1.756
H.L. TAYLOR, F. C BLOOM, R. B. l l c C . i i , ~ L. , A. HE'NDMAN AND H. D. ANDERSON Vol. 78 [CONTRIBUTION FROM
THE
DIVISIONOF LABORATORIES, MICHIGAN DEPARTMENT OF HEALTH]
The Preparation of 7-Globulin from Placental Blood by Ethanol Fractionation' BY HAROLD L. TAYLOR, F. C. BLOOM, K. B. MCCALL,LEE A. HYNDMAN AND H. D. ANDERSON RECEIVED OCTOBER 13, 1955
A procedure is described for the separation and purification of 7-globulin from human placental extracts and postpartum blood. Frozen placentas together with the postpartum blood are thawed, chopped and extracted with isotonic saline. The initial separation of a fraction containing 7-globulin is accomplished by the addition of ethanol in the cold. A large part of the a-globulins are separated from the y-globulin fraction by the precipitation of the former a t pH 4.8, and ethanol 8%. This is followed by the precipitation of a y-globulin-rich (70%) fraction a t pH 7.0-7.2 and ethanol 25%. A fraction rich (91%) in a- and &globulins is separated a t pH 5.1 and ethanol 17%, leaving a supernatant fluid from which the 7-globulin is again precipitated with a final purity of 96%. moles of sodium chloride and then diluted with an additional 4.0 1. of water. After a uniform suspension has been obA procedure was presented earlierz,3applicable tained, the p H is lowered to 4.8 f 0.05 by the slow addition to the large scale preparation of albumin from pla- of a mixed buffer7j8in an amount determined by an initial titrati0n.O After 30 minutes stirring, the system is diluted cental extracts and postpartum blood. In the prep- with 12.9 1. of water (0') per kg. of precipitate I. The mixaration of this albumin, a fraction (precipitate I) is ture is stirred 2 hours and then 2.18 1. of 9570 ethanol ( - 10') separated which contains most of the y-globulin, per kg. of precipitate I is added through jets to bring the and this has served in our study as the starting ma- ethanol concentration to 8%. During the addition the is lowered to and held a t -2 to -3". The terial. The general principles of the ethanol frac- temperature suspension is stirred for one hour before removing precipitionation procedure, as described by Cohn, et a1.,4 tate I1 by centrifugation.'O The supernatant fluid I1 is reand as applied previously to the production of y- tained. Precipitate III.-Tbe supernatant fluid I1 is adjusted to globulin from venous blood by Deutsch, et u I . , ~ H 7.2-7.4 with a solutionll of sodium bicarbonate and soand Oncley, et a1.,6 were followed in our study. Al- pdium chloride. Then 243 ml. of precooled (-10') 95% though the ethanol methods have been adapted for ethanol, per kg. of solution, is added through jets as the the production of y-globulin from placental blood, temperature is gradually lowered to and held a t - 5 to -7". no descriptions of methods have appeared in the The suspension is stirred for one hour before the y-globulin fraction (precipitate 111) is removed by centrifugation. literature. Precipitate 111-1.-A fraction consisting principally of aand @-globulinsis precipitated from the ?-globulin by susMaterials and Methods pending each kg. of precipitate I11 in 10 liters of water (0') The methods of collecting and handling placentas and and then adjusting the pH to 4.80 f 0.05 by adding a mixpostpartum blood were described previously,*)* as well as ture of 680 ml. of 0.05 M dibasic sodium phosphate and 4.17 the methods of electrophoretic analysis, and the determina1. of 0.05 M acetic acid. After stirring overnight, the pH tions of nitrogen and hemoglobin.* is next raised to 5.10 f 0.05 by the addition of about 390 ml. of 0.05 M sodium acetate per kg. of solution. This Directions for the Preparation of -,-Globulin from Placentas solution is then diluted with 10 1. of water (0") per kg. of (Scheme I) precipitate 111, followed by raising the ethanol concentraThe recovery of placental extracts and postpartum blood tion to 17y0. This requires adding 6.97 liters of precooled (-10') 95% ethanol per kg. of precipitate 111. During is accomplished by (a) thawing the placentas at 2-4' after removal from -30' storage; (b) chopping with a Hobart the addition the temperature is lowered to and held a t - 5 The suspension is stirred for one hour before food cutter; (c) stirring each kg. of tissue plus blood into to -6'. 1,100 ml. of saline (0.8%) containing 22.4 g. of paper pulp; removing precipitate 111-1 by centrifugation. The resulting (d) separating the extract a t 0"; and (e) clarifying by fluid (supernatant 111-1) is clarified by filtration through Sharples centrifugation. This extract contains approxi- S1Seitz-type pads12 to remove the remainder of Precipitate mately 19 g. of plasma and tissue proteins and 23 g. of 111-1. hemoglobin per liter. (7)The p H 4.0 acetate buffer bas a mole ratio of sodium acetate to Precipitate 1.-Precipitate I is formed by the addition of 351 ml. 0f095% ethanol (precooled t o -10') per kg. of ex- acetic acid of 0.2 and is conveniently made up by taking 200 ml. of 4 M sodium acetate and 400 ml. of 10 M acetic acid and water to make one tract a t 0 , giving a final ethanol concentration of 25%. The alcohol is added with stirring and a t such a rate that the liter. This buffer, diluted with water 80 times, has a pH of 4.00 f temperature is gradually lowered to -5" where it is held 0.02, as measured with a glass electrode potentiometer at 25'. (8) The pH 4.8 acetate buffer has a mole ratio of sodium acetate to during the remainder of the addition. The PH is uncontrolled but lies in the range 6.6-6.9. The suspension is acetic acid of 1.1 and is conveniently made by taking 250 ml. of 4 M stirred for a t least one-half hour after completion of the sodium acetate and 90 ml. of 10 M acetic acid and water to make one ethanol addition. Precipitate I is removed by centrifuga- liter. This buffer, diluted with water 100 times, bas a pH of 4.80 i 0.02 with a glass electrode potentiometer a t 25'. tion a t -5' and stored frozen. (9) T h e amount of p H 4.0 buffer is determined by a preliminary Precipitate 11.-An initial purification is achieved by separating a fraction rich in a-globulins. Each kg. of pre- titration to p H 4.80. The amount of pH 4.8 buffer is calculated to make the sodium acetate contribution of the mixed buffers 0.25 M cipitate I is suspended in 4.0 1. of water (0') containing 1.75
Introduction
(1) Presented before the Biological Chemistry Division of the 128th National Meeting of the American Chemical Society, Minneapolis, September, 1955. (2) F. H. Gordon, L. A. Hyndman, F. C. Bloom, H. D. Anderson, H. L. Taylor, K. B. McCall, Tms JOURNAL, 76, 5859 (1953). (3) €I. L. Taylor, F. C. Bloom, IC. B. McCall, L. A. Hyndman, THIS JOURNAL, 78, 1353 (1956). (4) E. J. Cohn, L. E Strong, W. L. Hughes, Jr., D. J. Mulford, J. N. Ashworth, M . Melin and H. L. Taylor, ;bid., 68,459 (1946). (5) H. F. Deutsch, L. J. Gosting, R . A. Alberty and J. W. Williams, J. Biol. Chcm., 164, 109 (1946). (6) J. L. Oncley, M. Melin, D. A. Richert, J. W. Cameron and P. M. Gross, Jr., THIS JOURNAL, 71, 541 (1949).
per kg. of precipitate 1. The buffers are mixed and diluted with sufficient water ( O O ) to give an additional volume of 2 1. per kg. of precipitate I. (10) W e have employed a number 16 Sharples Supercentrifuge with a three-wing insert and a rate of 20-30 liters per hour. (11) The amount of sodium bicarbonate is determined by a preliminary titration with care t h a t all samples are aerated before the pH is determined. To the calculated amount of sodium bicarbonate is added an amount of sodium chloride such that the addition of these two substances will be in an amount of 0.023 mole per liter of supernatant 11. Both substances are dissolved together in water a t 0' and are added as a 0.5 M solution. (12) Filter pads purchased from the Republic Seitz Filter Corporation, 17 Stone Street, Newark, New Jersey.
7-GLOBULIN FROM PLACENTAL BLOOD BY ETHANOL FRACTIONATION
April 5, 1956
+
SCHEME I Ppt. I redissolved Ethanol
8%
4 . 8 f 0.05
PH
Temp. Protein Salt concn. Sodium chloride Sodium acetate
-3O
1% 0.08 M 0.07 M 0.01 M
Ppt. I1 (discard)
Supernatant I1 Ethanol 25% 7.0-7.2 PH 5 O Temp. 0 . 0 M . 10 Ionic strength Protein 0.4%
-
Supernatant I11 (discard) Ppt. I11 redissolved Ethanol 17% 5.1 f 0.05 PH -6OC. Temp. Protein 0.7% Ionic strength 0.01
Ppt. I11 (save)
method employed for fraction I1 I11 from plasma resulted in a satisfactory product, but a very poor yield. Instead, conditions for the extraction of yglobulin from precipitate I were selected on the basis of data presented in Table I. TABLE I EFFECT OF pH AND IONICSTRENGTH O N THE EXTRACTION OF Y-GLOBULIN FROM PLACENTAL PRECIPITATE 1'
I
II
1337
Exp. no.
Extraction conditions b Ionic pH strength
Extracted material Total y-Globulin, protein, y-Globulin. % of that g. g. in ppt. I
(Starting material for (38.9) (5.44) (100) Experiments 14) 1 4.85 0.08 11.4 3.82 70.1 5.38 .08 14.0 3.18 58.5 2 3 5.70 .08 11.8 2.94 54.1 4 5.40 .04 4.8 0.24 4.4 843 328 65 37 5.38 .08 38 4.85 .08 981 520 100 Experiments 1-4 represent 150 g. precipitate I ; ExperiAll exments 37 and 38 represent 9.8 kg. precipitate I . tractions performed a t 8% ethanol, -2".
These small scale experiments, 1 through 4, indicated that the extraction of y-globulin was more Ppt. 111-1(discard) Supernatant 111-1 nearly complete at the lower pH (4.85). In exp. 4, a Filter lower ionic strength reduced the solubility of all Ethanol 25% proteins in precipitate I. When large-scale nms fiH 7.0 f 0.01 (37-38) verified the findings of the small-scale Temp. -5OC. work (experiment l), we adapted the conditions of Protein 0.6% p H 4.8 for this step. It was also determined that Ionic strength 0.05 less hemoglobin was extracted a t this PH. I It The conditions for the precipitation of the bulk Ppt. 111-2 Supernatant 111-2 (discard) of the 7-globulin from supernatant I1 are essenPrecipitate 111-2.-A final precipitation of the 7-globulin tially those employed by Oncley, et u Z . , ~ for the refraction from supernatant 111-1 is accomplished by first moval of a total 7-globulin fraction. They differ raising the PH to 7.2-7.4 by adding a sufficient amount of only in ionic strength and protein concentration. a 0.5 M solution of sodium bicarbonate. The ethanol conSince Oncley, et u Z . , ~ had reported that 7-globulin centration is raised to 25% by the addition of 114 ml. of precooled (-10") 95% ethanol per 1. of solution. During was more soluble in the presence of sodium acetate the addition the temperature is lowered to and held at - 10'. than sodium chloride, our original experiments on The suspension is centrifuged a t - 10" to remove precipitate the extraction of 7-globulin from precipitate I were 111-2. This 7-globulin fraction is suspended uniformly in about 1.5 vol. of water-ice mixture ( 2 : l ) and, after the ice performed with sodium acetate. However, the melts, the suspension is then frozen in suitable containers quantity of acetic acid required in the p H 4.8 aceand dried from the frozen state. tate buffer used earlier for the extraction of precipitate I nearly doubled the ionic strength at the pH Discussion of 7.2-7.4 employed for the subsequent precipitaPreliminary Purification.-We have employed a tion of 7-globulin (as precipitate 111). We then resaline extract of the placentas as a starting ma- placed of the sodium acetate with sodium chloterial for the preparation of both albumin and y- ride to determine whether or not the extraction of globulin. The presence of large amounts of hemo- y-globulin from precipitate I was as complete as globin in this extract rendered electrophoresis use- sodium acetate alone and would still increase the less in the early steps of purification. However, by recovery of y-globulin in precipitate I11 (Table 11). using infrared film and a tungsten lamp,'* i t was TABLE I1 possible to determine the 7-globulin electrophoretiTHEEXTRACTION AND PRECIPITATION OF GLOBULIN^ cally in supernatant I1 and the succeeding fractions. Extraction solution A fair estimate of the amount of y-globulin in the 0.01 M sodium ncetatec and 0.07 M sodium extract and in precipitate I was possible by super0.08 M sodium acetate b chloride imposing ascending and descending electrophoretic Protein. Protein, g./kg. -1-Globulin g./kg. y-Globulin patterns. T h e clarified extract was estimated ppt. I % G. ppt. I % G. to contain 1.2 t o 1.3 g. of 7-globulin per liter Precipitate I 226 16 43.4 211 16 40.6 (or per placenta) by this method. Supernatant I1 106 .. .. 97 59 The removal of precipitate I from the extract Supernatant 111 40 .. . . 39 9 was accomplished under the same conditions as Precipitate I11 64 53 33.9 55 70 38.5 employed by Cohn, et al.,4for the preparation of a (78%) (95%) similar fraction (IT 111) from human plasma. extractions were performed at pH 4.80 f 0.05; Attemp$s to further purify the y-globulin by the 8%"All ethanol, and -2". Average values from three runs.
+
(13) G . I.. Miller, S r i c n r r . 116, 687 (1952)
c
Average values from ten runs.
1358
H. L. TAYLOR,F. G. BLOOM,K. B. MCCALLL. A. HYNDMAN AND H. D. ANDERSON Vol. 78
The data indicate that although the sodium acetate -sodium chloride solution yielded a lesser quantity of precipitate 111, the total amount of y-globulin recovered was significantly greater (13%). Final Purification.-Analyses of precipitate 111 have shown a 7-globulin content of approximately 7070 (Table 111). For further purification we have employed the extraction procedure of Deutsch, et aL.,5 for our precipitate 111, with slight modifications. The procedure of Deutsch, et ~ l .for , ~fraction I1 111 of plasma employs a mixture of dibasic sodium phosphate and acetic acid to adjust the p H to 4.80 =t0.05. After stirring overnight, that system is raised to 5.10 f 0.05 by the use of additional dibasic sodium phosphate and acetic acid. We have modified this method by employing sodium acetate for the pH adjustment from 4.8 to 5.1. Our preliminary experiments with acetate indicated that the 7-globulin extracted from precipitate 111 was equal or slightly greater in amount than that obtained with the phosphate. Except for this change, all conditions of the method of Deutsch, et ~ l .have , ~ been applied without modification. Distribution of y-Globulin.-The results of electrophoretic analysis of the several precipitates and supernatant fluids are presented in Table 111. The data for the several precipitates are considered more reliable than those for the supernatant fluids. since all the supernatant fluids were concentrated before electrophoretic analysis by drying from the frozen state. In our experience, this often leads to error because of altered solubilities of many of the proteins.
+
ure for the 7-globulin content of precipitate I, since we feel confident that the electrophoretic data on the original extracts of precipitate I are less reliable. The data do indicate that a good separation of the y-globulins and the a- and @-globulins was accomplished by the removal of precipitate 11. The greatest losses of y-globulin apparently occur during the removal of precipitate 111 and the filtration of supernatant 111-1; these losses may be due to occlusion of y-globulin by the filter pads and fi1ter medium. Recently we have employed an alcohol-buffer wash which tends to minimize this loss. TABLE I\' DISTRIBUTIOK O F TOTAL PROTEIN ASD y-CLOBULIN PLACENTAL FRACTIOXS
IN
yGlobulin N X 6.25, Nitrogen, Protein, g./plac. g./plac. factor
7.19 48.6 Extract 42 3 6.66 5 50 7.53 Precipitate I .. .. 36 8 Supernatant I 3.66 Precipitate I1 2 76 8.30 2 36 Supernatant I1 1.41 1 34 6.60 Precipitate 111 0.96 Supernatant 111 0 91 (6.60) . 50 Precipitate 111-1 .42 7.49 69 6.25 69 Supernatant 111-1-F .63 Precipitate I 11-2 .63 6 25 .063 .06 (6.25) Supernatant 111-2
n./
%
plac.
2.5 16 1 3 59 70 9 7 95 96 90
1.22 1.06 0.11
0.99 .086 .035 ,655 .605 .05
The yield of y-globulin is 0.8 g. per kg. of placental tissue and postpartum blood. This represents an over-all recovery of approximately 50%. TABLE I11 The product is 96-98% pure; the other major comELECTROPHORETIC ANALYSISOF PLACENTAL FRACTIONS ponent is reported as @-globulin. The purified yGlobulins, % globulin has been prepared in final solution containAlbumin, d f ing 16.5 g. per 100 ml. and this solution has a light % a 81 Y 82 amber color. Although this globulin has not been Precipitate I* 7 47 22" 16 8 tested clinically, i t has been subjected to all the apPrecipitate I I ~ 8 70 14" 3 5 propriate laboratory tests for safety, pyrogenicity, 3 12 2Y 59 3 Supernatant II* stability and content of diphtheria antitoxin, In70 6 1 13 10 Precipitate III* fluenzae A and polioniyelitis virus neutralizing an6 25 55" 9 5 Supernatant JII* tibodies. In all these tests this product was found Precipitate 111-1" 2 17 24 7 20 to be indistinguishable from the y-globulin pre0 0 4 9 5 1 Supernatant III-l-Fc pared from fresh human plasma. 4 96 0 0 0 Precipitate 111-2 0 0 6 90 0 Acknowledgments.-We wish to acknowledge Supernatant 111-2' the assistance of the staff of the Michigan Departa Hemoglobin included in 8, 1-globulin peak. * Data ment of Health fractionation laboratory which inplacentas. are means of ten analyses, batch size-527 Data are means of five analyses; batch size-2,464 pla- cluded, besides the authors, F. C. Coryell, William centas. Gay, R. Murray, A. Kennedy, G. Nonhof, R. Johnson, J. Sgouris and F. Gordon. For assistance The distributions of total protein and 7-globulin are presented in Table IV. The most serious dis- in the many analyti'cal determinations and biologicrepancy would appear to be that the sum of the 7 - cal testing we are indebted to L. Rasmussen, E. globulin recovered in precipitate 11, precipitate I11 Hagenbuch, R. Yasin, J. Collins, A. Forell and V. and supernatant 111 is about 8% greater than that Ku . MICHIGAN in precipitate I. This is probably due to a low fig- LANSING,
