Countercurrent Distribution as a Tool for Purification of Hypothalamic Hormones on a Preparative Scale A. V. Schally, R. M. G. Nair, and W. H. Carter Endocrine and Polypeptide Laboratories, Veterans Administration Hospital and Department of Medicine, Tulane University School of Medicine, New Orleans, La. 70140
THEISOLATION of hypothalamic-releasing hormones involves tremendous efforts in which hundreds of thousands of hypothalami have to be processed to obtain even meager amounts of material for use in the study of composition and structure (1-3). Consequently, such an endeavor requires the selection of several purification techniques, the consecutive use of which would reduce the bulk of several kilograms of extracts (2, 3) to quantities more easily handled by usual laboratory procedures. In connection with such projects Guillemin et al. ( 4 ) stated that partition chromatography on Sephadex, by the method of Yamashiro (9, has much higher limits of capacity and produces a better degree of resolution than the countercurrent distribution technique of Craig (6). We have successfully used the technique of partition chromatography for the purification of thyrotropin-releasing hormone (TRH) (2) and luteinizing hormone-releasing hormone (LHRH) (7, 8), and we find it particularly useful when small amounts of material of the order of a few milligrams are involved. We wish to correct here the report ( 4 ) about the so-called limitations of counter-current distribution and relate our recent experiences with the use of this valuable and elegant method for the purification of LH-RH on a preparative scale. EXPERIMENTAL
Materials. Fragments of 250,000 ventral hypothalami of pigs (dry weight = 5.67 kg) were defatted with acetone and petroleum ether and then extracted with 2N acetic acid as described previously (2), yielding 2.05 kg of lyophilized extracts. The LH-RH activity in this extract was concentrated by gel filtration on a column of Sephadex G-25 (15.5 X 180 cm) in batches of 80 grams (2). The fractions with LHR H activity were lyophilized (yield 731 grams) and extracted with 4 1. of phenol (2, 7). The LH-RH active material was recovered from phenol by re-extraction into the aqueous phase, after the addition of 35 1. of redistilled diethyl ether (2, 9). The phenol extract (yield 179.9 grams), which showed LH-RH activity at doses of 10-100 pg was used as the starting material for countercurrent distribution. (1) A. V. Schally, A. Arimura, C. Y. Bowers, A. J. Kastin, S. Sawano, and T. W. Redding, Recent Progr. Horm. Res., 24, 497
Apparatus. Countercurrent distribution (CCD) was carried out in a n automatic all glass apparatus (H.O. Post Scientific Co.). The model C-2 liquid-liquid fractionator (CCD) containing 100 cells with 50-ml capacity in each phase was utilized. The time periods allowed for decantation and transfer were both increased from 20 sec to 45 sec. This was accomplished by substituting a new adjustable three-piece metal flap attached to the timing disk (10) driven by the Haydon timing meter (We are grateful to Mr. K. Schuerger, H.O. Post Scientific Instrument Co., Inc., for the construction of the metal flap with 3 adjustable fingers which we substituted in place of the original three-pronged metal strip). The mixing was accomplished in 12 strokes. Procedure. The CCD system consisted of 0.1 acetic acid-1-butanol-pyridine (1 1 :5 :3) as recommended by Craig et al. (11) and utilized previously for purification of T R H (2). The partition coefficient K of LH-RH in this solvent system is 2.0 (7). Analytical grade pyridine (Baker) was redistilled over sodium hydroxide pellets. 1-Butanol (Baker) was treated with zinc powder and redistilled. Glass distilled water was used for making up the solvent and for washing all glassware. After the run, both phases remaining in the CCD train were removed by suction. The active fractions were then recovered by adding 2-4 volumes of redistilled benzene (2) to displace all of the materials into the lower phase, which was then flash-evaporated to a small volume and lyophilized. The separation pattern brought about by the C C D run was followed by dry weight, Folin-Lowry reaction (12) and bioassays for LH-RH and pressor activity. The migration of the maximal concentration of activity (center of gravity of solute), N , was calculated according to an equation proposed by Williamson and Craig (13) N = nKr/Kr
-+ 1
where n is the number of transfers applied, r is the ratio of the upper and lower phase, and K is the partition coefficient. The whole operation was performed at a temperature of 70 f. 1 OF. Assays. The LH-RH activity was measured by stimulation of release of LH in ovariectomized rats pretreated with estrogen and progesterone (14, 15). Serum LH-levels were estimated by a radioimmunoassay procedure (16). The pressor activity was determined as recommended by Dekanski
(1968). (2) A. V. Schally, T. W. Redding, C. Y.Bowers, and J. F. Barrett, J . Biol. Chem., 244,4077 (1969). (3) R. Burgus, T. F. Dunn, D. Desiderio, D. N. Ward, W. Vale, and R. Guillemin, Nature, 226, 321 (1970). (4) R. Guillemin, R. Burgus, E. Sakiz, and D. N. Ward, C. R. Acad. Sci, 262, 2278 (1966). ( 5 ) D. Yamashiro, Naiure, 201,7677 (1964). ( 6 ) L. C. Craig, J. Bioi. Chem., 155, 519 (1944). (7) A. V. Schally, A. Arirnura, A. J. Kastin, J. J. Reeves, C. Y. Bowers, and Y. Baba, “Mammalian Reproduction,” H. Gibian and E. J. Plotz, Ed., Springer-Verlag, Berlin, 1970, pp 45-83. (8) A. V. Schally, A. Arimura, Y.Baba, R. M. G. Nair, H. Matsuo, T. W. Redding, L.Debeljuk, and W. F. White, Biochim. Biophys. Res. Comm., 43, 393 (1971). (9) A. V. Schally, C. Y. Bowers, W. F. White and A. I. Cohen, Endocrinology, 81, 77 (1967).
(10) L. C. Craig and D. Craig, “Technique of Organic Chemistry,” A. Weissberger, Ed., Vol. 111, Interscience New York, N. Y . , 1956, PP 149-332. (11) L. C: Craig, T. P.King, and W. Konigsberg, Ann. N. Y . Acad. Sci., 88, 571 (1966). (12) 0. H. Lowry, N. J. Rosebrough,A. L. Farr, and R. J. Randall, J. Bioi. Chem., 193, 265 (1951). (13) B. Williamson and L. C. Craig, ibid., 168,687 (1947). (14) V. D. Ramirez and S. M. McCann, Endocrinology, 73, 193 (1963). (15) A. V. Schally, A. Arimura, C. Y. Bowers, I. Wakabayashi, A. J. Kastin, T. W. Redding, J. C. Mittler, R. M. G. Nair, P. Pizzolato, and A. J. Segal, J. Clin. Endocrinol. Merab., 31, 291 (1970). (16) G. D. Niswender, A. R. Midgley, Jr., S. E. Monroe, and L. E. Reichert, Jr., Proc. SOC.Exp. Bioi. Med., 128,807 (1968).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
e
1527
2.8 2.4
P 2.0
---- LH-RH activity (plasma L H level) -Dry weight - - Folin color
-k
Q
P
3 C
1.6
I
8
9 L.
\-
1.2
9
8
.s
e \
0.8 I
i\
0.4
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0 0 40 80 Tube Number
250
210
170 I30 90 Transfer Number
Figure 1. Preparative countercurrent distribution of 171.3 grams of LH-RH concentrate in a system ofO.12 acetic acid : 1-butano1:pyridine = 11:5:3, by the single withdrawal method 171.3 grams of material was loaded in tubes No. 0-19. 100-cell train was filled with 50 ml lower phase and 25 ml upper phase. 250 transfers were performed. Folin-Lowry analyses were carried out on 10 p l lower phase (L) and 25 p1 upper phase (U). LH-RH activity was determined in 1-pI aliquots of upper phase, equivalent to approximately 0.8 rg dry weight
Table I. Biological Activity of Hypothalamic Fractions before and after CCD LH-releasing activity, ng Fraction No. Dose,pg LH/ml f SE P Before CCD , . . 12.21.1.2 ... Control 10 27 0 f 2 . 4 0.01 Phenol extract Phenol extract 100 75.0 1. 6 . 6 0.001 After CCD
Control Fraction 149-157, Figure 1 Fraction 189-197, Figure 1 Fraction 20-42, Figure 1 (inactive)
...
0.2 0.2 20
...
9.4zk1.2 43.9 1. 0 . 9 27.2 i 2 9
0.001 0.01
11.6 1. 2 . 3
N.S.
(17). Follicle stimulating hormone-releasing hormone (FSHRH) activity was measured as described previously (7, 8). RESULTS AND DISCUSSION
As the starting material (179.9 grams) for the countercurrent distribution was incompletely soluble, it was extracted 7 times with lower phase (140 ml each time) and upper phase (70 ml each'time) of the CCD solvent. This was followed by centrifugation. The residue (8.6 grams) showed no LH-RH activity at doses of 100 pg. The extracts were combined, and the upper phase was made up to 500 ml and the lower phase to 1000 ml, so that the average concentration of the solute was 11.42. This was introduced into the first 20
cells of the CCD train so that each of these cells contained 50 ml of lower phase and 25 ml of upper phase. The fore-run consisted of pre-equilibration with upper phase of about 20 tubes in front of the advancing solute bands. Co-current was set at about 0.5 ml. The distribution for 250 transfers was started using the single withdrawal method (18) to remove the LH-RH activity from the train and increase the chances of separation. The settling time was set at 1 hour for the first 20 transfers, then reduced to 29 min for the remaining 230 transfers. After 80 transfers were completed and the upper phase began to emerge from the CCD train, eight to 10 fractions were collected together into 1000-ml conical flasks, using the exit tube for manual operations. These flasks were changed every 4 hours. The collected fractions were immediately acidified with glacial acetic acid to pH 4. The pattern of separation as determined by dry weight, peptide analyses by the Fohn-Lowry method (12), and bioassays is shown in Figure 1. In agreement with calculations the LH-RH activity was transferred out of the train with the emerging upper phases and was found in fractions No. 130230. The band width and the location of LH-RH activity were in good agreement with theoretical computations. The increase of decantation and transfer periods to 45 sec allowed for an adequate draining of the viscous upper phase. The K value for LH-RH, determined to be 2.0 in two analytical runs in this solvent system, did not change in this run even in spite of very large amounts of material used. When the CCD separation of TRH was carried out on a preparative scale with 16-gram amounts, some change in K value was (18) T. P. King
(17) J. Dekanski, Brir. J . Plmrmucol., 7, 567 (1952). 1528
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and L. C. Craig, "Methods of Biochemical Anal-
ysis," D. Glick, Ed., Vol. IO, Interscience, New York, N. Y . , 1964. pp 201-228.
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found in this solvent system as compared with analytical runs with 1 gram of material (2). This was thought to be possibly due to incomplete draining during the decantation and transfer stages. When the fractions were flash-evaporated and lyophilized, the combined dry weight of the LH-RH active area was 2.7 grams. Thus a purification of over 60fold resulted in this run, with an excellent recovery of biological activity which was estimated to be essentially quantitative. The proportionate increase in the specific biological activity was evident from bioassays for LH-RH shown in Table I. The pressor activity was found in fractions 60-99 (mean K = 0.76). Since LH-RH contains an inherent activity of FSH-releasing hormone (FSH-RH) (7, a), this activity was also found in tubes No. 130-230. The fractions which remained in the CCD train contained the bulk of the dry weight and were devoid of LH-RH and FSH-RH activity at doses of 20 kg. This experiment is an example of the successful application of the technique of countercurrent distribution for the purification of a biologically active material from the hypothalamus on a preparative scale. The versatility of the apparatus is also apparent as the approach can be conveniently modified to suit one’s purpose.
Similar CCD techniques could be used for the purification of other biologically active substances as stated many times by Craig and associates (6, 10, 11, 13, 18). The advantages and suitability of the CCD method for this type of separation are once more confirmed. It may be pertinent also to mention that a modification of CCD called the “counter double countercurrent distribution” (CDCD) has a still much larger capacity (19). ACKNOWLEDGMENT
We are deeply grateful to Professor Lyman C. Craig, Rockefeller University, New York, for generous advice on the techniques of CCD. We wish to thank Mr. K. Schuerger, H. 0. Post Scientific Instrument Co., Inc., for his cooperation in constructing the adjustable metal flap for the timing disk. RECEIVED for review April 14, 1971. Accepted May 28, 1971. Supported in part by USPHS Grant AM-07467 and The Population Council, New York, N. Y . (19) 0. Post and L. C. Craig, ANAL.CHEM., 35, 641 (1963).
Sclvent Isotope Effects on Decomposition of N, N- DiaIky Idithiocarbamic Acids K. I. Aspila, S. J. Joris,‘ and C . L. Chakrabarti* Department of Chemistry, Carleton University, Ottawa, Ontario, KIS 586, Canada
STUDIES ON THE DECOMPOSITION of dithiocarbamates in DzO have appeared recently in the literature ( I , 2). It was shown that the rate of decomposition of C-N,N-tetramethylenedithiocarbamic acid at pH 1.0 is 2.6 times faster (1) in DzO than in H 2 0 ; and that the p D dependence of the decomposition of N,N-diethyldithiocarbamic acid is similar to its pH dependence (I, 2). In this paper, solvent isotope effects o n decomposition rates of several dialkyldithiocarbamates are compared. The synthesis of the dithiocarbamates used in this study was described in an earlier paper (3). D 2 0was 97 % pure. Rate measurements were done spectrophotometrically (3) at 15 i 0.1 “C and at pH (or pD) values of 1 =k 0.2. In this range of acidity the rates of decomposition of dithiocarbamates are independent of pH (pD) since nearly 99% of the dithiocarbamate anions are converted to the unstable acid form ( I , 3). It appears from Table I that the magnitude of the solvent isotope effect is related to the substituent in the dithiocarbaPostdoctoral research fellow. Present address, Noranda Research Centre, 240 Hymus Boulevard, Pointe Claire, Quebec,
Canada. Correspondence should be addressed to this author. (1) K. I. Aspila, V . S. Sastri, and C. L. Chakrabarti, Tularrta, 16, 1099 ( 1969). (2) S. W. Dale and F. Fishbein, J . Agr. Food Chem., 18, 713 (1970). (3) S. J. Joris, K. I. Aspila, and C . L. Chakrabarti, J . Phys. Chem., 74, 860 (1970).
Table I. Solvent Isotope Effects on the Decomposition of N,N-DialkyldithiocarbamicAcids (Temp = 15.0 f 0.1 “C, pH = pD = 1) Ratio of decomposition Decomposition rate rate Dithiocarbamic constants, set+ constants acid KD,O Kn2o KD,o/KH20 Dimethyl 0.0506 0.0180 2.80 0.0922 0.0363 2.54 Diethyl 0.0150 1.81 Dibutyl 0.0272 0,0352 0.0827 0.42 Diisopropyl
mate molecule. An inversion of isotope effect is observed in the case of diisopropyl-dithiocarbamate for which the rate of decomposition is slower in DzOthan in H20. It was shown (4) that dithiocarbamic (DTC) acids are formed by protonation of a sulfur atom of the dithiocarbamate anion (models I and 11). Through subsequent redistribution of electron densities in the acid molecule (5). the nitrogen atom accepts (3) a hydrogen bond. As the dithiocarbamate anion picks up only one proton (4,the hydrogen bond formed at the nitrogen atom is either intramolecular (4) S. J. Joris, K. I. Aspila, and C. L. Chakrabarti, ANAL.CHEM., 41, 1441 (1969). ( 5 ) D. M. Miller and R. A. Latimer, Can. J . Chern., 40,246 (1962).
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