InlmumJ~hemistry, 1976.

Vol, 13, pp. 135 139.

Pergamon Press.

Printed in Great Britain

PURIFICATION OF HUMAN IgA BY SALT-MEDIATED HYDROPHOBIC CHROMATOGRAPHY* GEORGE J. DOELLGAST and ANDREW G. PLAUT Tufts Cancer Research Center and the Department of Pathology (Oncology), Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA and Gastroenterology Unit, Department of Medicine, Tufts-New England Medical Center Hospital. Boston, MA 02111, U.S.A. [Received 25 August 1975}

Abstract- Salt-mediated hydrophobic chromatography, a technique by which hydrophobic interactions of a protein with a given ligand coupled to Sepharose are enhanced by specific salts, was used for human IgA purification. The IgA of both normal and myeloma serum undergoes binding to L-phenylatanine Sepharose in the presence of 1 M ammonium sulfate, and is eluted in 0.8 M ammonium sulfate. For IgA myeloma serum, the preparations were homogenous after salt-mediated hydrophobic chromatography and gel filtration chromatography.

INTRODUCTION The precipitation of plasma proteins in high concentrations of ammonium sulfate ('salting out') is a time-honored technique commonly used as a preliminary step in purification before other high resolution techniques such as molecular sieve and ion exchange chromatography (Dixon & Webb, 1961). Salt precipitation is particularly useful for immunoglobulin purification, because these proteins become insoluble at ammonium sulfate concentrations substantially lower than those required to precipitate most other plasma proteins (Heide & Schwick, 1973). Recently, reports from a number of laboratories (Doellgast, 1973; Doellgast & Kohlhaw, 1972; Doellgast & Fishman, 1973, 1974, 1975; Doellgast et al., 1974: Porath et al., 1973; Rimerman & Hatfield, 1973: Holmes et al., 1975: Memoli et al., 1975) have described the use of specific salts in chromatography on column supports such as Sepharose to which derivatives of nonpolar amino acids are covalently bound. In this technique, the high concentrations of sulfate or phosphate salts enhance interaction of the protein with the hydrophobic ligand, so that binding or adsorption occurs at high concentrations, and elution or desorption occurs as salt concentrations are reduced. In all cases, the proteins are initially bound to the columns at a salt concentration below that at which they precipitate from solution. Because it is the salt that mediates or controls the degree of interaction of the protein of interest with the hydrophobic column support we have named this technique 'salt-mediated hydrophobic chromatography' to distinguish it from hydrophobic chromatography using ligands of varying hydrophobicity to control binding * Supported by Grants CA-13332 and CA-12924 (to W. H. Fishman) and AM-17194 from the National Institutes of Health, U.S.P.H.S., and from the U.S. Army Medical Research and Development Command (DAMD 17-74-C-4022). G. Doellgast holds a Postdoctoral Research Fellowship (5-F22-CA00064-02) and A. Plaut, a Research Career Development Award (AI70420) both from the National Institutes of Health.

and elution (Shaltiel & Er-E1, 1973: Yon, 1974; Hjerten, 1973; Hofstee, 1973). The present report describes the purification of human immunoglobulin A (IgA) from normal serum and serum of patients with multiple myeloma using salt-mediated hydrophobic chromatography. The technique, which is rapid and reproducible, involves the binding of IgA to Sepharose bearing covalently bound L-phenylalanine as a ligand, and the subsequent elution of the protein by reduction in the salt concentration of the eluting solutions.

MATERIALSAND METHODS Materials Sepharose 4B was obtained from Pharmacia Fine Chemicals Co., L-phenylalanine from Sigma (Cat. No. P-2126t, ammonium sulfate and Tris salts from Mann Chemical Co. tMann ultra-pure reagents), sodium barbital from Merck and Co. and Noble agar from Difco. Rabbit and goat anti-human serum proteins and anti-immunoglobulin sera were either prepared in our laboratory or obtained commercially from Meloy Laboratories. lmmunoelectrophoresis Performed in 2°0 agar in 0.04 M veronal-HCI buffer, pH 8.4, by the micromethod of Scheidegger (1955). Slides were developed with antisera of defined specificity for human serum proteins. Ouehterlony double d!ffusion Ouchterlony double diffusion was carried out in 2°,;,agar in saline using antisera of characterized specificity. Immunoglobulin quantitation Immunoglobulin quantitation was undertaken by radial immunodiffusion using Behring tri-partigen plates and appropriate immunoglobulin standards.

Protein Protein was estimated by O.D.28o or determined by the method of Lowry et al. (1951) using Sigma protein standard (Sigma Cat. No. 540-d0). 135

136

GEORGE J. DOELLGAST and ANDREW G. PLAUT

Fractionation of normal human serum proteins on L-phenylalanine sepharose L-phenylalanine was covalently bound to cyanogen bromide-activated Sepharose 4B at pH 9.8 by a method previously described (Doellgast & Fishman, 1974). For purification of IgA from normal serum, 25 rnl of serum from a fasting individual was mixed with 16.7ml of 2.5M ammonium sulfate in 0.10M Tri~acetate, pH 8.0; this resulted in a final ammonium sulfate concentration of 1.0 M. Following centrifugation at 27,000 g for 30 rain at 4°C, the clarified serum was applied to a 2.6 × 30 cm column of L-phenylalanine-Sepharose previously equilibrated with 1.0 M ammonium sulfate in 0.04 M Tri~acetate, pH 7.6. After application of the sample, stepwise elution was undertaken by the sequential application of at least 200 ml of each of the following solutions: A. 1'0 M ammonium sulfate in 0-04 M Tris-acetate, pH 7.6; B. 0'8 M ammonium sulfate in 0'032 M Trisacetate, pH 7.6; C. 0.6M ammonium sulfate in 0.024 M Tris-acetate, pH 7.6; D. 0.4 M ammonium sulfate in 0.016 M Tribacetate, pH 7'6; E. 0'25 M sodium chloride in 0'05 M Tris-acetate, pH 8-0; F. 0"25 M Tris base, pH 10.5. Eluates were collected in 5 ml fractions and the elution was performed at 4~C. The location of IgA in the e[ution profile was determined by Ouchterlony anabsis using IgA-specific antiserum. Pooled fractions were concentrated by ultrafiltration (Amicon PM-30) to a volume equivalent to that of the starting serum before being analyzed for protein or immunoglobulin content. Isolation of human IyA myeloma proteins This was by conditions identical to those used with normal human serum with the exception that the sera • o/ were diluted with equal volumes of 09/0 sodium chloride prior to use. With any given myeloma serum, once the fractions in which the IgA eluted were identified, the subsequent eluting solutions were omitted in future runs. The column was regenerated for the next use by 'stripping off' the remaining proteins with Tris base followed by washing with 1.0 M ammonium sulfate. Once the lgA myeloma proteins were eluted from the column they were pooled, concentrated by ultrafiltration, and chromatographed on a 2.6 x 60 cm column of Sephadex G-200 equilibrated with 0'9°;; sodium chloride in 0'01 M Tris-HC1, pH 7'6, in order to remove traces of contaminating protein. RESULTS

An elution profile of normal human serum fractionated by chromatography on L-phenylalanine Sepharose is shown in Fig. 1. The designated pools (A-F) correspond to buffer changes. Pool A contained all the proteins which did not bind to L-phenylalanine in the presence of 1.0 M ammonium sulfate and therefore contained the bulk of the serum protein; at higher ammonium sulfate concentrations additional pool A proteins would also bind. Pools B-F represent proteins which were eluted by successive reduction in salt concentration or increase in pH. Pool F material was removed in Tris base at pH 10'5

I

B

Iolo

I E

I

tO

0D280

5

50

tOO 150 200 TUBE NUMBER

250

300

Fig. 1. Elution profile determined by absorbancy at 280nm of normal human serum chromatographed on L-phenylalanine-Sepharose as outlined in the text. The pools correspond to changes in eluting solutions. The large amount of protein in pool A represents that not binding to L-phenylalanine under the starting conditions selected (1'0 M ammonium sulfate).

and therefore represented 'stripping' of proteins not previously removed by the other solutions, indicating that they were tightly bound. Immunoelectrophoresis of each pool developed by unabsorbed goat anti-normal human serum is shown in Fig. 2. Pool A contained many protein species including virtually all of the albumin but was free of immunoglobulin including IgA, as shown by the slide at the bottom of the photograph. Pools B and C contained IgA apparently free of significant contamination by the other major immunoglobulin classes IgG and IgM, and pools D - F again had several protein bands including the bulk of the immunoglobulins. It should be emphasized that for this study elution conditions were specifically selected to separate l e a from IgG. The actual protein and immunoglobulin content of pools A - F of normal human serum are shown in Table 1. The radial immunodiffusion method for estimation of immunoglobulins showed that Pool A was very low in immunoglobulin content, indicating that the major Ig classes were initially bound to L-phenylalanine in 1 M ammonium sulfate. Secondly, IgG and IgA were widely separated in that most IgG was in pools E and F while IgA predominated in pools B and C. Thirdly, pool B contained no detectable IgG or IgM, although immunoelectrophoresis showed a number of other plasma proteins present. We have been able to further purify normal human serum IgA from Pool B material by passage over Sephadex G-200 molecular sieve chromatography followed by anion exchange chromatography; the product yielded a single precipitin line by Ouchterlony double diffusion using antiserum to normal human serum and IgA. Figure 3 shows a typical elution profile of a serum from a patient with multiple myeloma of IgA with kappa type light chains. The myeloma protein eluted in Pool B, where it was represented by a dominant spike which was readily identified as IgA by Ouchterlony analysis. The elution of the IgA myeloma protein in Pool B correlated with the etution position of IgA

Purification of Human IgA

137

POOL A

4

A N T I - NHS

B

C ANTI- NHS D E

ANTI-NHS F

A

4

ANTI-

19A

Fig. 2. Immunoelectrophoretic analysis of pools of normal human serum (A F) depicted in Fig. 1. Unabsorbed goat anti-normal human serum (anti-NHSt demonstrates the markedly different protein composition of the various pools. IgA is localized to pools B and C: it was bound to the column as indicated by its absence in Pool A as shown in the bottom slide developed with goat anti-human IgA. Anode is to the right.

in normal human serum, but as expected the protein content of the pool B of normal serum was far lower (Fig. 1). Pools C and D from the myeloma serum contained IgA by Ouchterlony analysis, but as shown by the elution profile the bulk of the paraprotein was localized in Pool B. In order to achieve final purification of the IgA paraprotein, pool B was concentrated and chromatographed on a Sephadex G-200 molecular sieve column. IgA eluted as a symmetrical peak; when examined by Ouchterlony analysis as shown in Fig. 4, the IgA paraprotein was free of contamination by Table 1. Total protein and immunoglobulins of pools from the chromatography of normal human serum on L-phenylalanine Sepharose. Pools as indicated in Fig. 1 Pool

Protein

(mg) IgA (mg)

A

1020 43

16.7

C

38

17.2

5.3

8.2

D

81

9.6

48.3

B.3

E

156

7.8

136.4

7.7

F

16__~Z

6.6

142,4

15.__.11

59,9

332.4

39,3

*Not Detectable

1500

---*

IgM (mg)

B

Total

2.0

IgG (mg)

---

. . . . . .

both IgM and IgG and showed a line of immunologic identity with another myeloma IgA and with IgA in normal human serum. To date, we have purified 12 IgA myeloma proteins of both subclasses and light chain types by salt-mediated hydrophobic chromatography. The proteins all eluted in the Pool B and C regions and were immunologically pure following passage over Sephadex G-200. DISCUSSION

The principle of salt-mediated hydrophobic chromatography is derived from the classic form of precipitation of protein by salt originally discovered by Hofmeister in 1888 and used and studied extensively since that time. A review by Von Hippel & Schleich (1969) has brought together in comprehensive form the large literature on this subject. Attempts have been made to adapt the precipitation of protein in salt solution for use on a column support: these methods involve precipitation of proteins by high ammonium sulfate concentrations on the columns, followed by their being dissolved and hence eluted by decreasing the salt concentration (King, 1972). The technique we are reporting here differs from all previous precipitation-dissolution procedures in that the protein is applied to the phenylalanine Sepharose column in a concentration of ammonium sulfate below its precipitation point, and thus binding

138

GEORGE J. DOELLGAST and ANDREW G. PLAUT POOL A

]

B

I C I D

J E

IFI

6

NHS

IgM

0D280 4

iJ 50

onti-lgM

I00

TUBE

150

200

PO0

250

L

NUMBER

Fig. 3. Elution profile of the serum of a patient with multiple myeloma, IgA1, kappa type applied to L-phenylalanine Sepharose. Conditions are the same as in Fig. 1, but note slight difference in absorbancy scale. The large peak in Pool B is the bulk of the paraprotein; lesser amounts of IgA were also demonstrated immunologically in pools C and D.

to the column is through direct interaction with the phenylalanine ligand. It should be noted that this technique also differs from systems of hydrophobic chromatography which rely on the use of a number of different hydrophobic ligands to effect the binding of different proteins. Because phenylalanine-Sepharose can bind any protein at a sufficiently high ammonium sulfate concentration, identification of optimal conditions for binding and elution is convenient since only one type of column would have to be used. The value in using other hydrophobic ligands occurs when the protein of interest binds too strongly or too weakly with phenylalanine~Sepharose but in our experience phenylalanine is adequate for the bulk of plasma proteins. The requirement for a hydrophobic ligand to accomplish protein binding can be demonstrated by using underivatized Sepharose in 1 M ammonium sulfate or Sepharose bound to a nonhydrophobic amino acid such as glycine. Binding of protein does not occur under these conditions, since applied protein is not retained and elutes directly from the column. While a number of other nonpolar ligands covalently coupled to Sepharose can be used for hydrophobic chromatography, the exclusive use of phenylalanine has allowed us to better define the relationship between binding and elution of a number of enzyme and protein species. Once the elution conditions for lgA were accurately defined, we were able to rapidly separate and purify multiple IgA myeloma proteins. As mentioned above, in most cases a second purification step involving molecular sieve chromatography was necessary to remove small amounts of other proteins. The purity of IgA prepared by this method is demonstrable by disc gel electrophoresis and Ouchterlony analysis of the preparations using a battery of antisera to human serum proteins. In addition, proteolytic fragments of these IgA preparations subjected to limited N-ter-

NHS

IgA

anti

- IgA

POOL

NHS

l QG

anti - I g G Fig. 4. Ouchterlony analysis of human myeloma IgA found in pool B (See Fig. 3). Prior to analysis the protein was passed over Sephadex G-200 molecular sieve chromatography, lgM, IgA and IgG are human serum containing those paraproteins, NHS is normal human serum, and Pool is the IgA purified by salt-mediated hydrophobic chromatography and gel filtration. Antisera specific for the three major Ig classes show the absence of lgM and IgG in the preparation and its immunologic identity with normal serum IgA and an unrelated myeloma IgA.

minal amino acid sequence analysis for other studies have yielded homogenous data, further indicating that contamination with non-IgA protein is at an insignificant level. The purification of IgA by the hydrophobic technique has several advantages; the cost of preparation of the column is low, the column has a high adsorption capacity, and elution is achieved rapidly under very mild conditions. Further, the columns can be

Purification of Human IgA easily regenerated and have been used repeatedly over a period of one year without substantial loss in their adsorption capacity for IgA. Using various combinations of salt concentration and pH, the technique could be used for partial purification of nearly any plasma protein. The hydrophobic technique, in comparison to immunoabsorption chromatography commonly used for IgA purification, does not of course have the obvious advantage of the antiserum specificity available in immunoabsorption. Aeknowled.qements The authors appreciate the encouragement and support given this work by Dr. W. H. Fishman, Director of the Tufts Cancer Research Center, and acknowledge the technical assistance of Ms. Joanne Gilbert. REFERENCES

Dixon M. & Webb E. C. (1961) Adv. Protein Chem. 16, 197. Doellgast G. J. Ph.D. Thesis, Purdue University. (December 1973). Doellgast G. J. & Fishman W. H. (1973) Fedn. Proc. 32, 582 Abs. Doellgast G. J. & Fishman W. H. (1974) Biochem. J. 141, 103. Doellgast G. J. & Fishman W. H. (1975) Isozymes--I Molecular Structure (Edited by Markert C. L.), p. 293. Academic Press. NY.

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Doellgast G. J. & Kohlhaw G. B. (1972) Fedn Proc. 31, 424 Abs. Doellgast G. J., Memoli V. A., Plaut A. G. & Fishman W. H. (1974) Fedn Proc. 33, 1906 Abs. Heide K. & Schwick H. G. (1973) in Handbook of Experimental Immunol. (Edited by Weir D. M.), Chap. 6. Blackwell, Oxford. Hjerten S. (1973) J. Chromato9. 87, 325. Hofstee B. H. J. (1973) Biochem. biophys. Res. Commun. 50, 751, Holmes W. M., Hurd R. E., Reid B. R., Rimerman R. A. & Hatfield G. W. (1975) Proc. natn Acad. Sci. U.S.A. 72, 1068. King T. P. (1972) Biochemistry 11, 367. Memoli V. A., Miyayama H. & Doellgast G. J. (1975) J. Histochem. Cytochem. 23, 325 (abstract). Porath J., Sundberg L.. Fornstedt N. & Olsson I. (1973) Nature, Lond. 245, 464. Rimerman R. A. & Hatfield G. W. (1973) Science 182, 1268. Scheidegger J. J. (1955) Int. Archs Allergy appl. lrnmunol. 7, 103. Shaltiel S. & Er-EI Z. (1973) Proc. natn Acad. Sci. U.S.A. 70, 778. Shaltiel S., Ames F. L. G. & Noel K. D. (1973) Archs Bioehem. Biophys. 159, 174. Von Hippel P. H. & Schleich T. (1969) in Structure and Stability of Biolo¢lical Macrom,lecules (Edited by TimasheffS. N. & Fasman G. D.) Chap. 6. Marcel Dekker, New York. Yon R. J. 11974) Biochem. J. 137, 127.

Purification of human IgA by salt-mediated hydrophobic chromatography.

InlmumJ~hemistry, 1976. Vol, 13, pp. 135 139. Pergamon Press. Printed in Great Britain PURIFICATION OF HUMAN IgA BY SALT-MEDIATED HYDROPHOBIC CHRO...
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