Fractionation of Blood Plasma Proteins sing Ion Exchange Resins REVISED TECHNIQUES ALLEN F. REID AND FRANCES JONES Baylor Graduate Research I n s t i t u t e , Baylor University, a n d Southwestern Medical School, University of Texas, Dallas. Tex.
T
H E fractional precipitation of blood plasma proteins has been accomplished by three general methods: (1) decrease of the ionic strength of the solution by removal of salts or by dilution, (2) increase of the ionic strength of the solution by addition of salts, and (3) decrease of the dielectric constant of the solution by dilution with a water-miscible organic solvent. An excellent review of these methods is given by Edsall ( 3 ) . In the application of the first method salts have been removed by dialysis; difficulty is encountered in proper control of the pH, and the techniques have not been adapted to large scale operations. The use of ion exchange resins in these laboratories for salt removal has been reported (8); further investigation has led to the application of new and improved techniques. PROCEDURE
In the previously reported work, a small amount of the cations was first removed with a cation exchange resin, then a small amount of the anions was removed with an anion exchange resin, and this alternating sequence was continued until a sufficient amount of salts was removed. It was necessary to limit the amount of cations or anions removed a t any time in order to keep the p H close to neutral, and thereby avoid denaturation of the proteins. It was not feasible a t that time to use a mixture of cation and anion exchange resins, because no high-capacity highly basic anion exchange resin was available that was stable enough for extended contact with materials designated for therapy. If an anion exchange resin which is not strongly basic is used in a mixture with a strong cation exchange resin, it is very difficult to get rapid anion sorption while still keeping the pH above a denaturing value. I-Iowever, in the case of a mixture such as the one given, the mutual buffering action of the tFTo resins is great enough so that the p H will stay satisfactorily constant, even though the ratio of the t x o resins varies to a considerable extent. In the operation of the fractionation method, a number of ion exchange resins are suitable. However, a large ploportion of the work reported here was done using two specific resins, and the procedure for these is described.
Former methods of large scale fractionation of blood plasma proteins have involl ed low temperature organic solvent techniques which were somewhat difficult to control. The work reported here was done in an attempt to develop a method of fractionation more easily controlled. A mixture of cation and anion exchange resins is used at room temperature to remove the salts at a constant pH from a solution of blood serum proteins. Upon the progressive removal of the salts, the various globulin fractions are precipitated. The y - , 8-, and a-globulin fractions are precipitated, respectively, at ionic strengths of 0.0.50,0.017, and 0.0010. Solubility, electrophoretic, and serological tests indicate that the serum protein fractions are not denatured in this procedure. The mixture of anion and cation exchange resins may be implemented by- a batchwise or multiplute column arrangement on a scale which is feasible for mmniercial application.
0,0010. At this Concentration the a-globulin fract.ion is precipitat.ed, leaving a solution of water-so!uble globulins and albumin. The water-soluble globulins may be removed from the albumin solution by pH adjustment (8), by salting out, or by heat treatment after the addition of an albumin-protect,ing chemical such as those used by Boyer et aZ. ( 1 , 8). If sodium caprylate is added t.o 0.050 molarity and the temperature is raised to 68" C. and held there for 30 minutes, the globulins will be denatured without any apparent effect on the albumin. Upon subsequent cooling of the solution and reduction of the pH to 5.6, the denatured globulins are q u a n t i t a h e l y precipitated. If more than traces of hemoglobin were in the solution, these are removed by the caprylate and heat t,reatinent. If the solution iz then treated with t,he resin mixture, the sodium caprylate is i~?movedin the same manner as the physiological salts. Electrophoretic studies were made of the fractions from the process. Results of a typical run are shown in Figure 1 and Table I, which show the electrophoretic patterns and analyses of the original serum and of the respective precipitates and supernatants after each stage. The small amount of coprecipitated proteins (the albumin shows prominently in the patterns) can be removed by washing with a solution of ionic strength from which the globulin was precipitated. Figure 2 shows the pattern of the solution after heat treatment.
Table I .
A mixture of 1 part of frwhly regenerated Amberlite I R 100-H, a cation exchange resin, and 2 parts of freshly regenerated Amberlite IRA-400, an anion exchange resm (Rohm & Haas Co., Philadelphia, Pa.), is washed with distilled mater until the salt concentration is decreased to less than the equivalent of 15 p.p.m. of sodium chloride as measured by a conductivitj. meter. One volume of this resin mixture is stirred with 4 volumes of human blood serum for several minutes a t room temperature until the ionic strength of the solution becomes 0.050. The solution is then strained off from the resin and centrifuged. The precipitate is primarily y-globulin, which is immediately dissolved in a phosphate-buffered saline solution and set aside for further processing. The supernatant solution is again treated with about one fourth its volume of the mixed resin until its ionic strength becomes 0.017, a t which concentration the p-globulin fraction is precipitated. After separation of the resin and precipitated globulins, the solution is again treated as before but with about double the previous quantity of resin until the ionic strength falls beloiv
Ionic Strength 0.20 0.050 0.017
0.0010
Analyses of Serum, Precipitates, and Supernatant Solutions yc of Total Protein (Elertrophoretic) m-
Fraction Selnin Supernatant Precipitate Supernatant Precipitate Supernatant Precipitate
Albumin 72.0
Globulin
71.3
2 2 0
1.5
79.4 3.4 89.1 0.5
2.4
1.8 0.4 0 2.1
o-
Y-
Gloh~llin Globulin 11.7 13.9 10.8
0
2.0
12.1
5.4
2.4 0.1 2.6
0.1
0.1
4.4
8.1
In this particular sample before heat treatment there was an appreciable concentration of water-soluble globulins. In other cases a comparatively low concentration of thwe proteins is found. The water-soluble globulins will be about 8 to 15% of the 1074
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
May 1951
total globulins. The patterns were obtained by analysis with a Perkin-Elmer Model 38 Tiselius electrophoresis apparatus. The buffer used was the veronal solution of ionic strength 0.088 described by Longsworth (7). An alternative procedure, which is more satisfactory for many types of work, is to utilize the ion exchange resins in a multiplate column arrangement, rather than in the batch procedure described. The same mixture placed in a column 40 cm. high will effectively remove the salts t o an ionic strength of less than
1075
tests. To appraise the stability of the fractions further, serological tests were made. To determine the retention of the biological activity, R h and typhoid antibodies were used as tracers, because they are known to follow globulin fractions. It has been shown (4, 6) that different orders of R h antibodies are included in different globulin fractions. Upon fractionating a serum fo which these antibodies were added, using the techniques described, quantitative recovery of the most labile of these was obtained. Typhoid-0 antibodies were found to be quantitatively recovered in the P-globu]in fraction. [This is a t a variance with the results reported by
1 I
I
I
I Jk
ORIGINAL
SERUM
JL IONlC,$TRENGTH 0.20-0.050 P r e c i p l t e t e ana S u p e r n a t e n t (LorerI (Upper J
Figure 1.
I O N I C STRENGTH 0.050-0.017 P P e C I p l t e t e and S u p e r n a t a n t (Lower) (Upper
PIONIC r e c i pSTRENGTH i t a t e a n d 0.017-0.001O supernatant
(Lower)
I Upper 1
Electrophoretic Patterns
0.0010. If blood serum is poured through a t a rate not exceeding 2 ml. per sq. em. per minute, most of the globulins precipitated in its passage will be carried through with the solution. The globulins may then be centrifuged off and fractionated by successive leaching with progressively higher concentrations of buffered saline solution. If it is so desired, the column arrangement may be such that the solution may be tapped off a t various points and the individual fracI tions centrifuged off. Using the column arrangement as described, one volume of mixed resin will effectively remove the salts from about one volume of solution. In the operation of the process the resin should always be wet before coming in contact with the solution, as a certain amount of moisture is absorbed by the resin partides. After a protein solution has been in contact with the resin, a small amount will cling to the resin as a surface film and should be washed off. Generally, sodium Figure2. Alchloride solution will work satisfactorily. bumin after Heat TreatAfter the protein has been washed off, the ment resin is regenerated in the conventional manner by treatment of the cation exchanger with an acid and the anion exchanger with a base. It is necessary to separate the mixture for this type of regeneration. With the two resins described, this may be done conveniently by hydraulic backwashing, as their densities are very different, Various schemes for routine operation of this separation are standard practice in resin deionization.
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DISCUSSION
It was desired to know (1) whether a quantitative yield of the
fractions could be obtained by these techniques, (2) whether the resulting protein fractions were native, and (3) whether any contaminants were added by the treatment. Analysis of the fractions for protein nitrogen showed 95 to 98% recovery in the effluent consisting of the serum treated combined with a rinse solution of 10% of the serum volume. Up to 1% of the albumin and proportionately less of the globulins may be left on the resin. The ionic strength necessary for solution of the globulins did not change even after several reprecipitations. The electrophoretic mobilities of the fractions were unchanged. This showed there was negligible denaturation, a t least within the scope of these
Edsall (5)and by Deutsch et al. (2), who found the typhoid-0 antibody in the Cohn 111-1fraction and the gamma fractions, respectively. The method of fractionation was different. ] The absence of appreciable denaturation is not surprising, in view of the experience of others in sorbing and eluting proteins without loss of biological activity (6, 9). I n all these experiments there was some overlapping of one group with another; the amount depended in each case on the ionic strength used for solution, but was generally not over 5 to 7%. Upon refractionation of the fractions any contamination of one group with another became negligible. After a third fractionation, no trace of contamination of one globulin fraction with another was detectable by any means at the authors’ command. When the usual precautions for cleanliness and sterility are observed, no toxic contaminants can be detected in the products. Certain very soluble anion exchange resins may be unsuitable in this regard. On the other hand, yrogens present in the serum may be removed by the procedure YS). ACKNOWLEDGMENT
The authors wish to acknowledge t,he help and participation of Finis Robbins, Elsie Harris, Marjorie Orr, and David Black in the procedures for separation and analysis. They wish to express their appreciation to Sol Haberman, R u t h Guy, and Sheila Gillis, who made the R h antibody tests, and to Richard Dickens and John Jackson for technical assistance. The wish in particular to thank Joseph M. Hill and the staff of the saylor University Hospital Laboratory and the William Buchanan Blood Center, without whose cooperation and help this work could not have been carried out. LITERATURE CITED
(1) Boyer, P. D., Lum, F. G., Ballard, G. -4., Luck, J. M., and Rice, R. G., J . Bid. Chem., 162, 181 (1946). (2) Deutsch, H. F., Alberty, R. A., Costing, J. L., and Williams, -J. W., J . Immunol., 56, 183 (1946). (3) Edsall, J. T., Advances in Protein. Chem., 3, 383 (1947). (4) Hill, J. M., Haberman, S., and Guy, R., A m . J . Clin. Path., 19, 134 (1949). ( 5 ) Hill, J. M., Reid, A. F., and Haberman, S., Tezas S t d e J . M e d . , 4 5 , 4 7 7 (1949). (6) Lo Grippo, G. A., Proc. Soc. Ezptl. Biol., 74, 208 (1950). (7) Longsworth, L. G., Chem. Rev.,30, 323 (1942). (8) Reid, A. F., and Jones, F., Am. J . Clin. Path., 19, 10 (1949). (9) Rohm & Haas Co., private communication from ion-exchange
laboratory. RECEIVED November 2, 1950. Presented before the Division of Colloid Chemistry, Symposium on Applications of Ion Exchange, at the 118th SOCIETY, Chicago, 111. Studies aided Meeting of the AMERICANCHEMICAL by grants from J. K. W a d l e ~ a n dthe Dick Price hirotor Co.
