholytes. The gels were stained for protein with Coomassie blue3 Top: anode.


FIGURE 5. Isoelectric focusing of L-amino acid oxidase from single snakes. Venom sam- les from three snakes were submitted to isoelectric focusing as in FIGURE 4 and stained

For enzymatic activity. The venom used in gel 1 was shown by electrophoresis to contain isoenzymes A, B, and C; the venom in gel 2 had only C; the venom in gel 3 had only A f


such an experiment are shown in FIGURE 6. It can be seen that some radioactivity was bound to the protein. Assuming an average molecular weight of 450 for the ampholytes, this would amount to 1.5 mole of ampholyte per mole of enzyme. When the radioactive protein peak was subsequently dialyzed against unlabeled ampholytes, however, most of the radioactivity appeared in the dialysate, showing

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FIGURE 6. Bindin of tritium-labeled carrier amphol tes to L-amino acid oxidase. En- zyme crystals were &solved in 2 Am holine, Batch 4 pH 3-10) labeled with tritium b the procedure of Wilzbach.14 J k e sofition was passed t 6 rough a column (20 X 0.9 an) or Sephadex G-100 equilibrated with 1% unlabeled ampholytes. Radioactivity was daer- mined by scintillation counting and protein by absorbance at 275 nm (bottom). A con- trol experiment without enzyme is also shown (top).

chat the binding was not irreversible and therefore could not account for the multiple components observed. Several amino acids that were found to be present in that particular batch of ampholytes may have accounted for the bound radioactivity.

Artifacts due to interactions between the protein or the ampholytes and the gel could also be eliminated by observing the similarity between isoelectric focusing


50

60 I

U I

FIGURE 7. Separation of L-amino acid oxidase isoenzymes by isoelectnc focusing in a suaose adient. A final potential of 500 V was applied f o r 36 hr at 4" C to a solution containing & mg crystalline enzyme and 1% pH 3-10 camer ampholytes. Fractions of 2.1 ml were collected.2

I I 1 I 1 1

- I 1

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7 0 - c - -


in a sucrose gradient and in a gel (FIGURES 7 and 8). Similary, although the isoenzymic patterns were found to differ among individual snakes within the same species, no difference in pattern was found between whole venom and the crystalline enzyme obtained from it. We therefore conclude that the multiple forms are preexistent in the venom and that their distribution is a characteristic of each individual snake.

Experiments in our laboratory on other enzymes also demonstrate the ability of gel isoelectric focusing to reveal the presence of multiple molecular forms. For example, in studies with D-amino acid oxidase of hog kidney, we can separate four enzymatically active components.1~ Similarly, sheep liver threonine deaminase can be separated by this method into at least seven enzymatically active proteins.16 Thus, it appears that isoelectric focusing in polyacrylamide gels is a method of high resolving power by which it has been possible to demonstrate the hitherto unsuspected existence of great heterogeneity in several enzyme systems. The struc- tural basis for the heterogeneity of L-amino acid oxidase is presently under inves- tigation in our laboratory.

REFERENCE^

1. HAYES, M. B. & D. WELLNER. 1968. Abstract no. 151. 156th American Chemical So- ciety Meeting. Division of Biological Chemistry.

2. HAYES, M. B. & D. WELLNER. 1969. J. Biol. Chem. 244: 6636. 3. VESTERBERG, 0. & H. SVENSSON. 1966. Acta Chem. Scand. 2 0 829. 4. AWDEH, Z. L., A. R. WILLIAMSON & B. A. ASKONAS. 1968. Nature (London) 219 66. 5. CATSIMPOOLAS, N. 1968. Anal. Biochem. 2 6 480. 6. DALE, G. & A. L. LATNER. 1968. Lancet i: 847. 7. FAWCEIT, J. S. 1968. FEBS Lett. 1: 81. 8. LEABACK, D. H. & A. C. R I J ~ R . 1968. Biochem. Biophys. Res. Commun. 32: 447. 9. RILEY, R. F. & M. K. COLEMAN. 1968. J. Lab. Clin. Med. 72: 714.

10. WRIGLEY, C. W. 1968. J. Chromatogr. 36 362. 11. DAVIS, B. J. 1964. Ann. N.Y. Acad. Sci. 121: 404. 12. WELLNER, D. 1971. Anal. Chem. 43: 59A. 13. WELLNER, D. & A. MEISTER. 1960. J. Biol. Chem. 235: 2013. 14. WILZBACH, K. E. 1957. J. Amer.Chem. Soc.79 1013. 15. SIVAKOFF, M. C. & D. WELLNER. Un ublished results. 16. GREENFIELD, R. S. & D. WELLNER. &published results.

DISCUSSION

DR. P. G. RIGHETTI: I am impressed with your results on L-amino acid oxidase. I wonder if you have investigated whether your enzyme’s FAD moiety is in the oxidized or reduced stage? If it is in the oxidized form, as it becomes increasingly reduced, it takes on the conformation of butterfly wings. Therefore, you have pro- gressive changes in the conformation of the protein moiety. Also, because the two FAD moieties are probably not covalently bound to the protein, could YOU have a partial loss of one or both of the moieties? Finally, I want to ask if your FAD enzyme could contain an iron-sulfur bridge? If it did have an iron-sulfur bridge, a partial loss of this component might produce the heterogeneous system that YOU have demonstrated.

DR. WELLNER: To answer your first question, all the FAD is in the oxidized state. This is apparent by the yellow color of the bands. When FAD is reduced it becomes colorless. These experiments were not performed under anaerobic condi- tions. FAD is very easily autooxidizable, and unless extremely rigid precautions are taken to exclude oxygen, FAD will remain in the oxidized state.


Concerning your second question about the partial dissociation of FAD; it is true that FAD is noncovalently bound, but it is extremely tightly bound. The enzyme can be dialyzed for long periods without a loss in FAD. It can be put through columns and crystallized, and during the entire purification procedure there is no loss of FAD, and it is not necessary to add any FAD to reactivate the enzyme. I n fact, FAD is so tightly bound that no one has yet succeeded in removing FAD from the enzyme without denaturing the protein.

And, your third question. This is not a metal-containing enzyme. It contains FAD as its only cofactor. However, I should mention that it is a glycoprotein. It contains carbohydrate including sialic acid, and we believe that some variations in the amount of carbohydrate might, in part, account for the heterogeneity.

isoelectric focusing in polyacrylamide gels

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