[9]

BIAFFINITY CHROMATOGRAPHYOF FGF

[9] B i a f f i n i t y C h r o m a t o g r a p h y By

91

of Fibroblast Growth Factors

YUEN SHING

Heparin affinity chromatography was first used to purify a tumorderived angiogenic endothelial mitogen in 1984.1 It has since been widely used for the purification of fibroblast growth factors (FGF) from a large variety of tissue sources (for review, see Refs. 2-4). We subsequently found that F G F also contains a separate binding site for copper. This led to the development of heparin-copper biaffinity chromatography, 5'6 which was an improvement over previous methods owing to its capacity to resolve multiple forms of FGF. This chapter describes the procedures for the purification of basic and acidic F G F using this novel chromatographic technique. Principle of Method The principle of heparin-copper biaffinity chromatography is illustrated diagrammatically in Fig. 1. The success of the method depends on thorough rinsing of the biaffinity column with alternate solutions of 2 M NaCl and l0 m M imidazole in the presence of 0.6 M NaCl before starting the NaCl/imidazole gradient. In addition, the optimal gradient concentrations of NaC1 and imidazole for purifying a certain specific form of F G F might have to be determined empirically based on the individual affinities for heparin and copper. Growth Factor Assays Fibroblast growth factors stimulate DNA synthesis in both mouse BALB/c 3T3 cells and bovine capillary endothelial cells. Measurement of the stimulation of DNA synthesis in 3T3 cells is a relatively simple but nonspecific assay. Methods for this assay have been reviewed in a previous volume in this series 7 and are not discussed here. The capillary endothelial l y. Shing, J. Folkman, R. Sullivan, C. Butterfield,J. Murray, and M. Klagsbrun, Science 223, 1296(1984). 2j. Folkman and M. Klagsbrun, Science 235, 442 (1987). 3 D. Gospodarowicz, Crit. Rev. Oncogenesis 1, 1 (1989). 4 W, H. Burgess and T. Maciag, Annu. Rev. Biochem. 58, 575 (1989). 5 y. Shing, J. Biol. Chem. 263, 9059 (1988). 6 y. Shing, J. Folkman, and M. Klagsbrun, Ann. N.Y. Acad. Sci. 556, 166 (1989). 7 y. Shing, S. Davidson, and M. Klagsbrun, this series, Vol. 146, p. 42. METHODS IN ENZYMOLOGY, VOL. 198

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

92

FIBROBLAST GROWTH FACTOR

Elufion Reagents

[9]

Biaffinity Column

2 M NaCI

~Heparin C/opper-

0.6 M NaCi

[ "'rar~

0.6M NaCI • I0 mM Imidazole L

L

.,

i.

0.6 M NaCI

l""ar'~

I

7"rPer"

~opper.

r=

_7 rper"

(Gradlent)

2

M NaC l • I0 mM Imidazole ~l.lenl___~arin I

Coppe.Ir~ I

FIG. 1. Illustration of the principle of heparin-copper biaffinity chromatography. When the column as shown at right is initially rinsed with 2 M NaCl after the FGF-containing sample has been loaded, most of the heparin-binding proteins, including FGF, are detached from the heparin moiety but FGF remain bound to the column because of their affinity for copper. This step eliminates most of the heparin-binding proteins except those which are being bound to copper, such as FGF. On the other hand, when the column is subsequently rinsed with l0 mM imidazole in 0.6 M NaCl, most of the copper-binding proteins including FGF are detached from the copper moiety but FGF remain bound to the column because of their affinity for heparin. This step eliminates most of the copper-binding proteins. At this point, virtually all of the non-FGF proteins should have been eluted from the column. The remaining FGF can then be eluted with a linear NaCl/imidazole gradient, and the various forms of FGF are eluted according to their respective affinity for both heparin and copper. (From Shing. 5)

cell DNA synthesis assay is much more specific. This method is particularly useful in the study of endothelial cell-specific growth factors and is presented here. Measurement of DNA Synthesis in Capillary Endothelial Cells. Capillary endothelial cells are prepared from bovine adrenal glands and grown

[9]

BIAFFINITYCHROMATOGRAPHYOF FGF

93

on gelatin-coated dishes as described by Folkman et al. 8 For assay, endothelial cells are trypsinized and resuspended in Dulbecco's modified Eagle's medium (DMEM, 1 g/liter glucose) supplemented with 10% calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100/xg/ml streptomycin (Irvine Scientific, Santa Ana, CA) and plated sparsely (10,000 cells/0.4 ml/ well) into gelatinized 48-well microtiter plates (Costar, Cambridge, MA). On the following day, the medium in each well is replaced by 0.4 ml of DMEM containing 2% calf serum, bovine serum albumin (BSA; 5 mg/ml), and thymidine (0.2/zg/ml). One day later, 5-30/xl of test samples is added. (Samples from the biaffinity column are usually diluted 20- to 40-fold with 0.1% BSA in saline before assaying.) Sixteen hours later, 10 tzl (0.6 ~zCi/ well) of [methyl-3H]thymidine (85 Ci/mmol, NEN, Boston, MA) is added. Four hours later, cells are fixed by washing the microtiter plates consecutively with phosphate-buffered saline (PBS), methanol (twice, 5 min each), 5% cold trichloroacetic acid (twice, 10 min each), and water. The cells are lysed by addition of 200 tzl of 0.3 N NaOH, and the lysates are transferred to scintillation vials. Three milliliters of scintillation fluid (Ecolume, ICN, Costa Mesa, CA) is added, and the vials are counted in a Beckman Model LS1800 scintillation counter. Tritiated thymidine incorporation in this assay varied from batch to batch of cultures for reasons that are not totally clear. In general, the background incorporation with saline is about 2000 counts/min (cpm), and the maximal stimulation obtained by FGF is about 24,000 cpm. Isolation of Growth Factor Bovine hypothalami (100 g) obtained from Pel-Freez (Rogers, AR) are homogenized in 300 ml of 0.15 M (NH4)2SO 4 at pH 6 and extracted by stirring at 4° for 2 hr. The crude extract is centrifuged at 15,000 g for 1 hr, and the supernatant solution is diluted with about 100 ml of 0.15 M (NH4)2SO 4 and centrifuged once again to remove the remaining residue. The clear supernatant solution is loaded directly onto a heparin-Sepharose column (1.5 × 12 cm) preequilibrated with 0.6 M NaCI in 10 mM Tris, pH 7. The column is rinsed with 300 ml of 0.6 M NaC1 in 10 mM Tris, pH 7. FGF are subsequently eluted with 40 ml of 2 M NaCI in the same buffer. This represents the starting material for the purification procedures described in this chapter.

s j. Folkman, C. Haudenschild, and B. R. Zetter, Proc.Natl. Acad. Sci. U.S.A. 76, 5217 (1979).

94 A.

FIBROBLAST

GROWTH

[9]

FACTOR

7 6

2 M NaCl + 10 m M I m i d a s o l e

T= "~ 5

I

[

I Q

fJ m m

1.$ M NaC1 + §mM ImJd~ole

3

~2

Q N I

0.6 M NaCI +

0 mM Imiduole

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zt 2

3

4

5

B

7

8

g I0 It 12 13 14 15 t5 17 18 19 20 MW -___=COO0_

I

' -21000

B. Silver Stain

-14300

C. W e s t e r n B l o t ( A n t i - b a s i c FGF)

D. W e s t e r n B l o t ( A n t i - a c i d i c FGF) 9 I0 11 12 13 14 15 15 17 18 19 20 FRACTION

NUMBER

FIG. 2. Heparin-copper biaffinity chromatography of FGF isolated from hypothalamus. Samples of bovine hypothalamic FGF partially purified by heparin-Sepharose were loaded directly onto a heparin-copper biaffinity column. The column was rinsed with alternate solutions and then eluted with a linear NaCl/imidazole gradient. Aliquots of the eluates were assayed for growth factor activity by measurement of DNA synthesis in 3T3 cells (A). Proteins contained in the indicated fractions were analyzed by SDS-gel electrophoresis followed by silver staining (B). Identical samples from all indicated fractions were also analyzed by immunoblotting (Western blot), being probed with antibodies raised against basic FGF (C) and with antibodies raised against acidic FGF (D). (Adapted from Shing))

H e p a r i n - C o p p e r Biaffinity C h r o m a t o g r a p h y

The heparin-copper biaffinity column is prepared by mixing 3.5 ml each of heparin-Sepharose (Pharmacia, Piscataway, N J) and chelating S e p h a r o s e ( P h a r m a c i a ) that has b e e n s a t u r a t e d with c o p p e r ( I I ) chloride.

The sample (40 ml) of FGF partially purified by batchwise adsorption to

[9]

BIAFFINITY CHROMATOGRAPHYOF FGF

95

heparin-Sepharose is applied directly to the blue-colored biaffinity column (1 × 9 cm) preequilibrated with 2 M NaC1, 10 mM Tris, pH 7. The column is rinsed consecutively with 40 ml each of the following four reagents in 10 mM Tris, pH 7: (i) 2 M NaCI, (ii) 0.6 M NaCI, (iii) 0.6 M plus 10 mM imidazole, and (iv) 0.6 M NaCI. Finally, FGF are eluted at a flow rate of 20 ml/hr with a linear NaC1/imidazole gradient from 100 ml of 0.6 M NaC1 without imidazole to 100 ml of 2 M NaC1 plus 10 mM imidazole in 10 mM Tris, pH 7. Fractions eluted from the column are analyzed by sodium dodecyl sulfate-gel electrophoresis 9 with silver staining 1°and immunoblotting jl using site-specific antibodies against basic and acidic FGF. 12 A typical result as shown in Fig. 2 demonstrates that it is possible for this biaffinity chromatography to resolve from hypothalamus at least two basic FGF species (with M r values of 19,000 and 18,000) and three acidic FGF species (with Mr values of 18,000, 16,400, and 15,600). Comments The principle of biaffinity chromatography apparently can also be applied to purify many other proteins which have affinities for more than one ligand. For example, isolation of three urokinase-related proteins has been reported by the use of benzamidine-zinc biaffinity chromatography. 13 Furthermore, it has been shown recently that basic FGF can be purified from a tumor source by the use of tetradecasulfated/3-cyclodextrin-copper biaffinity column. 14The physiological significance of the existence of structurally similar FGF in various tissues is not clear. The observation that they can be separated based on their differential affinities for heparin and copper may eventually lead to the development of chromatographic conditions that allow the further identification and purification of varied FGF molecules in various tissue and species sources.

9 U. K. Laemmli, Nature (London) 227, 680 (1970). 10 B. R. Oakley, D. R. Kirsch, and N. R. Morris, Anal. Biochern. 105, 361 (1980). u M. Klagsbrun, J. Sasse, R. Sullivan, and J. A. Smith, Proc. Natl. Acad. Sci. U.S.A. 83, 2448 (1986). 12 M. Wadzinski, J. Folkman, J. Sasse, K. Devey, D. Ingber, and M. Klagsbrun, Clin. Physiol. Biochem. 5, 200 (1987). 13 D. Zhu, J. Fu, J. Wu, and Y. Shing, "Intracellular Proteolysis, Proceedings of the 7th ICOP Meeting," p. 150. 1989. i4 y. Shing, J. Folkman, P. B. Weisz, M. M. Joullie, and W. R. Ewing, Anal. Biochem. 185, 108 (1990).

Biaffinity chromatography of fibroblast growth factors.

[9] BIAFFINITY CHROMATOGRAPHYOF FGF [9] B i a f f i n i t y C h r o m a t o g r a p h y By 91 of Fibroblast Growth Factors YUEN SHING Heparin af...
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