Fractionation of mucopolysaccharides by countercurrent distribution in aqueous polymer two-phase systems

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Biochimica et Biophysica Acta, 404 ( 1 9 7 5 ) 2 8 9 - - 2 9 9 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 2 7 7 3 0

FRACTIONATION OF MUCOPOLYSACCHARIDES BY cOUNTERCURRENT DISTRIBUTION IN AQUEOUS POLYMER TWOPHASE SYSTEMS* D A V I D G. PRITCHARD**, R I C H A R D M. HALPERN, JAMES A. HALPERN, B A R B A R A C~ H A L P E R N and R O B E R T S A. SMITH

Department of Chemistry and the Molecular Biology Institute,The Universityof California at Los Angeles, Calif. 90024 (U.S.A.) ( R e c e i v e d J u n e 3rd, 1975)

Summary A new procedure for the fractionation of mucopolysaccharides based upon differences in their partition behavior in aqueous polymer two-phase systems has been devised. Systems containing dextran, poly(ethylene glycol), trimethylamino-poly(ethylene glycol), potassium bromide and sodium phosphate buffer were employed. Countercurrent distributions were performed with a miniature countercurrent distribution device designed especially for use with aqueous polymer two-phase systems. An advantage over the widely used procedures involving precipitation of rnucopolysaccharides as their quaternary a m m o n i u m detergent complexes is that the countercurrent distribution pattern of a particular mucopolysaccharide is not affected by the simultaneous presence of other mucopolysaccharides. Preliminary distributions of labelled mucopolysaccharides isolated from the cells and culture medium of monolayer cultures of rat tumor cells demonstrate that the procedure is particularly well suited for the fractionation of very minute quantities of mucopolysaccharides. Introduction The use of cell cultures has numerous advantages in studies of the metabolism and biological functions of the mucopolysaccharides. Cell culture flasks, however, contain only very small amounts of mucopolysaccharides and the efficient isolation and fractionation of the compounds present a considerable problem. The most widely employed procedures used for very minute amounts * Chemistry Department c o n t r i b u t i o n N o . 344"/. ** Current address: Department of Immunology, City of H o p e National Medical Center, Duarte, Calif. 91010.

290 of mucopolysaccharides involve precipitation of the compounds as their quaternary ammonium detergent complexes [1--3] w h i c h are usually adsorbed onto an inert support such as celite or cellulose, powder; then fractionally eluted with various salt solutions. Unfortunately, these procedures have several major disadvantages. There is always considerable overlap between the various fractions. The salt concentration at which a particular mucopolysaccharidedetergent complex redissolves often depends upon the type and amount of other mucopolysaccharides present. Aqueous polymer two phase systems containing poly(ethylene glycol) and dextran Were introduced by Albertsson [4] and have been employed successfully for the isolation and fractionation of small quantities of nucleic acids [5--7]. Previously we reported [8] that DNA prepared by an aqueous polymer two-phase method was contaminated with mucopolysaccharides, suggesting that aqueous polymer two-phase systems, with suitable modifications, might be useful for the fractionation of the different mucopolysaccharides. We describe a new procedure for the fractionation of minute quantities of mucopolysaccharides based upon differences in their partition behavior in aqueous polYmer two-phase systems. Materials and Methods Chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate and hyaluronic acid were obtained through Miles Laboratories, Elkhart, Indiana 46514. Heparan sulfate was a generous gift of Dr J.A. CifoneUi. Carrier free Na2 a s SO4 and D-[U -~ 4 C]glucosamine, D-[G-aH]glucosam ine and D-[6 -a H]glucosamine were obtained from International Chemical and Nuclear Corp. Hexadecylpyridinium chloride was obtained from Matheson, Coleman and Bell as was the carbazole, which was recrystallized from benzene before use. Celite Analytical Filter-Aid was obtained from Johns-Manville Co. and was exhaustively washed with 50% (v/v) sulfuric acid before use. Aquasol, a xylene-based liquid scintillation fluid, was purchased from New England Nuclear.

Biological Monolayer cell cultures of two rat tumor lines were employed; W12-4 cells, a malignant cell line originating from a primary culture of Walker-256 cancinosarcoma cells, and BslA cells, a very malignant single-cell clone originating from a Walker-256 carcinosarcoma metastisis. Modified McCoy's 5a medium [9] and a special sulfate-free medium containing 165 mg/l of MgCI~ • 6H~ O was purchased from Gibco. The final medium contained 15% fetal calf serum and 100 units of penicillin G per ml. Cell cultures were incubated at 37°C in a water-saturated atmosphere of 5% CO2 in air.

Radioactive labelling of mucopolysaccharides Radioactive mucopolysaccharides were prepared by allowing the cultured cells to grow for 24 h in the presence of either a s SO~- (25 pCi/ml) in regular

291 medium. Cell cultures were always labelled when they were approximately two days from confluence. At the end of the labelling period, the culture medium from each flask was d e c a n t e d and the cell monolayers were rinsed twice with cold 0.9% NaC1 solution. The saline rinses and the culture medium were pooled and added to four volumes of ice cold absolute ethanol. The cell layer was then lysed with 5 ml of 4% sodium dodecyl sulfate solution at 37°C for 1 h and then added to four volumes of ice cold absolute ethanol. Subsequently, cell and culture medium fractions were treated identically. After ethanol precipitation for at least 2 h in an ice bath, the precipitates were collected b y low speed centrifugation, then resuspended in absolute ethanol (25 ml) and once again collected b y a brief centrifugation. The precipitates were then extracted with 25 ml of chloroform followed b y two more ethanol extractions. Finally, they were suspended in 0.1 M Tris • HC1 buffer (usually 2 ml) and heated in a boiling water bath for 5 min. After cooling to r o o m temperature, pronase was added to the suspensions to give a final concentration of 1 mg/ml. After approximately 12 h at 37°C an additional 1 mg/ml of pronase was added to each sample and a b o u t 12 h later the tubes were cooled in an ice bath and ice-cold 30% (w/w) trichloroacetic acid was added to yield a final concentration of 7.5%. After 1 h at 4°C, the precipitates were removed by centrifugation. The supernatant solutions were dialyzed against five changes of distilled water. The retentate was lyophilized prior to use in the countercurre_lt distribution experiments.

Aqueous polymer two-phase systems Dextran T500 was obtained from Pharmacia Fine Chemicals, Inc. Poly(ethylene glycol) 6000 was purchased as 'carbowax 6000' from Union Carbide Chemicals Co. Trimethylamino-poly(ethylene glycol) was synthesized from poly(ethylene glycol) 6000 according to the procedure described by Johansson et al. [ 1 0 ] . The composition of the two-phase systems is expressed as a particular weight percentage of the aqueous polymers and a particular molarity of the electrolytes. All solutions containing dextran were stored frozen to prevent the growth of mold. Partition coefficients of mucopolysaccharides in various two-phase systems were determined using a novel microdistribution procedure. Aqueous polymer mixtures containing a particular mucopolysaccharide were placed in thin-walled transparent polystyrene tubes (4 mm × 160 mm) that had been heat-sealed at one end. After a brief centrifugation, the contents of the tubes were frozen in dry ice, and the tubes were cut at the interface to cleanly separate the t w o phases. Countercurrent distributions were carried out in a new t y p e of miniature countercurrent distribution device designed especially for use with aqueous polymer two-phase systems (Plate 1"). This new device was essentially a miniaturized version of the original stainless steel countercurrent distribution machine developed b y Craig and Post [ 1 1 ] . The advantages of this device are that it requires only a small volume of polymer mixture, and phase separations can * Engineering d r a w i n g s are available u p o n r e q u e s t f r o m D r R.A. S m i t h .

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P l a t e I . C o u n t e r c u r r e n t d i s t r i b u t i o n d e v i c e . A, The components; B , A s s e m b l e d .

be accomplished very quickly by briefly centrifuging the entire device. It was designed to fit into the large swinging buckets of a size 2, model V, International centrifuge. The miniature countercurrent distribution device was constructed from a solid lucite cylinder 6.35 c m in diameter. A circular arrangement of 18 holes (6.5 m m dia.) was bored into upper and lower sections of the device. The two sections were held together tightly be means of a stiff steel spring. The upper and lower sections could be rotated in opposite directions about a central stainless steel rod. Correct alignment of the sample holes in the two sections was achfeved by means of a spring-loaded register pin. A g u m rubber gasket attached to the lower section forms a leak-free seal at the junction of the two sections. A circular disk, also cut from a sheet of g u m rubber, was cemented to the stainless steel cap of the device. The sample wells in the lower section were

293 bored completely through the lucite cylinder and sealed with a gum-rubber covered steel baseplate that can be removed for cleaning. A single indexing line was engraved on the b o t t o m of the upper section and the sample wells of the lower section were numbered from 0 to 17. Nickel balls (4.5 mm diameter) are sometimes placed in every sample well to facilitate efficient mixing of the viscous polymer mixtures. Before loading the devices with the aqueous polymer mixtures, all gum rubber surfaces are lubricated with a thin layer of D o w Coming High Vacuum Silicone Grease. The two-phase polymer system chosen for a particular countercurrent distribution run was thoroughly mixed before being added to all 18 sample wells of a device. The volume ratio of the polymer systems is such that u p o n phase separation, the b o t t o m phase precisely fills the lower sample wells. The wells are normally filled to within a b o u t 0.5 cm of the top of the well. The small a m o u n t of air remaining in the sample wells aids mixing of the phases. Samples are dissolved in the polymer mixture present in sample well number "0". The loaded devices were cooled to approximately 7°C and the caps were tightly fastened. The device was thoroughly mixed (40 inversions) prior to centrifugation for 5 min at 300 × g. After each centrifugation step the upper section was rotated counterclockwise until the index line was centered over the adjacent lower sample well. The devices were once again repeatedly inverted, centrifuged to separate the phases, and the t o p section rotated one sample well to the right. This cycle was repeated until the index line became opposite lower sample well number '17'. The entire contents of each of the 18 sample wells was withdrawn and placed in appropriately numbered test tubes. Countercurrent distributions of commercially obtained (1--2 mg) samples of mucopolysaccharides were performed routinely. At the end of the distribution, the mucopolysaccharides in each sample well were precipitated b y the addition of an equal volume of a 1% (w/v) hexadecylpyridinium chloride solution containing 5% (settled volume) celite. The precipitates were washed twice with 0.5% (w/v) hexadecylpyridinium chloride to remove any remaining twophase system polymers. The mucopolysaccharices were estimated b y the carbazole procedure [ 1 2 ] . When radioactive mucopolysaccharides were distributed, aliquots of the aqueous polymer mixture in each sample well were added directly to Aquasol and measured in a liquid scintillation counter. Results

Experiments performed with this microdistribution procedure showed that raising the pH in two-phase systems containing poly(ethyle'ne glycol) and dextran increased the partition coefficients of the mucopolysaccharides. In addition, the alkaline chlorides were f o u n d to lower the partition coefficients of mucopolysaccharides in the order Li ÷ < NH4 ÷ < Na ÷ < Cs÷ < K ÷. The same order of effectiveness of the cations was observed b y Albertsson [15] in partition studies of native DNA. Substituting 0.1 M LiC1 for 0.1 M KC1 resulted in more than a 100-fold increase in the partition coefficient of chondroitin-6-sulfate. At a constant ionic strength, increasing the ratio of KC1 to LiCI resulted in an almost linear

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6

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Sample well number

Fig. I . C o u n t e r c t t r r e n t d i s t r i b u t i o n p a t t e r n for h y a l u r o n i c acid ( t u b e s 0 - - 4 ) a n d c h o n d r o i t i n - 6 - s u l f a t e ( t u b e s 1 0 - - 1 7 ) in a t w o - p h a s e s y s t e m c o n t a i n i n g p h o s p h a t e / c i t r a t e b u f f e r ( p H 3.5). D i s t r i b u t i o n s w e r e c a r r i e d o u t in t w o - p h a s e p o l y m e r s y s t e m s c o m p o s e d o f 2 . 6 7 % d e x t r a n , 5 . 1 5 % p o l y ( e t h y l e n c g l y c o l ) , 0 . 1 0 M N a 2 H P O 4 , a n d citric acid s u f f i c i e n t to b r i n g t h e p H to 3.5 at 2 5 ° C . See M e t h o d s f o r f u r t h e r e x p e r i m e n t a l details.

decrease in the partition coefficient of mucopolysaccharides. In this respect, the partition behavior of mucopolysaccharides is quite different from that of native DNA which Albertsson [13] has shown to undergo an abrupt change in its partition coefficient at a critical ratio of the two salts. On the basis of partition coefficients determined by the microdistribution TABLE I SUMMARY OF THE P A R T I T I O N C O E F F I C I E N T S OF MUCOPOLYSACCHARIDES IN A Q U E O U S POLYMER TWO-PHASE SYSTEMS OF VARIOUS COMPOSITIONS Partition coeffcients were d e t e r m i n e d for the m u c o p o l y s a c c h a r i d e s using the microdistribution p r o c e d u r e d e s c r i b e d in t h e M e t h o d s . All d i s t r i b u t i o n s w e r e c a ~ i e d o u t u s i n g t w o - p h a s e p o l y m e r s y s t e m s c o n t a i n i n g 5% d e x t r a n a n d a t o t a l p o l y e t h y l e n e g l y c o l c o n c e n t r a t i o n o f 4% ( p o l y ( e t h y l e n e g l y c o l ) a n d t r h n e t h y l -

amino-poly (ethylene glycol)). ReIative % trimethylaminop o l y ( e t h y l e n e glycol) 2 2 2 2 5 5 5 5

5 10 10 10 10 10 10 20 20 20 20

[KBr] (mM)

5 10 15 20 5 10 15 20 25 5 10 20 23 25 30 20 30 40 50

Hy aluronic acid

19 9.4 0.22 0.05 >20 >20 0.29 0.033 0.015 >20 14 0.13 0.048 0.032 0.024 9.2 0.037 0.023 0.018

Chondroitin4-sulfate

Chondroitin6-sulfate

Heparin sulfate

3,9 2.3 1.5 0.70 --

14 1.5 1.2 0.65 14 11 1.18 0.82 0.75 9.8 4.3 1.8 1.5 1.5 0.79 6.0 2.2 0.99 0.46

3.3 0.77 0.51 0.32 2.8 1.1 0.56 0,41 0.34 3.5 2.3 0.71 0.49 0.44 0.40 1.1 0.58 9.27 0.26

--

-----1.5 ---5.1 1.7 1.5 1.1

295 procedure, two-phase systems were selected for use in countercurrent distribution experiments. A system containing 0.1 M phosphate/citrate buffer (pH 3.5) completely separates hyaluronic acid from chondroitin-6-sulfate after 17 transfer steps (Fig. 1). The other sulfated mucopolysaccharides, unfortunately, partitioned in virtually the same manner as chondroitin-6-sulfate. It is significant that the distribution patterns of the two mucopolysaccharides were not altered when both were present simultaneously. The substitution of trimethyaminopoly(ethylene glycol) for a portion of the poly(ethylene glycol) in a two-phase system significantly increased the selectivity of the system. By varying the proportion of this modified poly(ethylene glycol) as well as the potassium bromide concentration, it was possible to exert considerable control over the partition coefficients for the different mucopolysaccharides. Table I lists the partition coefficients determined for the different mucopolysaccharides in two-phase systems of various composi-

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Fig. 2. Countercurrent distribution Patterns for h y a l u r o n i c acid (a), heparan sulfate (b), and chondroitinS-sulfate (e) in a two-phase s y s t e m containing 2 0 m M KBr and with 10% of the p o l y ( e t h y l e n e glycol) replaced with t r i m e t h y l a m l n o - p o l y ( e t h y l e n e glycol). Distributions were carried out in a two-phase s y s t e m c o m p o s e d o f 2.67% dextran, 4.64% p o l y ( e t h y l e n e glycol), 0.515% t r i m e t h y l a m i n o - p o l y ( e t h y l e n e glycol), 5 m M m o n o s o d i u m phosphate, 5 m M disodium phosphate and 2 0 m M KBr, See Methods for additional e x p e r i m e n t a l details.

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tions. Fig. 2 shows the countercurrent distribution patterns obtained for samples of hyaluronic acid, heparan sulfate, and chondroitin-6-sulfate in a twophase system containing 20 mM KBr and in which 10% of the poly(ethylene glycol) had been replaced with trimethyl-amino-poly(ethylene glycol). While hyaluronic acid and chondroitin-6-sulfate were once again well separated, the surprisingly broad peak of heparan sulfate slightly overlapped the peaks of the other two mucopolysaccharides. The separation, however, was considered to be adequate for analytical purposes and the two-phase system was used in a preliminary study of radioactive mucopolysaccharides isolated from cell structures. Fig. 3 shows the countercurrent distribution patterns obtained for the labelled mucopolysaccharides isolated from the cell layer and the culture medium of BslA cells grown in the presence of [3 H] glucosamine. The medium contained a higher proportion of hyaluronic acid than did the cells. Fig. 4 shows the results of a similar experiment in which W12-4 cells were grown in the presence of 3 s SO~-. Virtually identical countercurrent distribution patterns

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Fig. 3. C o u n t e r c u r z e n t d i s t r i b u t i o n p a t t e r n s o f t h e r a d i o a c t i v e m u c o p o l y s a c c h a r i d e s i s o l a t e d f r o m t h e cell l a y e r (a) a n d f r o m t h e c u l t u r e m e d i u m (b) o f B s l A cells g r o w n in t h e p r e s e n c e o f tritiated glueos,~mlne. B s l A cells ( a p p r o x . 3 • 1 0 6 cells/flask) w e r e a l l o w e d t o g r o w in t h e p r e s e n c e o f D - [ 6 - 3 H ] g l u e o s a m i n e ( 2 . 5 /~Ci/ml) for 8.5 h. T h e m u c o p o l y s a c c h a r i d e s w e r e t h e n i s o l a t e d f r o m b o t h t h e cell l a y e r and t h e c u l t u r e m e d i u m a n d d i s t r i b u t e d in a m i n i a t u r e e o u n t e r c u r r e n t d i s t r i b u t i o n device. D i s t r i b u t i o n s w e r e c a r r i e d o u t in t h e s a m e t w o - p h a s e s y s t e m as in Fig. 2. S e e t h e M e t h o d s f o r a d d i t i o n a l e x p e r i m e n t a l details.

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Fig. 4. Countercurrent distribution patterns o f the radi.oactive m u c o p o l y s a c c h a r i d e s isolated f r o m t h e cell layer (a) and t h e culture m e d i u m (b) o f W 1 2 - 4 cells g r o w n in t h e presence o f radioactive sulfate. W 1 2 - 4 cells ( a p p r o x , 3 • 106 cells/flask) w e r e a l l o w e d to g r o w in the presence of 35S02- ( 2 5 ~tCi/rnl) for 24 h. T h e m u c o p o l y s a c c h a r i d e s w e r e t h e n isolated f r o m b o t h the cell layer and t h e culture m e d i u m and distributed in a miniature c o u n t e r c u r r e n t d i s t r i b u t i o n device. D i s t r i b u t i o n s w e r e carrled o u t in t h e same t w o - p h a s e s y s t e m as in Fig. 2. See t h e M e t h o d s for additional e x p e r i m e n t a l details.

were observed for the a 5 S-labelled mucopolysaccharides isolated from the cells and culture medium of B s l A cells. In all cases there was a small, but reproducible, leftward shift in the distributionpatterns of the cell layer material compared with the labelled material isolated from the culture medium. Discussion The unique properties of aqueous polymer two-phase systems have been exploited for the fractionation of mucopolysaccharides. An important characteristic of this type of procedure is that it remains useful even when only very small amounts of material are being fractionated. Sensitivity of detection for the compounds of interest is often the limiting factor and when radioactive c o m p o u n d s are studied, the sensitivity may be very great. In general, the effect on the partition coefficients of mucopolysaccharides of pH, various cations, or the presence of charged polyethylene glycol was very

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similar to that observed for nucleic acids. This, of course, is not surprising since both groups of substances are acidic polymers of high molecular weight. However, unlike the case with native DNA, the partitiofl coefficients of mucopolysaccharides do not show abrupt changes with small changes in the composition of the two-phase system. This perhaps is the result of a much smaller average molecular weight of the mucopolysaccharides compared with native DNA. Partition coefficients for the different mucopolysaccharides in various two-phase systems are not sufficiently different to permit separation in a single distribution. Instead, it is necessary to employ a countercurrent distribution procedure, which we have shown in this work. A totally different type of countercurrent distribution machine for use with aqueous polymer two-phase systems has been described by Albertsson [14]. In his machine the time required for phase separations was reduced by using extremely shallow (4 mm) sample cavities. By contrast, phase separations in the device employed in this work were accomplished by means of a brief centrifugation. Some of the advantages of the new type of countercurrent distribution device are that only very small amounts of polymer mixture are required per run, phase transfers are carried out much more quantitatively, complete phase separations may be accomplished much more rapidly, and several devices may be operated simultaneously in one centrifuge. The fact that the distribution patterns of hyaluronic acid and chondroitin-6-sulfate were not altered when both were present simultaneously implies that no major interaction occurs between the two compounds under the conditions employed. This represents an important difference between mucopolysaccharide fractionation procedures based on partition behavior and procedures involving precipitation of the compounds as their quaternary ammonium detergent complexes. In the latter case, the elution behavior of a particular mucopolysaccharide is very much influenced by the simultaneous presence of other mucopolysaccharides. BslA cells were grown in the presence of [3 H]glucosamine. Countercurrent distribution of the labelled mucopolysaccharides isolated from the cell layer and culture medium revealed that the latter contained a considerably higher proportion of hyaluronic acid. This probably reflects a mudh higher secretion by these cells of hyaluronic acid compared with other mucopolysaccharides. Countercurrent distribution patterns of the 3s S-labelled mucopolysaccharides isolated from the cells and culture medium of W12-4 cells were also very similar to those observed from BslA cells.

Acknowledgments This work was supported by grants from the USPHS Service (GM 13407 and CA 13196) and the Julius and Dorothy Fried Research Foundation.

References 1 Scott, J.E. (1960) in M e t h o d s o f B i o c h e m i c a l Analysis (Glock0 D., ed.)1 Vol, VIII, pp. 145--197, Wiley, New York

299 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Svejcar, J. and Robertson, W. (1967) Anal. Biochem. 18, 333--350 Mier, P.D. and Wood, M. (1969) Clin. Chim. Acta 24, 105--110 Albertsson, P.A. (1960) Partition of Cell Particles and Maeromolecules, Wiley, New York Rudin, L. and Albertsson, P.A. (1967) Biochim. Biophys. Acta 134, 37--44" Alberts, B.M. (1967) Biochemistry 6, 2527--2532 Favre, J. and Pettijohn, D.E. (1967) Euz. J. Biochem. 3, 33--41 Pritchard, D.G., Halpern0 It.M. and Smith~ R.A, (1971) Biochim. Biophys. Acta 228, 127--134 Iwakata, S. and Grace, Jr, J.T. (1964) New York J. Med. 64, 2279--2282 Johansson, G., Hartman, A. and Albertsson, P.A. (1973) Eur. J. Biochem. 33, 379--386 Craig, L.C. and Post, H.O. (1949) Anal. Chem. 21,500--504 Bitter, T. and Muir, H.M. (1962) Anal. Biochem. 4, 330--334 Albertsson, P.A. (1965) Biochim. Biophys. Acta 103, 1--12 Albertsson, P.A. (1965) Anal. Biochem. 1 1 , 1 2 1 - - 1 2 5 Albertsson, P.A. (1971) Partition of Cell Particles and Macromolecules, 2nd edn, Wfley-Interscience, New York