Chemical heterogeneity of heparan sulfate from a human neuroblastoma cell line

The chemical heterogeneity of radiolabelled neuroblastoma heparan sulfate has been studied by ion exchange chromatography and by affinity chromatography on heparan sulfate-agarose. Although the entire population of chains shows considerable homogeneity in charge density, the deaminative cleavage products ranged in size from disaccharides to eicosasaccharides. Under appropriate conditions neuroblastoma heparan sulfate could be separated into two pools of low or high affinity for lung heparan sulfate-agarose. Analyses of periodate oxidation-alkaline elimination indicated that the high affinity chains contained larger proportions of heparin-like segments, i.e. iduronate-rich and N-sulfated ones.     

Detection of chondroitin sulfate-binding proteins on the membrane

Chondroitin sulfate is a ubiquitous component of proteoglycans that is present both in the extracellular matrix and at the cell surface of various tissues. Until recently, chondroitin sulfate has attracted less attention than heparan sulfate and dermatan sulfate, owing to the limited number of known chondroitin sulfate-binding proteins. To determine the biological function of chondroitin sulfate, biotinylated probes were prepared and used to search for binding proteins. Chondroitin sulfates A, C, D, and E were biotinylated through either the uronic acid or the residual core peptide. Lysates from mouse Lewis lung carcinoma (3LL) cells were blotted onto a nitrocellulose membrane after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the membrane was treated with the biotinylated chondroitin sulfates. Among the chondroitin sulfate variants, the E type showed the most intense bands upon visualization of the membrane with avidin-conjugated alkaline phosphatase and the appropriate substrates. The binding of chondroitin sulfate E to proteins in the cell lysate was not affected by the A, C or D variants but was reduced by treatment with dermatan sulfate. Lysates from 3LL cells were also treated with biotinylated chondroitin sulfate E and, after two-dimensional (2-D) electrophoresis and blotting, several chondroitin sulfate E-binding proteins including lamins and heterogeneous nuclear ribonucleoproteins were identified by mass spectrometry.     

Chemical and immunological studies of cell surfaces from normal and transformed cells.

Immunological and chemical studies of cell surfaces from normal and transformed BALB/c fibroblasts have shown alterations associated with transformation. The cells studied include normal lines which do not cause tumors when injected into BALB/c mice, viral transformants, and spontaneous transformants which cause tumors that either regress or grow progressively, killing the host. The spontaneously transformed progressors include cell lines which are immunogenic and nonimmunogenic as determined by the ability of tumor excision to protect an animal from subsequent rechallenge by tumor cells. Tumor-bearing mice produce lymphocytes which are nonspecifically cytotoxic for all the normal and transformed lines. Some of the cell lines induce specific antibody formation in BALB/hosts. Antisera have been prepared in rabbits which are specific for the transformed cell lines. These antisera can be used to determine specific surface changes on the transformed cells. Chemical studies have shown glycolipid alterations between the normal cells and some, but not all, of the transformants. Glycoproteins labeled by lactoperoxidase-125I or [3H] glucosamine were compared by SDS gel electrophoresis. Results from these studies do not show changes associated with malignancy. Individual glycoprotein regions from gels were treated with pronase, and the glycopeptides compared by Sephadex G50 chromatography. Alterations in glycopeptides from several cellular glycoproteins are the only changes which appear to be associated with malignancy.     

Synthesis and distribution of glycosaminoglycans in human leukemic B- and T-cells and monocytes studied using specific enzymic treatments and high-performance liquid chromatography

Identification of glycosaminoglycans (GAGs) synthesized by three human leukaemic cell lines-Jurkat (T-cell leukaemia), Daudi (Burkitt's lymphoma, B-cell leukaemia) and THP-1 (acute monocytic leukemia)-and normal peripheral blood mononuclear cells (PBMC) and their distribution among cell membrane and culture medium were studied. GAGs were isolated using ion-exchange chromatography on DEAE-Sephacel and their composition and fine chemical structure were studied using high-performance liquid chromatography with radiochemical detection. All cell lines synthesize chondroitin sulphate (CS) and heparan sulphate (HS) in both cell membrane and culture medium. No hyaluronan was detected using treatment with specific lyases and highly sensitive HPLC methodology. CS is the major secreted GAG in all cell lines tested and the major cell retained GAG in Jurkat and Daudi. HS is the major GAG in the cell membrane of THP-1. The amounts of distinct GAGs synthesized by all cancer cell lines differ from those produced by normal PBML indicating a major role of GAGs in malignant transformation of human lymphocytes and monocytes.     

Application of heparinized cellulose affinity membranes in recombinant adeno-associated virus serotype 2 binding and delivery

The microporous affinity membrane based on cellulose matrices offers minimal mass-transfer effects in membrane chromatography with low nonspecific adsorption. In this research, we tested a novel application of the microporous, heparinized cellulose membranes for their affinity toward recombinant adeno-associated virus serotype 2 (rAAV2, which uses heparan sulfate proteoglycans as the primary cellular receptor) to develop a controlled, substrate-mediated viral vector delivery. We conjugated rAAV2 to an epoxy-crosslinked heparin cellulose membrane, which led to vector transduction upon cellular adhesion. When adhered, human fibroblasts exhibited proliferation kinetics similar to those on the standard polystyrene tissue-culture surface. Using fluorescent proteins as the reporter, we showed that the heparin-bound rAAV2 particles remained active and that the rAAV2-heparin binding was reversible and capable of mediating transgene delivery in cell culture. In addition, we applied the affinity membrane to adsorb unpurified rAAV2 from the crude lysate of packaging cells via the ligand–receptor binding, avoiding the use of conventional ultracentrifugation or chromatography in preparation of infectious rAAV2 for transduction. Our work explores a new application of affinity cellulose membranes in substrate-mediated viral vector delivery, which can be a useful tool in developing protocols for localized gene transfer.     

Hyphenated techniques for the analysis of heparin and heparan sulfate

The elucidation of the structure of glycosaminoglycan has proven to be challenging for analytical chemists. Molecules of glycosaminoglycan have a high negative charge and are polydisperse and microheterogeneous, thus requiring the application of multiple analytical techniques and methods. Heparin and heparan sulfate are the most structurally complex of the glycosaminoglycans and are widely distributed in nature. They play critical roles in physiological and pathophysiological processes through their interaction with heparin-binding proteins. Moreover, heparin and low-molecular weight heparin are currently used as pharmaceutical drugs to control blood coagulation. In 2008, the health crisis resulting from the contamination of pharmaceutical heparin led to considerable attention regarding their analysis and structural characterization. Modern analytical techniques, including high-performance liquid chromatography, capillary electrophoresis, mass spectrometry, and nuclear magnetic resonance spectroscopy, played critical roles in this effort. A successful combination of separation and spectral techniques will clearly provide a critical advantage in the future analysis of heparin and heparan sulfate. This review focuses on recent efforts to develop hyphenated techniques for the analysis of heparin and heparan sulfate.     

Fibronectin and proteoglycans as determinants of cell-substratum adhesion.

When normal or SV40-transformed Balb/c 3T3 cells are treated with the Ca++-specific chelator EGTA, they round up and pull away from their footpad adhesion sites to the serum-coated tissue culture substrate, as shown by scanning electron microscope studies. Elastic membranous retraction fibers break upon culture agitation, leaving adhesion sites as substrate-attached material (SAM) (Cells leave "footprints" of substrate adhesion sites during movement by a very similar process.) SAM contains 1-2% of the cell's total protein and phospholipid content and 5-10% of its glucosamine-radiolabeled polysaccharide, most of which is glycosaminoglycan (GAG). By one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, there is considerable enrichment in SAM for specific GAGs; for the glycoprotein fibronectin; and for the cytoskeletal proteins actin, myosin, and the subunit protein of the 10 nm-diameter filaments. Fibrillar fibronectin of cellular origin and substratum-bound fibronectin of serum origin (cold-insoluble globulin, CIg) have been visualized by immunofluorescence microscopy. The GAG composition in SAM has been examined under different cellular growth and attachment conditions. Heparan sulfate content correlates with glycopeptide content (derived from glycoprotein). Newly attaching cells deposit SAM with principally heparan sulfate and fibronectin and little of the other GAGs. Hyaluronate and chrondroitin proteoglycans are coordinately deposited in SAM as cells begin spreading and movement over the substrate. Cells attaching to serum-coated or CIg-coated substrates deposited SAM with identical compositions. The proteoglycan nature of the GAGs in SAM has been examined, as well as the ability of proteoglycans to form two classes of reversibly dissociable "supramolecular complexes" - one class with heparan sulfate and glycopeptide-containing material and the second with hyaluronate-chondroitin complexes. Enzymatic digestion of "intact" SAM with trypsin or testicular hyaluronidase indicates that (1) only a small portion of long-term radiolabeled fibronectin and cyto-skeletal protein is bound to the substrate via hyaluronate or chondroitin classes of GAG; (2) most of the fibronectin, cytoskeletal protein and heparan sulfate coordinately resist solubilization; and (3) newly synthesized fibronectin, which is metabolically labile in SAM, is linked to SAM by hyaluronate- and/or chondroitin-dependent binding. All of our studies indicate that heparan sulfate is a direct mediator of adhesion of cells to the substrate, possibly by binding to both cell-surface fibronectin and substrate-bound CIg in the serum coating; hyaluronate-chondroitin complexes in SAM appear to be most important in motility of cells by binding and labilizing fibronectin at the periphery of footpad adhesions, with subsequent cytoskeletal disorganization.     

Analysis of allergens in tubeimu saponin extracts by using rat basophilic leukemia 2H3 cell‐based affinity chromatography coupled to liquid chromatography and mass spectrometry

An affinity two‐dimensional chromatography method was developed for the recognition, separation, and identification of allergic components from tubeimu saponin extracts, a preparation often injected to treat various conditions as indicated by traditional Chinese medicine. Rat basophilic leukemia‐2H3 cell membranes were used as the stationary phase of a membrane affinity chromatography column to capture components with affinity for mast cells that could be involved in a degranulation reaction. The retained components were enriched and analyzed by membrane affinity chromatography with liquid chromatography and mass spectrometry via a port switch valve. Suitability and reliability of the method was investigated using appropriate standards, and then, the method was applied to identify components retained from tubeimu saponin extracts. Tubeimoside A was identified in this way as a potential allergen, and degranulation assays confirmed that tubeimoside A induces RBL‐2H3 cell degranulation in a dose‐dependent manner. An increase in Ca²⁺ influx indicated that degranulation induced by tubeimoside A is likely Ca²⁺ dependent. Coupled with the degranulation assay, RBL‐2H3 cell‐based affinity chromatography coupled with liquid chromatography and mass spectrometry is an effective method for screening and identifying allergic components from tubeimu saponin extracts.     

Aluminium Mediated Glycosaminoglycan/Amyloid-β Association Mechanism

In Alzheimer’s disease, it has been proposed that glycosaminoglycans facilitate amyloid fibril formation and/or stabilize the plaque aggregates. Chondroitin sulfates are sulfated glycosaminoglycans represented an ideal distribution of charge for amyloid-β (Aβ) interactions. Recent studies have suggested a possible link between the neurotoxicity of aluminum and the pathogenesis of Alzheimer’s disease. In this paper, the interaction of Aβ with chondroitin sulfates immobilized on a chromatographic column and the role of aluminum had been studied using a biochromatographic approach (molecular chromatography). A novel biochromatographic column was developed in our laboratory for studying this interaction. This study demonstrated that the aluminum interacted with Aβ and played a role in the Aβ/chondroitin sulfates association. For a Al³⁺ concentration (x) in the medium less than 30 μmM, the Aβ/chondroitin sulfates binding decreased with x due to a decrease of the charge–charge interactions between Aβ and its chondroitin sulfates binding site. Above 30 μmM of Al³⁺ in the medium, the affinity of Aβ to chondroitin sulfates increased slightly with x because the net number of ions (n) (Al³⁺ or Cl^?) released or bound upon complex formation is low. As well, it was clearly demonstrated, that above 30 μmM the n value depend on the Al³⁺ concentration in the bulk solvent. This dependence was due to a gradual and conformational change of the Aβ which around 80 μmM adopted a less flexible structure; its binding site was thus less accessible to Aβ and Aβ/chondroitin sulfates association decreased slightly.     

Aluminium Mediated Glycosaminoglycan/Amyloid-β Association Mechanism

In Alzheimer’s disease, it has been proposed that glycosaminoglycans facilitate amyloid fibril formation and/or stabilize the plaque aggregates. Chondroitin sulfates are sulfated glycosaminoglycans represented an ideal distribution of charge for amyloid-β (Aβ) interactions. Recent studies have suggested a possible link between the neurotoxicity of aluminum and the pathogenesis of Alzheimer’s disease. In this paper, the interaction of Aβ with chondroitin sulfates immobilized on a chromatographic column and the role of aluminum had been studied using a biochromatographic approach (molecular chromatography). A novel biochromatographic column was developed in our laboratory for studying this interaction. This study demonstrated that the aluminum interacted with Aβ and played a role in the Aβ/chondroitin sulfates association. For a Al³⁺ concentration (x) in the medium less than 30 μmM, the Aβ/chondroitin sulfates binding decreased with x due to a decrease of the charge–charge interactions between Aβ and its chondroitin sulfates binding site. Above 30 μmM of Al³⁺ in the medium, the affinity of Aβ to chondroitin sulfates increased slightly with x because the net number of ions (n) (Al³⁺ or Cl⁻) released or bound upon complex formation is low. As well, it was clearly demonstrated, that above 30 μmM the n value depend on the Al³⁺ concentration in the bulk solvent. This dependence was due to a gradual and conformational change of the Aβ which around 80 μmM adopted a less flexible structure; its binding site was thus less accessible to Aβ and Aβ/chondroitin sulfates association decreased slightly.

     

Fibroblast surface antigen (SF): molecular properties, distribution in vitro and in vivo, and altered expression in transformed cells.

We have recently described a cell type-specific surface (SF) antigen that is deleted in chick fibroblasts transformed by Rous sarcoma virus, SF antigen is a major surface component and makes up about 0.5% of the total protein on normal cultured fibroblasts. The antigen is shed from normal cells and is present in circulation (serum, plasma), and in vivo, also, in tissue boundary membranes. The molecular equivalents of both cellular and serum SF antigen are distinct, large polypeptides, one of which (SF210, MW 210,000) is glycosylated and, on the cell surface, highly susceptible to proteases and accessible to surface iodination. Immunofluorescence and scanning electron microscopy have indicated that the antigen is located in fibrillar structures of the cell surface, membrane ridges, and processes. Human SF antigen is present in human fibroblasts and in human serum. We have recently shown that human SF antigen is identical to what has been known as the "cold-insoluble globulin" and that it shows affinity toward fibrin and fibrinogen. Our results also indicate that loss of the transformation-sensitive surface proteins is due not to loss of synthesis but to lack of insertion of the protein in the neoplastic cell surface. Both normal and transformed cells produce the SF antigen, but the latter do not retain it in the cell surface. The loss of SF antigen, a major cell surface component, from malignant cells creates an impressive difference between the surface properties of normal and malignant cells. The possible significance of SF antigen to the integrity of the normal membrane and its interaction to surrounding structures is discussed.     

Chromatographic behavior of IgM:DNA complexes

This study documents the presence of stable complexes between monoclonal IgM and genomic DNA in freshly harvested mammalian cell culture supernatants. 75% of the complex population elutes from size exclusion chromatography with the same retention volume as IgM. DNA comprises 24% of the complex mass, corresponding to an average of 347 base pairs per IgM molecule, distributed among fragments smaller than about 115 base pairs. Electrostatic interactions appear to provide most of the binding energy, with secondary stabilization by hydrogen bonding and metal affinity. DNA-dominant complexes are unretained by bioaffinity chromatography, while IgM-dominant complexes are retained and coelute with IgM. DNA-dominant complexes are repelled from cation exchangers, while IgM-dominant complexes are retained and partially dissociated. Partially dissociated forms elute in order of decreasing DNA content. The same pattern is observed with hydrophobic interaction chromatography. All complex compositions bind to anion exchangers and elute in order of increasing DNA content. A porous particle anion exchanger was unable to dissociate DNA from IgM. Monolithic anion exchangers, offering up to 15-fold higher charge density, achieved nearly complete complex dissociation. The charge-dense monolith surface appears to outcompete IgM for the DNA. Monoliths also exhibit more than double the IgM dynamic binding capacity of the porous particle anion exchanger, apparently due to better surface accessibility and more efficient mass transfer.     

Efficient one-pot synthesis of n-butyl levulinate from carbohydrates catalyzed by Fe_2(SO_4)_3

Butyl levulinate(BL) is a promising new candidate as diesel fuel and fuel additive. In this study, an efficient process for a one-pot synthesis of BL from biomass-derived carbohydrates in butanol medium with the catalysis of metal sulfates was developed. The catalytic activity of a series of metal sulfates for the synthesis of BL from fructose was investigated. Among various metal sulfates, ferric sulfate Fe_2(SO_4)_3 was found to be the most efficient catalyst, which gave a remarkably high BL yield of 62.8 mol% under the conditions of 463 K, 3 h, a catalyst dosage of 5.0 g/L, and fructose concentration of 25 g/L. Different carbohydrates including glucose, cellulose, inulin and sucrose were also used for one-pot synthesis of BL with the catalysis of Fe_2(SO_4)_3, showing the yields of 39.6, 30.5, 56.6 and 50.1 mol%, respectively. In addition,the recycling and reuse of Fe_2(SO_4)_3 was studied by characterizing them using powder X-ray diffraction(XRD), scanning electron microscope(SEM), X-ray photoelectron spectroscopy(XPS). A plausible reaction pathway for the one-pot synthesis of BL from fructose was proposed. This study provides a facile and feasible way for the synthesis of BL from biomass.     

ENHANCED SUSCEPTIBILITY TO HPD-SENSITIZED PHOTOTOXICITY AND CORRELATED RESISTANCE TO TRYPSIN DETACHMENT IN SV-40 TRANSFORMED IMR-90 CELLS

Abstract— The response of a normal human cell strain, IMR-90 and a line derived from it by SV40 transformation were compared after treatment in vitro with hematoporphyrin derivative (HPD) and red light. Transformed cells were inactivated at a significantly higher rate than normal cells when assayed by clonogenic survival. Co-treatment with HPD and light also induces cellular resistance to enzymatic cleavage by trypsin; transformed cells exhibit a greater resistance than the normal cells to detachment from the growing surface. These data indicate that transformed cells may possess intrinsic characteristics that render them more sensitive than normal cells to HPD-induced phototoxicity and that the plasma membrane may be the site of differential toxicity.     

HSulf-2, an extracellular endoglucosamine-6-sulfatase, selectively mobilizes heparin-bound growth factors and chemokines: effects on VEGF, FGF-1, and SDF-1

Background  

Heparin/heparan sulfate (HS) proteoglycans are found in the extracellular matrix (ECM) and on the cell surface. A considerable body of evidence has established that heparin and heparan sulfate proteoglycans (HSPGs) interact with numerous protein ligands including fibroblast growth factors, vascular endothelial growth factor (VEGF), cytokines, and chemokines. These interactions are highly dependent upon the pattern of sulfation modifications within the glycosaminoglycan chains. We previously cloned a cDNA encoding a novel human endosulfatase, HSulf-2, which removes 6-O-sulfate groups on glucosamine from subregions of intact heparin. Here, we have employed both recombinant HSulf-2 and the native enzyme from conditioned medium of the MCF-7-breast carcinoma cell line. To determine whether HSulf-2 modulates the interactions between heparin-binding factors and heparin, we developed an ELISA, in which soluble factors were allowed to bind to immobilized heparin.     

Two‐step chromatography purification of IgGs possessing sialidase activity from human blood serum

Sialation of cell surface is known to be tightly connected with tumorigenicity, invasiveness, metastatic potential and clearance of aged cells, while sialation of immunoglobulin G (IgG) molecules determines their anti‐inflammatory properties. Recently, we have found for the first time IgG‐antibodies possessing sialidase‐like activity (sialylic abzyme) in blood serum of multiple myeloma and systemic lupus erythematosis patients. This abzyme was detected in a pool of IgGs purified by a typical procedure including immunoglobulin's precipitation with ammonium sulfate and following chromatography on protein G–Sepharose column. Here we describe a novel matrix for affinity purification of sialylic abzyme that is based on using bovine submandibular gland mucin conjugated to Sepharose matrix (mucin–Sepharose). This matrix preferentially binds sialidase‐like IgGs from a pool of sialidase‐active fraction of proteins precipitated with 50% ammonium sulfate from blood serum of the systemic lupus erythematosis patients. That allowed us to develop a new scheme of double‐step chromatography purification of sialidase‐like IgGs from human blood serum. Copyright © 2014 John Wiley & Sons, Ltd.     

Effect of purified fractions from cell culture supernate of high‐density pre‐B acute lymphoblastic leukemia cells (ALL3) on the growth of ALL3 cells at low density

The mechanisms underlying the aberrant growth and interactions between cells are not understood very well. The pre‐B acute lymphoblastic leukemia cells directly obtained from an adult patient grow very poorly or do not grow at all at low density (LD), but grow better at high starting cell density (HD). We found that the LD ALL3 cells can be stimulated to grow in the presence of diffusible, soluble factors secreted by ALL3 cells themselves growing at high starting cell density. We then developed a biochemical purification procedure that allowed us to purify the factor(s) with stimulatory activity and analyzed them by nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS). Using nanoLC‐MS/MS we have identified several proteins which were further processed using various bioinformatics tools. This resulted in eight protein candidates which might be responsible for the growth activity on non‐growing LD ALL3 cells and their involvement in the stimulatory activity are discussed.     

Synthetic Molecules that Protect Cells from Anoikis and Their Use in Cell Transplantation

One of the major problems encountered in cell transplantation is the low level of survival of transplanted cells due to detachment‐induced apoptosis, called anoikis. The present study reports on the chemical synthesis and biological evaluation of water‐soluble molecules that protect suspended cells from anoikis. The synthetic molecules bind to and induce clusters of integrins and heparan‐sulfate‐bound syndecans, two classes of receptors that are important for extracellular matrix‐mediated cell survival. Molecular biological analysis indicates that such molecules prolong the survival of suspended NIH3T3 cells, at least in part, by promoting clustering of syndecan‐4 and integrin β1 on the cell surface, leading to the activation of small GTPase Rac‐1 and Akt. In vivo experiments using animal disease models demonstrated the ability of the molecules to improve cell engraftment. The cluster‐inducing molecules may provide a starting point for the design of new synthetic tools for cell‐based therapy.     

A possible modulation of acetylcholine receptors of embryonic chick muscle cells by alpha-bungarotoxin

Acetylcholine receptors were assayed with alpha-bugarotoxin on embryonic chick skeletal muscle growing in primary cell culture. Toxin was bound specifically to muscle cells and could be competed with D-tubocurarine. Two dissociation constants were obtained by equilibrium binding: 7.2 x 10(-9)M and 2.7 x 10(-7)M at 25 degrees C. Two sets of rate constants were also obtained from dissociation kinetics. There are five times more low affinity sites on cells than high affinity sites. The average density of high-affinity receptors is about 200/micrometers2. A time course of toxin binding to receptors at 37 degrees C in growth medium revealed that under conditions permitting growth and metabolism, toxin bound to cells was lost. The possibility that the growth medium was inactivating toxin molecules was ruled out by showing that unbound toxin molecules in the medium were fully capable of binding to fresh cultures.     

Capillary electrophoresis and enzyme solid phase assay for examining the purity of a synthetic heparin proteoglycan-like conjugate and identifying binding to basic fibroblast growth factor

Interaction of basic fibroblast growth factor (bFGF) with heparin/heparan sulfate proteoglycans protects the growth factor against proteolytic degradation and is essential for its cellular activity. Although the structural requirements of heparin and heparan sulfate for the high-affinity binding to bFGF have been extensively examined, studies on intact heparin proteoglycans are limited. In this report, the purity and the binding ability of a heparin proteoglycan-like molecule-the heparin-bovine serum albumin (heparin-BSA) conjugate-was examined using capillary zone electrophoresis (CZE). Furthermore, the affinity of bFGF binding to the heparin-BSA conjugate was studied using an enzyme solid-phase assay. Chondroitin sulfate, dermatan sulfate, hyaluronan, heparan sulfate and variously sulfated disaccharides derived from heparin and heparan sulfate were also studied for their ability to compete with the binding of bFGF to heparin. Heparin-BSA conjugate was synthesized by reductive amination and, following precipitation with 1.5 vols of ethanol-sodium acetate, it was obtained free of contaminating heparin. Heparin-BSA-bFGF conjugate was obtained following incubation of heparin-BSA with bFGF for 2 h at 37 degrees C. Intact heparin, heparin-BSA and heparin-BSA-bFGF conjugates were completely resolved by CZE using 50 mM phosphate, pH 3.5, as operating buffer, reversed polarity (30 kV) and detection at 232 nm. Competitive solid phase assay showed that, among the glycosaminoglycans tested, heparin exhibits the highest affinity binding to bFGF (IC(50) = 6.4 nM). Heparan sulfate showed a lower affinity as compared with that of heparin, whereas all other glycosaminoglycans and heparin/heparan sulfate-derived disaccharides tested showed minute effects. The developed CZE method is rapid and accurate and can be easily used to identify bFGF-interacting heparin preparations of biopharmaceutical importance.