the order of emergence from a cation exchange column is nearly the same when the developing agent is a strong acid. Here virtually all of the amino acids in solution and on the resin possess a positive charge and therefore move d o m the column mainly in that form. However, in the case of two amino acids held to a sulfonic acid resin, all else equal, a competing hydrogen ion will have a better chance of dislodging the amino acid that has the greatest tendency to become neutral by losing a proton from its own carboxyl group. It is not clear why the effect of cyclic or aromatic structure is so much greater with this type of development than with the other two. On columns developed Kith hydrochloric acid (IO,31) phenylalanine follows not only the neutral amino acids but the basic ones as well. ACKNOWLEDGMENT
The author is most grateful to R. T. hlarkiw for performing the chromatographic separations and to the U. S. Public Health Service for partial financial support. LITERATURE CITED
(1) Block,
R. J., “Ion Exchange” (Nachod, F. C., ed.), p. 303, Academic Press, Yew York, 1949.
(2) Block, R. J., Bolling, D., “Amino Acid
Composition of Proteins and Foods,” p. 375, Charles C Thomas, Springfield, Ill., 1951. (3) Brimley, R. C., Barrett, F. C., “Practical Chromatography,” pp. 22-3, Reinhold, ?Jew York, 1953. (4) Buchanan, D. L., ANAL. CHEM.29, 1877 (1957). (5) Buchanan, D. L., J. Biol. Chem. 229, 211 (1957). (6) Cassidy, H. G;: “Fundamentals of Chromatography, pp. 14, 246, Interscience, New York, 1957. (7) Davies, C. W., Biochem. J . 45, 38 (1949). (8) Hale, D. K., “Ion Exchangers in
Organic and Biochemistry” (Calmon, C., Kressman, T. R. E., eds.), p. 171, Interscience, New York, 1957. (9) Hall, D. A., Tiselius, A., Acta Chem. Scand. 5, 854 (1951). (10) Him, C. H. W., Moore, S., Stein, W. H., J . Am. Chem. SOC.76, 6063 (1954). (11) Hirs, C. H. W.,Moore, S., Stein, IT.H., J . Biol. Chem. 195, 669 (1952). (12) Holman, R. T., J . Am. Chem. SOC.73, 1261 (1951). (13) Kitchener, J. A4.,“Ion Exchangers
In Organic and Biochemistry” (Calmon, C., Kressman, T. R. E., eds.), p. 63, Interscience, Yew York, 1957. (14) Kunin, R., “Ion Exchange Resins,” 2nd ed., p. 115, Wiley, New York, 1958. (15) Kurtz, F. E., J . Am. Chem. Soc. 74, 1902 (1952). (16) Ledere;: E., Lederer, bI., “Chromatography, pp. 5, 56, 190, Elsevier, Amsterdam, 1955. (17) Li, C. H., Ash, L., Papkoff, J., J . Am. Chem. Soc. 74, 1923 (1952).
(18) Li, C. H., Tiselius, A., PederEen, K.
O., Hagdahl, L., Carstensen, H., J . B i d . Chem. 190, 317 (1951). (19) Moore, S., Stein, It7. H., Ibid., 192, 663 (1951). (20) Partridge, S. M.,Biochem. J . 44, 521 (1949). (21) Zbid., 45,459 (1949). (22) Partridge, S. N., Brimley, R. C., Zbid., 44, 513 (1949). (23) Zbid., 48,313 (1951). (24) Zbid., 49,153 (1951). (25) Zbid., 51, 628 (1952). (26) Partridge, S. hl., Brimley, R. C., Pepper, K. IT7., Ibid., 46, 334 (1950). (27) Partridge, S. M., Westall, R. G., Zbid., 44, 418 (1949). (28) Porath, J., Acta Chem. Scand. 6, 1237 (1952). (29) Reichenberg, D., “Ion Exchangers in Organic and Biochemistry” (Calmon, C., Kressman, T. R. E., eds.), pp. 89, 92, 94, Interscience, New York, 1957. (30) Samuelson, O., “Ion Exchangers in Analytical Chemistry,” p. 61, Wiley, New York, 1953. (31) Stein, W.H., Moore, S., Cold Spring Harbor Symposia Quant. Bid. 14, 179 (1949). (32) Tiselius, A, Advances in Protein Chem. 3, 67-93 (1947). (33) Tiselius, A., Arkiv Kemi Mineral. GeoZ. 16A, KO. 18 (1943). (34) Tiselius, *4., Hagdahl, L., Acta Chenz. Scand. 4,394 (1950).
RECEIVEDfor review December 1, 1958. Accepted February 18, 1959.
Isolation of Cytoplasmic Particulates from Animal Tissues by Density-Gradient Centrifugation JOHN F. THOMSON Division of Biological and Medical Research, Argonne National laboratory, lemont, 111.
+
Density-gradient centrifugation has been used to study the distribution of enzymes and other components of mammalian tissues. The technique permits the construction of curves relating intracellular distribution to particle size, and is particularly useful in comparing normal and abnormal tissues.
D
centrifugation (6) has been widely used during the past decade to obtain subcellular fractions of cells in sufficient quantity for chemical analysis and enzyme assay. A piece of tissue is homogenized in a suitable dispersing medium, and then centrifuged several times a t progressively increasing speeds to yield fractions containing principally nuclei, mitochondria, and microsomes. However, it is virtually impossible to prepare these subcellular fractions in a sufficient homogeneous
836
IFFERENTIAL
ANALYTICAL CHEMISTRY
state, and many techniques of subfractionation have been devised. Because the rate of sedimentation of the particles is a function of their size, a much better separation can be made by layering the homogenate over a column of liquid of slightly higher density than would be possible by conventional methods of differential centrifugation. The use of a density gradient to stabilize the boundaries of the sedimenting material is an essential part of this procedure. Anderson has discussed in considerable detail both the historical development and the principles of density-gradient centrifugation, or “gradient differential Centrifugation,” as he prefers to call it ( I ) . I n the work described here, the density of the particles was greater than the maximum density of the gradient column. If the maximum density of
the column is greater than that of any of the particles, after sufficiently long centrifugation a t high speed the particles will reach an equilibrium position in the tube where their density equals that of the suspending medium. This principle has been used to separate epinephrine-containing particles of the adrenal medulla from mitochondria (2); the two types of particles are similar in volume but differ in density. Nost of this work has been carried out with gradients prepared by successiyely layering 9-ml. portions of 30, 25, 20, 15, and 10% sucrose solutions into a 50-ml. plastic centrifuge tube (inside diameter 2.8 em.). Each solution contained 0.0003.Il disodium (ethylenedinitri1o)tetraacetate, p H 7.4, to decrease the possibility of agglutination by calcium ions. The tube mas stored a t 1’ C., usually for 48 hours; during this time
there was sufficient diffusion between layers to form a continuous gradient. Actually any time between 24 and 120 hours seemed to be satisfactory. The details of preparation and layering of the tissue homogenate, centrifugation, removal of layers, and assays for various cellular components have been described
(8,11). I n more recent work ( l o ) , solutions of sucrose plus polyvinylpyrrolidone (PVP) have been used. The sucrose concentration was kept constant at 0.25M, n-hile the polyvinylpyrrolidone concentration was varied from 6 to 18%. Because of its high molecular n-eight, polyvinylpyrrolidone does not appreciably alter the osmotic pressure of the medium. It has been recommended for use in preparation of cellular particulates for electron microscopy. In addition, extraction of enzymes from particulates is minimized by the use of polyvinylpyrrolidone. It has some disadvantages, however, in that assays for total protein are complicated by its presence, and its high viscosity necessitates the use of much higher centrifugal forces (20,000 X g instead of 2000 X g) to achieve the same rate of sedimentation observed in the sucrose gradient. Calculation of Particle Sizes. The rate a t which a particle sediments in a centrifugal field is given by:
Figure 1 . Effect of 600 r total-body x-radiation on intracellular distribution of cytochrome oxidase in rat thymus Rats sacrificed 1, 2, and 3 days after irradiation. Fractionation carried out using sucrose-PVP gradients
3.0
C Ad
2.0
I .o
0
0
+ a(L - Lo) (2) = + P(L - Lo) + r(L + S(L - (3) = P: + e(L - Lo) (4) d = d o [ l + T(L - Lo) + S(L - Lo)'] (5) Ps = pa
?I
'lo
Lo)2
La)3
dL - = dt
This equation was integrated graphically, and for various combinations of w and t the values of do were obtained for the 12 values of L chosen, the distances from the axis of centrifugation t o the boundaries of the zones. W h e n p o 1y v i n yl p y r r o lid o n e a a s used to produce the gradient, the changes in osmotic pressure were negligible; further, the change in viscosity could be described adequately by a binomial rather than a trinomial. Thus Equation 6 siniplifies to :
0.4
0.3
0.5
0.6
(p)
sumption is not completely warranted, as mitochondria are generally somewhat elongated. The departure from sphericity, horrever, is not so great that the error involved exceeds 5%. Particle sizes are presented as the equivalent spherical diameters-Le., the diameter of a sphere which would have sedimentation characteristics identical with those of the particles actually studied. On the basis of the sizes of particles predicted in each zone under the conditions employed, and of the concentration of a cellular component in each zone (after correction for side-wall sedimentation ( l l ) , one can construct curves relating the distribution of that component to the size of particles with which it is associated ( 5 ) . RESULTS
dL _ -dt
+ a(L + r ( L - .Lo)*
1 dZw*L(pp - [ P o -~ 18 7 0 P(L - Lo)
+
L0)21)
(7)
which is readily integrated to give:
Pp
where po, no, p i , and do are the densityof the medium, viscosity of the medium, density of the particle, and diameter of the particle a t a distance Lo from the axis of centrifugation t o the meniscus. The coefficients in Equations 2 and 3 were obtained from measurements of the viscosities and densities of sucrose solutions a t '1 C.; those for Equations 4 and 5 were estimated from data presented by Tedeschi and Harris (7). Substitution into Equation 1 gives:
0.2
DIAMETER
(6)
where d is the particle diameter, ~3 is the angular velocity, L is the distance from the axis of centrifugation to the particle, p p is the density of the particle, and p s and 9 are the density and viscosity of the suspending medium. I n the presence of a concentration gradient, however, empirical correction factors must be introduced to correct for the changes in density and viscosity of the medium, and for the effect of changing osmotic pressure on the particle density and diameter. Thus;
0.1
All the constants fAr evaluation of Equations 6 and 8 can be obtained with good accuracy except for the density of the particles. This problem is discussed elsewhere (8, 10, 11). In this treatment it is assumed that the particles are spherical. This as-
Observations on particulates of mammalian liver have been described in considerable detail (4, 8-15). The results may be summarized as follows. Distribution curves of various enzymes in rat liver are of a t least four types. The first, typified by succinic dehydrogenase and cytochrome oxidase, shows maximum activity a t an equivalent spherical diameter of 0.75 micron (mitochondria). The second, of which esterase is characteristic, is predominantly associated with particulates of about 0.12 micron diameter (microsomes). Bimodal distribution curves mere obtained for diphosphopyridine nucleotidecytochrome c reductase, arginase, and adenosine triphosphatase, the two maxima occurring a t 0.12 and 0.75 micron. Finally, uricase and catalase are associated with particles of 0.44 micron diameter; this cytoVOL. 3 1 , NO. 5, MAY 1 9 5 9
837
plasmic component has not been adequately characterized cytologically. Whether it is identical with the “lysosome” fraction (3) is not certain; the distribution curve for acid phosphatase is similar to that of uricase (12) but not identical. The principal usefulness of this method has been the comparison of tissue of normal animals with those that have been treated in various ways. Thus enzyme distributions in livers of rats have been studied after partial hepatectomy (11) or carbon tetrachloride treatment ( I S ), livers of hypoxic guinea pigs (C), and livers of tumor-bearing mice (9). As an example of the applicability to other tissues, Figure 1 shows the distribution of cytochrome oxidase in the thymus of normal rats and rats sacrificed 1, 2, and 3 days after exposure to 600 r totalbody x-radiation. The size associated with maximum activity was decreased slightly but consistently in the irradiated tissue, and the shape of the distribution curve was markedly altered. The mechanism by which x-radiation produces this change is not altogether clear. DISCUSSION
Density-gradient centrifugation has provided a method for estimating the intracellular distribution of enzymes and other cellular components in terms
of the size of the cytoplasmic particulates with which they are associated. Such a procedure obviates the necessity of assigning morphologic labels to subcellular fractions of known heterogeneity. The method has certain drawbacks. Only a limited amount of material can be handled in one centrifuge tube; the author has used homogenategradient volume ratios of 1 to 10 or 1 to 12. It is obvious that the lower the ratio the better the resolution will be; and the smaller the amount of material available for analysis. The necessity for making certain assumptions about osmotic pressure gradients, sphericity of the particulates, particle density, and side-wall sedimentation has been mentioned. It is clear, also, that the homogenization process common to all differential centrifugation techniques, involving the disruption of cells by shearing and the liberation of cellular components into solutions of sucrose, is decidedly unphysiological. The author has used the procedure to measure the sizes of polystyrene latex spheres of 0.5 to 3.0 micron diameter. These experiments, carried out in collaboration with H. E. Kubitschek of this laboratory, will be reported elsewhere; very close agreement has been obtained between sizes of particles estimated by this procedure with those measured optically.
LITERATURE CITED
(1) hnderson, N. G., in Oster, G. and Pollister, A. W. (eds.), “Physical !l!echniques in Biological Research,” Vol. 111, pp. 300-53, Academic Press, New York, 1956. (2) Blaschko, H., Hagen, J. M., Hagen, P., J . Physiol. ( L o n d o n ) 139, 316 (1957). (3) DeDuve, C., Pressman, B. C., Gianetto, R., Wattiaux, R., Appelmans, F., Biochem. J . 60, 60.4 (1955). (4) Klein, P. D., Thomson, J. F., Am. J . Physiol. 187, 259 (1956). (5) Nichols, J. B., Bailey, E. D., in Weissberger, A. (ed.), “Physical Methods of Organic Chemistry,’] 2nd ed., Part I, pp. 676-9, Interscience, New York, 1959. (6) Schneider, W.C., J . Biol. Chem. 176, 259 (1948). (7) Tedeschi, H., Harris, D. L. , Arch. Biochem. Biophys. 58, 52 (1955). (8) Thomson, J. F., Klipfel, F. J., Zbid., 70, 487 (1957). (9) Thomson, J. F., Klipfel, F. J., Cancer Research 18, 229 (1958). 10) Thomson, J. F., Klipfel, F. J., Erptl. Cell Research 14, 612 (1958). (11) Thomson, J. F., Mikuta, E. T., Arch. Biochem. Biophys. 51, 487 (1954). (12) Thomson, J. F., Moss, E. M., Zbid., 61,456 (1956). (13) Thomson. J. F.. Moss, E. ?VI.,Cancer ’ 8; 789 (1955). ’ RECEIVED for review December 29, 1958. Accepted February 5, 1959. Based on material presented before the Division of Analytical Chemistry, Symposium on Separation Processes through Differential Migration Analysis, 134th Meeting, ACS, Chicago, Ill., September 1958. Work performed under the auspices of the U.S. Atomic Energy Commission.
Isolation of Carotenoids, Coumestrol, Chlorogenic Acid, and Antibiotics Application of Countercurrent Distribution C. R. THOMPSON, A. L. CURL, and E. M. BICKOFF Western Regional Research laboratory, Albany 7 0, Calif.
b The application of countercurrent distribution technique to biologically active materials is reviewed briefly and examples showing its uses are given. These include carotenoids, isolated from oranges; a plant estrogen, coumestrol, isolated from Ladino clover; an isomer of chlorogenic acid from peaches; and the antibiotics, aterrimins, from Bacillus subtilis var. aterrimus.
T
isolation and identification of small amounts of biologically active constituents from plant or animal mateHE
838
ANALYTICAL CHEMISTRY
rials by the classic methods of extraction, precipitation, and crystallization are often next to impossible. Chromatography, or selective adsorption in the m-ider sense-column, paper, ion exchange-has served admirably with some substances, but the separation of similar entities by these techniques is often unsuccessful. Countercurrent distribution (CCD) may often accomplish the separation and, by prior knowledge of the different components’ behavior, also serve to identify each. This operation has been practical only recently, since the development of integrated and automatic
equipment for multiple transfers. Hundreds or even thousands of individual equilibrations are now possible within a few hours or days. This makes the high resolving power of the method usable for many separations and analyses. Craig and Craig (4)have been largely responsible for much of the development in this field. Compounds that have been resolved by countercurrent distribution include various glycerides in natural fats, steroid hormones, amino acids, protein fragments, organic acids, dyes, and chemical intermediates (10).
