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
of at lcnst six semipurified labile entities such as enzymes directly from a very crude preparation illustrates some of the preparative advantages of electrochromatography. I n addition, most complex preparations snch as this one present higlil?- colored hands in the pattern which immediately characterize that prepar:ition and facilitate checking the position and stability chxracteristics of the fractionating pxttern during the experimcnt. WORK WITH PLAGUE TOXIN
One of thc biological prcparations most thoroughl>-studied has heen crude and pnrificd plague toxin. Ajl and coworkers ( 1 ) first explored the possibility of preparative fractionation of plagiir toxin. Later studies hy Ajl and coworkers (Z) and by Spivack and Karler ( 1 1 ) represented a.n attempt to use continuous electrochromatography inore comprehensively as a single method for larger scale prrparations
pigment. As the work with various preparations progressed, this decrease in pigmentation Kas found to be an accurate measure of the degree of purification which had been effected with cach passage of plague toxin through the curtain. It was thus possible t.o minimize the use of expensive, time-consuming biological assay techniques. It hecsmr possible hy mere visual ohservation of the frartionating pattern to predict the purification factors and the biological activities (within accepted expcrimental limits) a t every stage in t.he purification procedurc. The fractionating characteristics of highly purified plague toxin are illustrated in Figure 5. This phase in purification was achieved just at that point
with higher rrcoverics. The work of Spivack and Karler is reviewed to illustrate several other features of electrochromatography. As with the crude bacterial homogenate descrihed above, the crude plague toxin preparations presented a wide spectrum of proteins and other constituents. Figurc 4 illustrat,es thin heterogeneity and localizes the position of all the protcin which is plague toxin. I n thPx crude prrparations a daylight visible yellow eomponcnt which gave a yellow fluorescence with an ultraviolct light was invariably found associated ITith the peak of the plagne toxin. The routine fractionation procedure involvrd pooling of the three fractions containing the bulk of the plague toxin and recycling this pool under the same or differcnt fractionating conditions until a toxin of required chemical, immunochemical, and biochemical purity was obtained. With each recycle of the toxin there was a corrcsponding diminution of the yellow
Figure 2. Fractionating pattern of the whole homogenate of Pseudomonas Ruarescens Arepresentative iomple w o i left
on the Curtoin and rtoined for protein using bromophenol blue
Figure 5. The fractionating properties of highly purified plague toxin (P. pestis) after four posses through a preparative electrochromatogrophic apparatus 6
M
)
,
,
,
I
I
I
I
I
I
I
'
PURIFICATION OF PLAGUETOXIN IF! pestis) First Cycle
O13 l DRIP POINT
Figure 3. Electrochromatographic distribution of six enzymes from the whole homogenate of Pseudomonos fiuorescenr
~ I5
~ 17 ~
- Lot
105 + 1%.
"
F)
DRIP POINT
" 21 '
-
"
" " 25 23
Figure 4. Distribution pattern of plague toxin ond associated proteins obtained from the first pass (or cycle) of crude plague toxin (P. pestis) through a preporative electrochromatographic apparatus VOL. 31,
NO. 5,
MAY 1959
849
Figure 6. Untreated curtain obtained by the electrochromatography of partially purified tetanus toxoid Irregularity of dark bond is a result of intermittent interference by a trace gellike Conltuent
when the yellow contaminating pigment had been completely removed from the observable fractionating pattern of the curtain. This early correlation of a physical fractionating pattern with biological activity and chemical and physical properties has proved to be a common characteristic in the electrochromatographic analysis and proces+ ing of nearly all complex samples. I n those few cases where natural pigments or other indicator compounds have been absent or nonspecific, some noninterfering harmless dye or other indicator may often be added to the system; it i s then possible to establish an artificial but valid physical picture for evaluating the stability and general operating characteristics of the electrochromatographic pattern and for correlating that physical pattern with the purification process as described above for plague toxin. TECHNIQUE
OF
the formation of the various components or bands and their crossing over to assume their expected, normal positions on the curtain. This technique works for other complex, crude preparations (such as whole homogenized red blood cells) during their initial passage through a preparative electrochromatographic apparatus. Attempts to extend it to semipurified and purified fractions, as might be done in recycling, thus far have not proved successful. Although the exact mechanism involved in the successful overfeeding of crude preparations has not been ascertained, the technique has frequently proved a convenient way to increase the throughput capacity of an electrochromatographic instrument. This has been especially helpful where the nature of the starting samples has necessitated repeated recycling of a pertinent component.
OVERFEEDING
I n addition to correlation of the fractionating patterns with biological activitirs, the work with plague toxin resulted in the development of the technique of overfeeding of a crude preparation in order to increase the throughput capacity of a given experimental system appreciably with nomina1 loss in over-all resolving power. With the whole crude plague toxin preparation it was possible to increase the apparent optimal rate of sample addition of 5 ml. per hour to 15 ml.per hour with approximately a 25% loss in resolving power. With this increase in the rate of eample addition the comparatively narrow area of application becomes three or four times as broad, thus forming a large elliptically shaped area or “blob” within which it is possible to observe 850
Figure 7. Untreated curtain obtained with tetanus toxoid after removal of interfering gel constituent [cf. Figure 6)
ANALYTICAL CHEMISTRY
WORK WITH TOXOIDS
Work with toxoids and toxins as a group has provided much information which has helped develop further the methodology of continuous electroChromatography. Work with tetanus toxoid preparations provides useful illustrations of the difficulties that may be encountered in this type of fractionation. Figure 6 is a phot.ograph of a partially purified tetanus toxoid preparation having a gray color. The irregular pigmented band is illustrative of the interference by trace gel constituents frequently found in crude biological preparations. Their effect upon the fractionating pattern may be a simple physical one, in which the gel either increases, continuously carrying with it increasing amounts of the sample,
or builds up intermittently with subsequent movement down the curtain in the direction of the general pattern. The latter case is illustrated by this tetanus toxoid preparation. Frequently these gel constituents can be removed by centrifugation or by more careful and complete dissolution of the crude preparation. The result of such a successful removal from this tetanus toxoid preparation is illustrated in Figure 7. Subsequent analysis showed that the experiment was unsuccessful, in that there was no rqal fractionation of the protein, the toxoid, and the contaminating gray pigment. Examinntion of this starting sample showed it was composed of not less than 30% of a buffer salt. This order of concentration of a given ion or salt may sometimes interfere seriously with the fractionation mechanism of continuous electrochromatography. The simplest way to handle such a possibility was to dialyze away the bulk of the buffer impurity and repeat bhe fractionation without further experimental changes. The result of this huffer removal is illustrated in Figure 8: The analytical data describing the fra.ctions obtained from this curtain are summarized in Figure 9. The contaminating pigment has now been removed from the bulk of the protein and the toxoid protein has been coming off at the other side of the protein spectrum. This extreme type of interference by one or more associated small ions or salts bas usually proved to be minimal, or a t least less extensive than was the case with this partially purified toxoid preparation. Normally the electrochromatograpbic process automatically effects the necessary desalting of the sample shortly after application to
30,
,
, ,
,
,
,
,
,
,
TETANUS TOXOID FRACTIONATWN
Lf mik
Pwity I-
b
v)
t
> z
" Y
P Y
-PVm
*
L
*R P *4 *A
L I I 1 1
d 0 r M mhrbl
Figure 9, Distribution of protein (nitrogen), toxoid activity (lf units), and purity (ratioof Lf to protein) obtained from curtain illustrated in Figure 8
Figure 8. Untreated curtain obtained with tetanus toxoia after removal of both interfering gel constituent and contaminating buffer salt (cf. Figures 6 and 7)
'
the curtain. Where desqlting is inadequate under a given set of experimental conditions, the sample may he dialyzed as descrihed above-a pr* cedure initially recommended by Durrum (3). Where dialysi? is inconvenient or intolrrable, the sample may still be processed, using more power, or the unresolved fractions may be recycled until true fractionation becomes operative. Presumably the explanation for such a breakdown in the fractionation process lies in the establishment of a limited region of high conductivity or very low voltage drop and hence little or no fractionation; perhaps it is best to visualize the movement of current across a region of minimal voltage drop -a region corresponding to the restricted position of the unfractionated sample band on the curtain. The work with toxoids is also illustrative of the preparative potential of continuous electrochromatography. Extrapolation of the 24hour preparative experiments upon which Figure 9 is hasrd shows that with suitable modification of the laboratory apparatus illustrated in Figure 1 it should be possible to process a 500-gram hat.ch of a crude toxoid or vaccine with unprecedented purification factors within 2 to 4 weeks. Although on a weight basis such an amount of sample may sccm nominal, the toxoid or vaccine procrssed may be equivalent to one-half billion units of biological activity. After making allowancrs for discarding 10 or 20% of the biological activity, to secure unusually pure toxoid or vaccine, and keeping in mind that only four or five units of activity may he required per dose of vaccine, it can he seen t,hat sufficient vaccine has been produced to vaccinate approximately 100,000,000 people. With this type of preparative
experiment we are only one step removed from routine, automatic continuous processing using large scale industrial types of fractionating equipment. The significance of the stable equilibrated pattern so necessary for successful continuous electrochromatography takes on added importance when the method is projected into full scale industrial production. Such stability provides inherent modern quality control of product, irrespective of the length of the processing period. This high degree of quality control should eliminate the variability found in customaQ7 hatch processing and minimize expensive, time-consuming assays. Unlimited processing rvith continuous, automat.ic quality control will almost certainly lead to large scale industrial usage of continuous electrochromatography. Nost of the fractionations thus far effected by continuous electrochromatography have been restricted to comparatively highly charged or ionic sample systems processed through a curtain washed with an ionic medium such as buffer solutions. Recently, however, Kirk (7) fractionated successfully cerhin type-specific antigens using distilled or redistilled water as background medium on a thoroughly washed filter paper curtain. Although Paul and Durrum (10) and McDonald and Willia,mson (9) have established the feasibility of one-dimensional (paper strip) electrophoretic separations of highly charged part,icles in low ionic systems, this preliminary work repre sents an extension of continuous electrochromatography to the fractionation of noncharged molecules or particles in a comparatively nonionic medium. Current work in this laboratory involves the fractionation of nonpolar samples in
nonpolar media mch as the hydrocarbons or esters. On the basis of such work done by Lipsky and Landowne (8) in the chromatographic fractionation of fatty acids with ester solvents and in view of the pioneering work of Haugaard and Kroner (6),who used standard paper chromatographic solvent systems with their electrochromatographic system, there are no a prwn' or theoretical reasons why continuous electrochromatography cannot he extended to hydrocarbon, lipide, and other nonpolar substances within a reasonable time. LITERATURE CITED
RECEIVED for review December 17, 1958. Accepted March 17, 1959. VOL. 31, NO. 5, MAY 1959
851
