Heparin-Copper Biaffinity Chromatography of Fibroblast Growth Factors*

A novel method is described to separate and identify the various forms of fibroblast growth factor (FGF) based on their differential affinities for both heparin and copper. FGFs were extracted from bovine hypothalamus and purified by batchwise adsorption to hep-arin-Sepharose. The partially purified FGFs were then applied to an affinity column prepared by mixing equal portions of heparin-Sepharose and copper-Sepharose. The column was rinsed consecutively with the following four reagents: (i) 2 M NaCl, (ii) 0.6 M NaCl, (iii) 0.6 M NaCl plus 10 mM imidazole, and (iv) 0.6 M NaCl. FGFs were then eluted with a linear NaCl/imidazole gradient (from 0.6 M NaCl without imidazole to 2 M NaCl plus 10 mM imidazole). Fractions eluted from the column were analyzed by sodium dodecyl sulfate-gel electrophoresis with silver staining and electrophoretic immunoblot using site-specific antibodies against basic and acidic FGF. The results demonstrate that it is possible to resolve from hypothalamus at least two basic FGF species (with M, values of 19,000 and 18,000) and three acidic FGF species (with M, values of 18,000, 16,400, and 15,600). These findings indicate that heparin-copper biaffinity chromatography may have wide applicability in the study of the structure and activity of FGFs. intervals with 200 pg of KLH-peptide conjugate emulsified in incomplete Freund’s adjuvant. The titer of the antisera after the second booster injection was about 1:15,000 to 1:50,000 as determined in an enzyme-linked immunosor-bent assay using unconjugated peptide as the antigen. Zmmunoblotting (Western Blot)-Proteins were separated by elec- trophoresis on SDS-15% polyacrylamide gels and transferred electro-phoretically to a nitrocellulose sheet as previously described (35). The nitrocellulose sheet was incubated with antiserum against either bFGF or aFGF and visualized by successive incubations with bioti- nylated goat anti-rabbit IgG, streptavidin-biotinylated pe:oxidase complex, and enzyme substrate (4-chloro-1-naphthol) (36).

Heparin affinity chromatography has been widely used for the purification of endothelial cell growth factors from a large variety of tissue sources since we first reported it (1,2) (for review see . Virtually all endothelial cell growth factors bind avidly to heparin. On the basis of their isoelectric points and their respective affinities for heparin, the heparinbinding endothelial cell mitogens can be subdivided into two classes, typified by basic fibroblast growth factor (bFGF)' and acidic fibroblast growth factor (aFGF) (6,7). Recently, the amino acid sequences of bFGF (8) and aFGF (9)(10)(11)(12) have been established. However, both bFGF (13)(14)(15)(16)(17) and aFGF (18)(19)(20) have been shown to exist in multiple molecular weight forms. All of them are heat-and acid-labile. This precludes the use of reverse-phase high performance liquid chromatography for their isolation in their native form (5). To date, the physiological significance of these structurally similar FGFs in various tissues is not clear, and it has been difficult to isolate them in sufficient quantities for further investigation.
* This work was supported by a grant to Harvard University from Takeda Chemical Industries, Ltd. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
FGFs that have a broad spectrum of target cells in culture have been reported to induce vascular growth (angiogenesis) in vivo (8,21). In attempting to further understand the mechanism of these activities and the heparin affinity of FGFs, Folkman and Klagsbrun (3) speculated that the binding of FGFs to heparin might be copper-dependent. This idea was based on several reports in the literature indicating that copper levels in tissue could somehow modulate the intensity of the neovascular response to a given angiogenic stimulus. Such reports included the observations that (i) copper ions augment endothelial locomotion in vitro (22), (ii) rabbits on a copper-deficient diet are unable to mount an angiogenic response to prostaglandin El (23) and to implants of a variety of human brain tumors (24), (iii) both ceruloplasmin and heparin become angiogenic when complexed to copper, but not when deprived of copper (25), and (iv) heparin can act as a copper chelator (26, 27). To test this hypothesis, I found instead that while copper was not required for the binding of FGFs to heparin, FGFs appear to have separate binding sites for copper and for heparin. By taking advantage of the unique interactions between heparin, copper, and FGFs, I was able to develop a novel method to resolve the multiple forms of FGFs, based upon biaffinity chromatography, the details of which are presented here.

EXPERIMENTAL PROCEDURES
Isolation of FGFs from Hypothulnmus-Bovine hypothalami (100 g) obtained from Pel-Freez were homogenized in 300 ml of 0.15 M (NH1)2S04 at pH 6 and extracted by stirring at 4 "C for 2 h. The crude extract was centrifuged at 15,000 X g for 1 h, and the supernatant solution was loaded directly onto a heparin-Sepharose column (1.5 X 12 cm) pre-equilibrated with 0.6 M NaCl in 10 mM Tris, pH 7. The column was rinsed with 300 ml of 0.6 M NaCl in 10 mM Tris, pH 7. FGFs were subsequently eluted with 40 ml of 2 M NaCl in the same buffer.
Heparin-Copper Biuffinity Chromtography-The heparin-copper biaffinity column was prepared by mixing 3.5 ml each of heparin-Sepharose (Pharmacia LKB Biotechnology Inc.) and chelating Sepharose (Pharmacia LKB Biotechnology Inc.) that had been saturated with copper(I1) chloride. The sample (40 ml) of FGF partially purified by batchwise adsorption to heparin-Sepharose was applied directly to this blue color biaffinity column (1 X 9 cm) pre-equilibrated with 2 M NaC1, 10 mM Tris, pH 7. The column was rinsed consecutively with 40 ml each of the following four reagents in 10 mM Tris, pH 7 (i) 2 M NaCl, (ii) 0.6 M NaC1, (iii) 0.6 M NaCl plus 10 mM imidazole, and (iv) 0.6 M NaCl. Finally, FGFs were eluted at a flow rate of 20 ml/h with a linear NaCl/imidazole gradient from 100 ml of 0.6 M NaCl without imidazole to 100 ml of 2 M NaCl plus 10 mM imidazole in 10 mM Tris, pH 7. Fractions (10 ml) were collected and assayed for growth factor activity.
Growth Factor Assay-FGF activity was assessed by measuring the incorporation of f'Hlthymidine into the DNA of quiescent, confluent monolayers of BALB/c mouse 3T3 cells in 96-well plates as previously described (28). One unit of activity was defined as the amount of growth factor required to stimulate half-maximal DNA synthesis in 3T3 cells (about 10,000 cells/0.25 ml of growth medium/well). For 9059 determination of specific activities, protein concentrations of crude extract and the active fraction eluted from heparin-Sepharose column were determined by the method of Lowry et al. (29). Protein concentrations of the pure FGFs eluted from biaffinity column were estimated by comparing the intensities of silver-stained FGF bands on SDS-polyacrylamide gel electrophoresis to those of the protein standards, which were prepared by a serially 2-fold dilution of @-lactoglobulin from 800 ng to 50 ng.
SDS-Polyacrylamide Gel Electrophoresis-FGF-containing samples were analyzed by electrophoresis on 15% polyacrylamide gels as described by Laemmli (30). The polypeptide bands were visualized by a silver stain (31).

RESULTS
Hypothalamus is a rich source of mitogens. Approximately 7 X lo6 units of growth factor activity were extracted from 100 g of tissue. More than 95% of this activity bound to heparin-Sepharose in 0.6 M NaCl and were eluted with 2 M NaC1. A single passage of the bovine hypothalamic crude extract over 20 ml of heparin-Sepharose resulted in approximately 100-fold purification. FGFs were further purified by chromatography on a heparin-copper biaffinity column as described under "Experimental Procedures.'' The results were summarized in Table I. This purification was possible because of the unique affinities of the multiple forms of FGF for heparin and copper. The principle of this biaffinity chromatography is diagrammatically illustrated in Fig. 1. The success of this method depended on thorough rinsing of the column with alternate solutions of 2 M NaCl and of 10 mM imidazole in the presence of 0.6 M NaCl before starting the NaC1/ imidazole gradient. After the rinses, FGFs were eluted with a linear NaCl/imidazole gradient and the growth factor activities in the eluates were measured (Fig. 2, panel A ) . growth factor activity was detected starting at fraction 9, which corresponded to about 1.3 M NaCl and 5 mM imidazole.
Analysis of the eluates from fraction 9 to fraction 20 by SDSpolyacrylamide gel electrophoresis followed by silver staining revealed the existence of multiple protein bands (Fig. 2, panel  B ) . Further analysis of these protein bands by electrophoretic immunoblot (Western blot) using site-specific polyclonal antibodies raised against either bFGF (Fig. 2, panel C), or aFGF ( Fig. 2, panel D) demonstrated that it was possible to resolve from hypothalamus at least two bFGF species with M, values of 19,000 and 18,000 and three aFGF species with M, values of 18,000, 16,400, and 15,600. Basic FGF (fractions 10 to 13) and acidic FGF (fractions 14 to 20) triggered a half-maximal response at concentrations of 0.24 and 1 ng/ml, respectively. Interestingly, they appear to elute from the heparin-copper biaffinity column in an order opposite of that from the heparin affinity column alone (28). This result indicates that aFGF species probably have stronger affinities for copper than those of the bFGF species.

DISCUSSION
Basic FGF was first sequenced and initially reported as a 146-amino acid polypeptide (8). Subsequently, both aminoterminally extended (13, 14) and truncated (15)(16)(17) forms of bFGF were demonstrated in a variety of cells and tissues. Recently, the gene for bovine bFGF has been cloned (37), and the nucleotide sequence predicts a 155-amino acid bFGF translation product. This is consistent with the finding of the existence of a 154-amino acid form of bFGF which is extended by 8 residues on the amino-terminal side (13). Based on these facts and the apparent molecular weights of FGFs shown in Fig. 2, it is likely that the M, = 18,000 band of hypothalamic bFGF species corresponds to the 154-amino acid form of bFGF. This raises an interesting possibility that the M, = 19,000 band of hypothalamic bFGF species might correspond to a larger precursor of bFGF.
A primary structure of 140 amino acids has been determined for acidic FGF (9)(10)(11)(12). Similar to bFGF, both amino-terminally extended and truncated forms of aFGF have been demonstrated (19,20). Amino acid sequence analysis of a 154amino acid extended form and a 134-amino acid truncated form of aFGF (19) and the original 140-amino acid of aFGF itself are all in agreement with the gene sequence data (38). These are consistent with the possibility that the M, = 18,000, 16,400, and 15,600 bands of the hypothalamic aFGF species shown in Fig. 2 actually correspond to a 154-amino acid precursor of aFGF, the 140-amino acid aFGF and a 134-amino acid truncated form of aFGF, respectively.
We have previously reported the separation of hypothalamic bFGF and aFGF by heparin-Sepharose affinity chromatography (28). With heparin-copper biaffinity chromatography, the multiple forms of both basic and acidic FGFs can further be resolved and identified. An additional advantage of using the biaffinity column includes its capability of purifying FGFs from the heparin-contaminated sample. Furthermore, the principle of this biaffinity chromatography appar-  When the column as shown in the right panel is initially rinsed with 2 M NaCl after the FGF-containing sample has been loaded, most of the heparin-binding proteins including FGFs are detached from the heparin moiety, but FGFs remain bound to the column because of its affinity for copper. This step eliminates most of the heparin-binding proteins except those which are being bound to copper such as FGFs. On the other hand, when the column is subsequently rinsed with 10 mM imidazole in 0.6 M NaCI, most of the copper-binding proteins including FGFs are detached from the copper moiety, but FGFs remain bound to the column because of its affinity for heparin. This step eliminates most of the copper-binding proteins except those which are being bound to heparin such as FGFs. A t this point, virtually most of the non-FGF proteins should have been eluted from the column. The remaining FGFs can then be eluted with a linear NaCl/imidazole gradient and the various forms of FGFs are eluted according to their respective affinity for both heparin and copper. ently can be applied to purify many other proteins which have affinities for more than one ligand. For example, a lysine-zinc biaffinity column would probably be very useful in identifying and purifying plasminogen activators which have been reported to have affinities for both lysine (39) and zinc (40).
Acidic FGF species appear to have a stronger affinity for copper than those of the bFGF species. This unique characteristic of aFGF allows them to bind more avidly to the copper moiety of the biaffinity column and therefore greatly facilitates their separation from bFGF. However, copper-Sepharose column alone following heparin-Sepharose column is not sufficient for the complete purification of either bFGF or aFGF (data not shown). Recently, copper-Sepharose affinity chromatography has been reported in the purification of a human mammary tumor-derived growth factor, which binds to heparin and has a molecular weight of 16,000 (41). The relationship of this growth factor to the previously reported M, = 18,000 heparin-binding mitogen purified from rat chondrosarcoma (2,42) and the hypothalamus-derived FGFs demonstrated here is presently unknown. However, in view of their Heparin-copper biaffinity chromatography of FGFs isolated from hypothalamus. Samples of bovine hypothalamic FGF partially purified by heparin-Sepharose as described under "Experimental Procedures" were loaded directly onto a heparin-copper biaffinity column. The column was rinsed as described under "Experimental Procedures." FGFs were then eluted with a linear NaCI/ imidazole gradient, and aliquots of the eluates were assayed for growth factor activity of which the result is shown in panel A. The inset to panel A is the photography of a silver-stained gel showing the protein profile of a FGF sample prior to the biaffinity chromatography. The protein markers (phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, @-lactoglobulin, and lysozyme) were shown in the right lane of the inset. Proteins contained in the indicated fractions shown in panel A were analyzed by SDSgel electrophoresis followed by silver staining which is shown inpanel E . Identical samples from all of the respective indicated fractions were also analyzed by immunoblotting (Western blot) probed with antibodies raised against bFGF as shown in panel C and with antibodies raised against aFGF as shown in panel D.
reported characteristics, it is conceivable that they are structurally related although their relationship can only be ascertained by direct comparison of their amino acid sequences. Despite the apparent importance of FGF, its exact structure has been the subject of some controversy since its identification. Due to the differences in tissue and species sources as well as purification procedures, it has been difficult to make direct comparisons of the various forms of FGFs isolated in separate laboratories. Furthermore, although it has been well established that both basic and acidic FGFs exist in multiple forms, it has been difficult to isolate them in their native forms in a reproducible manner. The observation that various FGF species can be separated and eluted from the biaffinity column in an order of descending molecular weight as shown in Fig. 2 may eventually lead to the development of a chromatographic condition that allows the further identification and complete separation of the native forms of the varied FGF molecules in various tissue and species sources.