(53) &to, T. R., Sorris, \I-. F.> Strain, H. H., Abstracts of Papeis, p. 43, 124th meeting, .ICs, Chicago, Ill., 1953. (54) Sato, T. R., Norris, IV. P., Strain, H. H., Ahr.4~.CHEL 27, 521 (1955). (55) Sellers, P.A , , Sato, T. It., Strain, H. H., J . IrkOTg. and 1 7 u c k i ?Chem. 5 , 31 (1857). (56, Strain, H. H., Sato, T. R., A 4 x . \ ~ . CHEN.28. 687 11956). (57) Strain,”. H:, Sato, T. R., conference on use of isotopes in agiiciilture, p. 175, IT. 8. -4tomic Energy Comm., TID-7512 (1956). (58) Strauss, T’. P., Trwtler, T . I-..J . Am. C’hern. SOC.77, 1473 (1065).
( 5 9 ) Thilo, E., Grunze, J., 2. anorg. u . allg e m . Chenr. 290, 209 (1957). (60) Van \Yazer, J. R., “Encyclopedia of Chem. Technology,” T’ol. 10, Kirk and
Othmer, ed., p. 413, Interscience, 1053.
161) 1-an Wazer. J . R.. J . A m . Chenr. SOC. ‘ 72, 906 (1950)’. (62) Tan \Taxer, J. R., Goldstein, AI., Farber, E., Ihid., 75, 1503 (1953). (63) Tan TYazer, J. R., Griffith, E.J., RIcCulloueh. J. F.. A x . 4 ~ .CHEX 26. 1755
11854’l.” ,- - - , ‘ (64) Wade, H. E., Aforgan, D. >I., Biochem. J . 60, 264 (1955). (65) Waggaman, \I7. H., “Phosphoric Acid, Phosphate and Phosphatic Ferti-
lizers,” 2nd ed., Reinhold, XeTv York, 1952. (66) FTeiser, H. J., Jr., . ~ . ? - A L .CHEJI. 28, 477 (1956). (67) Restman, -4. E.R., Scott, A. E., Pedley, J. T., Cheniist,y in Can. 4,35 (1952). (68) +Kiame,J. >I , J . Bzol. Chem. 17, 919 (1949)
\ - - - - / .
(69) Wnteringham, F. P. IT., Harrison, A , , Bridges, R. G., Analysf 7 7 , l Q(1952). ( 7 0 ) Zettlemover, -4.C., Schneider, C. H., ’ Anderson, H , I-,, Fuchs, R. J.. J . Am. Chem. SOC.61, 991 (1957). RECEIVED for reviexv January 12, 1959. Accepted February 26, 1959.
Separation of Antibodies by Starch Zone Electrophoresis PETER STELOS‘ and W. H. TALIAFERRO Department o f Microbiology, University of Chicago, Chicago 37, 111.
b Rabbit antisera against sheep red cells were fractionated by starch zone electrophoresis to ascertain the distribution of antibody in the serum proteins and ascertain if the number of antibody species was dependent on the immunization schedule. In antisera obtained early in immunization the antibody activity migrated as a single peak between p- and y-globulins and sedimented with a globulin fraction of molecular weight about 1,000,000; late in immunization a second antibody migrated in the more slowly moving y-globulins and sedimented with a fraction of molecular weight about 160,000. Physically distinct antibodies of the same serological specificity may be induced by a given antigen and the ratio in which they appear i s a function of the immunization schedule.
Z
ELECTROPHORESI~ in a starchsupporting mcdiuni is usefiil in resolving a complex mixture of proteins into its individual components. The chief advantages of the method over other preparative electrophoretic methods are: the relatively large amounts of material that can be separated in a single operation, and the comparative ease and simplicity of the experimental procedure involved. As with all electrophoretic methods, the degree of resolution attainable depends on the nature of the proteins involved and, particularly, the net electrical charge that these bear in a buffer of given p H and ionic
strength. Since its introduction by Kunkel and Slater ( 4 ) to study the lipoproteins of blood qeruni. the method has been used in the analysis of rirus inhibitors ( I O ) , bacterial enzymes ( 6 ) , antibodies against diphtheria toxoid @), serum proteins ( I ) , and many others. The work reported here involved the characterization of rabbit antibodies against sheep rcd blood cells formed after inimuniaation with sheep red cells
or other material such as guinea pig kidnry. These antibodies, hemolysins, have the property of lysing sheep red cells when mixed Ivith complement. a normal serum component. Hemolysins are particularly useful in immunological studies, because the amount of hemoglobin released by the lysed red cells can be measured aecuratelJ- and conveniently by photometric methods. I n general, hemolysinf of two types occur; one has a molecular weight of
-300
osc
Present address, Sterling Chemistry Laboratory, Yale University, New Haven, Conn.
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VOL. 3 1 . NO. 5, M A Y 1959
845
about 1,000,000and is highly hemolytic, whereas the other is a smaller globulin of ordinary size (about 160,000) and is poorly hemolytic. The latter is often measured by its ability to combine with the red cell, as expressed in 50% combining units (see Figure 3). The purpose of this work was twofold: to ascertain the electrophoretic distribution of antibody activity in the serum proteins, and to ascertain whether antisera obtained during the early and late periods of immunization contained the same number of antibody species. The results illustrate the applicability of starch zone electrophoresis to a particular problem. It is hoped, however, that they also illustrate the generality of the method to problems involving the separation of biologically active substances in complex mixtures. RESULTS AND DISCUSSION
The procedures used in preparing early and late rabbit antisera against various antigens and the measurement of antibody activity have been described (7-Q). The lytic activity of antibody is defined in terms of 50% hemolysin units. One 50% hemolysin unit is that amount of serum or serum fraction which, in the presence of four 50% guinea pig complement units, will lyse 50% of a standardized number of red cells in 30 minutes a t 37" C. All starch zone electrophoresis experiments were performed in pH 8.6 veronal buffer, 0.1 ionic strength, The apparatus used differs only in minor respects from that described by Kunkel and Slater (3, 4). I n a typical experiment, about 2 ml. of undiluted serum were mixed with dry potato starch to form a thin slurry and mere carefully poured into a rectangular slit cut transversely 8 cm. from one end of a starch block 46 X 18 cm. in area and about 0.5 cm. thick. The starch block was equilibrated with buffer for about 2 hours a t 5" C., after which an electromotive force of 300 volts was applied for 48 hours. At the end of electrophoresis the starch block was allowed to dry partially and was partitioned into 1-cm. segments. Each segment was eluted Lvith veronal buffer and the eluates were tested for protein content by Lowry et al. (6) method and for antibody activity. Protein recovery ranged from 90 to 102% of the starting material, and antibody recovery from 70 t o 95%. Figure 1 shows the results with a normal rabbit serum. As compared to moving boundary electrophoresis of the same serum, the starch method yielded a greater number of subfrnctions and higher percentage values for y-globulin and lower ones for albumin. The first difference is undoubtedly a result of the longer migration path of the starch block as compared t o the path length of the Tiselius cell. The second difference 846
ANALYTICAL CHEMISTRY
Figure 2. Starch zone electrophoresis of an antiserum against sheep red cells obtained during early stage of immunization Antibody activity represented by a single peak centered between
8- and
y-globulin fractions (7)
x
t: 100. 5 C.
3,
Figure 3. Starch zone electrophoresis of antiserum against sheep red cells obtained during late stage of immunization Two peaks of antibody activity. Curve marked combining units parallels distribution of second antibody and indicates that two antibodies are serologically very closely related ( 9 )
activity was found in only a small fraction of the total protein in spite of the great increase of the yglobulin fraction. This conclusion was further supported by the observation that absorption with specific antigen removed only about O.2Y0of the total protein ( 7 ) . During the late stage of immunization against sheep red cells, the electrophoretic distribution of antibody activity differs markedly from that observed in the early stage (Figure 3). The most striking feature is the appearance of a second antibody in the more slowly migrating yglobulins. The tn-o antibodies are serologically very closely related, as evidenced by the ability of the second antibody to inhibit the reaction of antigen with the first antibody. This relationship is shoan in Figure 3 by the curve of antibody activity measured in combining units. In Table I are summarized the properties of the two antibody fractions which
reflects the fact that all serum proteins do not contain the same number of tyrosine and tryptophan residues and hence would not be expected to give equal color reactions 1%-iththe phenol reagent used in determining the amount of protein. Figure 2 illustrates the distribution of antibody activity in a rabbit antiserum obtained during the early stage of immunization against sheep red cells. The salient features are: the single, symmetrical peak of antibody activity which was distributed betn een the pand 7-globulins, and the peak of nntibody activity which was not associated with a corresponding peak of protein concentration. The first point is significant because it demonstrates that a n antibody may be distributed in more than one globulin fraction simply because its electrophoretic distribution overlaps neighboring fractions. The second point shows that the antibody
Anti
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Antibody Components Faster Slower migrating migrating
Property Appearance in immunization Early Late Electrophoretic -2 1p -1.2p mobilitya Approximate sedimentation constantb 18 S 6s a In pH 8.6 veronal buffer, 0.1 ionic strength. Mobility, p, expressed in units of p X lOssq. em. volt-' set.-'. b In pH 8.6 veronal buffer, 0.1 ionic strength. Fractions adjusted to contain about 1% protein. were further characterized by moving boundary elertrophoresis and ultracentrifugal analysis. It is possihle to increase the rcsolving power of the electrophoretic method by using it m-ith another differential migration method. Thus electrophoretic separation of fractions o h tained by preliminary high-speed centrifugation can furnish information about the relative size of two or more different proteins. Figure 4 presents some results obtained with late antisheep cell sera produced by immunizing vith guinea pig kidney antigen and human Type A red blood cells (8). Each antiserum was diluted with a n equal volume of pH 8.6 veronal buffer and subjected to an average centrifugal force of 105,000 X g for 210 minutes. Each 11-ml. centrifuge tube mas then partitioned into top (5-ml.) middle (5-m1.), and bottom (1-ml.) fractions. The three fractions nere adjusted to the same protein concentration and were simultaneously subjected to electrophoresis in a starch block divided into three compartments.
0 10
Table I. Some Properties of Rabbit Antibodies against Sheep Red Cells
I
I
Figure 4. Starch zone electrophoresis of antisera formed after immunizing with guinea pig kidney (anti-GKP) and human Type A red blood cells (anti-A) obtained during late stage of immunization Antisera first fractionoted by high-speed centrifugation and three centrifugal fractions simultaneously Separated in same starch black. Large and small antibody present in anti-GPK and only large antibody In anti-A (8)
T o conserve space, only the globulin portions of interest are shown in Figure 4. I n antiserum produced by immunizing with guinea pig kidney antigen, two antibody peaks may be seen. The faster moving antibody is clearly associated with a fraction of large molecular weight as evidenced by the progressively greater increase in antibody activity per microgram of protein nitrogen from top to bottom fractions (0.3 t o 10.0). The more slowly moving antibody is similarly observed to be associated with a smaller protein, as the ratio of antibody activity per microgram of protein nitrogen was about the same in all three fractions. On the other hand, the antibody activity of human antiserum appears to be associated solely with the fraction of large molecular weight. The immunological significance of these findings is not discussed here; they may be taken as illustrations of the VOL. 31, NO. 5, MAY 1959
847
type of information that can hc obtained by a r e l a t i d y simple and useful electrophoretic method. LITERATURE CITED
\‘”“7,.
(3) Kunkel, H. G., in “Methods of Biochemical Analysin,” D. Qlick, ed., Vol.
Separations
by
I, pp. 141-70, Interscience, New York, 1954. (4) Kunkel, H. G., Slater, R. J., Proc. SOC.Ezptl. Bid. & Med. SO. 4 2 4 (1952). (5) Lowry, 0. H., Rosehrough, N. J., Farr, A. L., Randall, R. J., J . Bid. Chen. 193, 265-75 (1951). (6) Rotman, B., Spiegelman, S., J . Baol v i o l . 68,419-29 (1954). (7) Stelos, P., J . Immunol. 77, 396-404 (1956). (8) Stelos, P., Taliaferro, W. H., J . Infectious Diseases 104, 105-18 (1959).
(9) Stelos, P., Talmage, D. W., Ibid., 100,12635 (1957). (10) Tamm, I., Tyrell, D. A,, J . Immunol. 72,424432 (1954).
tract AT(l1-lj-175 between the U. S. Atomic Energy Commission and the University of Chicago and aided by grants from the Dr. Wallace C. and Clara A. Ahhot Memorial Fund of the TJniversitv of Chicago.
Continuous Electrochromatography
ARTHUR KARLER Karler laborofories, Berkeley, Calif.
b Electrochromatography (continuousflow curtain electrophoresis) is a comprehensive method in the analysis and preparative fractionation of crude labile biological materials and other complex systems. The operating principles, illustrations of typical experiments, and a discussion of some special problems emphasize its special properties. Particular emphasis is placed on the individuality of a given sample pattern and its value in correlation with special biological activities and other properties of the sample or feed stock; such a correlation ensures quality control in large scale industrial processing.
T
HE over-all operating principles and methodology of electrochromatography have been discussed by many investigators, including Grassmsnn and Hannig (d), Durruni (S), Strain (fg), and Karler (6‘). Although “electrochromatography” i s used throughout this paper, the longer eltpression “continuous-flow curtain electrophoresis” i s more accurate; “electrochromatography” is shorter and emphasizes the fact that chromatographic or surface forces are always operative in the curtain or stabilized medinm. Electrochromatographic methods thus far drveloped are embodied in a variety of instruments consisting of a filter p8,pe.r curtain or a packed cell, down which a fluid medium or background electrolyte is continuously fed and across which a continuous direct rlcctricd current is passed; thus, we have an experimental situation in which the electrophoretic forces (electrical current flow or voltage gradient) act a t right angles to the chromatographic or adsorptive forces (fluid flow). If a
848
ANALYTICAL CHEMISTRY
molecule or particle is now introduced in the upper part of this curtain Sr ccll, i t will move down the curtain tr-ith a definite reproducible angle under the influence of these two forces and will come off the drip points or take-offs provided a t the bottom. Under proper conditions, different compounds will move through the curtain or cell a t different angles, depending on their respective over-all physicochemical characteristics; thus, each substance will build up its own path and come off free of the other constituents in the original sample. The various constituent.s or fractions are collected, and the individnal fractions can he put through this apparatus again (recycled) for further purification, or subjected to other physical, chemical, biological, or clinical methods of study. I n contrast to analytical electrcchromatography, continuous electrochromatography involves the continuous addition of samples, the establishment of E stable unchanging pattern through the cell or curtain, and its maintenance throughout the course of fractionation. This stable pattern or “spectrum” presented by a given sample under B specific set of experimental conditions i s distinctively individnalistic for each sample preparation; Figure 1 illustrates such a pattern as obtained with the biological stain acid fuchsin. Any change in over-all physicochemical propertics of the samplc or in the experimental conditions will be ultimately reflected in changes in the specific fractionating pattern estahlished in the curtain or cell. Shifts or changes in an established pattcrn will of course detract from the efficiency or resolution of the frsctionation process. The individuality and stability of a specific fractionating pa& tern are the key to the full realization of the potential of continuous electro-
Figure 1. Stable fractionating pattern or “spectrum” of componenk produced b y continuous electrochromatogrophy of acid fuchsin stain using a modern preparative apparatus
chromatography as a research and conimercial processing operation. Some of the preparative aspects of continuous electrochromatography may be illustrated by an early experiment involving the fractionation of a whole crude bacterial homogenate with the simultaneous recovery of no less than six biologically active components (enzymes). The over-all fractionating pattern in SO far as the protein distribution on the curtain is concerned is illustrated in Figure 2. The complexity of the sample provides an overlapping, irregular continuum of the many protein as well as other constituents exiyting in this protoplasmic system. A summary of the analytical and a m y results (Figure 3), however, emphasizes the fractionating effectiveness of continuous electrochromatography. The simultaneous isolation with high yields
