Size and conformational stability of the hepatitis A virus used to prepare VAQTA, a highly purified inactivated vaccine.

A variety of biophysical techniques have been employed to examine the size and conformational integrity of highly purified hepatitis A virus (HAV) in solution (purified HAV particles are subsequently formalin-inactivated and adsorbed to aluminum salts for use as the vaccine VAQTA). The size of HAV particles was assessed by a combination of electron microscopy, sedimentation velocity, and dynamic light scattering. The effect of ionic strength and temperature on the overall conformational stability of HAV was determined by a combination of intrinsic HAV protein fluorescence, fluorescent probes of both RNA and protein, and UV-visible spectroscopy. A major structural change in HAV occurs near 60 degrees C with the addition of 0.2 M magnesium chloride enhancing the thermal stability of HAV by approximately 10 degrees C. Salt concentrations above 0.2 M, however, decrease the solubility of HAV. The effect of pH on the physical properties of HAV particles was monitored by dynamic light scattering, analytical size exclusion HPLC, and interaction with fluorescent dyes. HAV particles undergo a substantially reversible association/aggregation at pH values below 6 with the concomitant exposure of previously buried hydrophobic surfaces below pH 4. These results are in good agreement with previous studies of HAV thermal stability under extreme conditions in which the irreversible inactivation of the viral particles was measured primarily by the loss of viral infectivity. The wide variety of biophysical measurements described in this work, however, directly monitor structural changes as they occur, thus providing a molecular basis with which to monitor HAV stability during purification and storage.

Deamidation of polyanion-stabilized acidic fibroblast growth factor.

The deamidation of polyanion-stabilized acidic fibroblast growth factor (aFGF; FGF-1) can be induced by prolonged storage under accelerated conditions of elevated pH and temperature. A urea-isoelectric focusing (urea-IEF) method has been developed to monitor aFGF deamidation in the presence of highly negatively charged polyanions which are required to maintain the conformational stability of the protein. The kinetics of aFGF deamidation have been established by a combination of urea-IEF and an enzymatic ammonia assay. Native, non-deamidated aFGF (complexed with heparin) has a half-life of 16 weeks at pH 7, 30 degrees C, and 4 weeks at pH 8, 40 degrees C. The mitogenic activity and biophysical properties of deamidated aFGF were compared to the non-deamidated protein. These initial deamidation events have no significant effect on the protein's overall conformation, thermal stability, interaction with heparin, or bioactivity. At longer times, however, limited aggregation of the protein was observed after prolonged storage under some conditions. N-terminal protein sequencing of the protein's first 21 amino acid residues have identified one of the deamidation sites in a flexible, peptide-like region of the protein (Asn8-Tyr9).

Interaction of nucleotides with acidic fibroblast growth factor (FGF-1).

A wide variety of nucleotides are shown to bind to acidic fibroblast growth factor (aFGF) as demonstrated by their ability to (1) inhibit the heat-induced aggregation of the protein, (2) enhance the thermal stability of aFGF as monitored by both intrinsic fluorescence and CD, (3) interact with fluorescent nucleotides and displace a bound polysulfated naphthylurea compound, suramin, (4) reduce the size of heparin-aFGF complexes, and (5) protect a reactive aFGF thiol group. The binding of mononucleotides, diadenosine compounds (ApnA), and inorganic polyphosphates to aFGF is enhanced as the degree of phosphorylation of these anions is increased with the presence of the base reducing the apparent binding affinity. The nature of the base appears to have much less effect. Photoactivatable nucleotides (8N3-ATP, 2N3-ATP, 8N3-GTP, and 8N3-Ap4A) were employed to covalently label the aFGF nucleotide binding site. In general, Kd's in the low micromolar range are observed. Protection against 90% displacement is observed at several hundred micromolar nucleotide concentration. Using 8N3-ATP as a prototypic reagent, photolabeled aFGF was proteolyzed with trypsin and chymotrypsin and labeled peptides were isolated and sequenced resulting in the identification of 10 possible labeled amino acids (Y8, G20, H21, T61, K112, K113, S116, R119, R122, H124). On the basis of the crystal structure of bovine aFGF, eight of the prospective labeled sites appear to be dispersed around the perimeter of the growth factor's presumptive polyanion binding site. On residue (T61) is more distally located but still proximate to several positively charged residues, and another (Y8) is not locatable in crystal structures. Using heparin affinity chromatography, at least three distinct photolabeled aFGF species were resolved. These labeled complexes display diminished affinity for heparin and a reduced ability to stimulate mitogenesis even in the presence of polyanions such as heparin. In conclusion, nucleotides bind apparently nonspecifically to the polyanion binding site of aFGF but nevertheless are capable of modulating the protein's activity. Evidence for the presence of a second or more extended polyanion binding site and the potential biological significance of these results in terms of potential natural ligands of aFGF are also discussed but not resolved.

Sucralfate and soluble sucrose octasulfate bind and stabilize acidic fibroblast growth factor.

The actions of the anti-ulcer drug sucralfate have been proposed to be mediated through interaction with fibroblast growth factors (Folkman, J., Szabo, S., Strovroff, M., McNeil, P., Li, W. and Shing, Y. (1991) Ann. Surg. 214, 414-427). We show here that acidic fibroblast growth factor (aFGF; FGF-1) binds in vitro to both the soluble potassium salt and the insoluble aluminum salt of sucrose octasulfate, as demonstrated by a variety of biophysical techniques. Similar to the well-described interaction and stabilization of aFGF by heparin, soluble sucrose octasulfate (SOS) stabilizes aFGF against thermal, urea and acidic pH-induced unfolding as determined by a combination of circular dichroism, fluorescence spectroscopy and differential scanning calorimetry. In addition, SOS also enhances the mitogenic activity of aFGF and partially protects the protein's three cysteine residues from copper-catalyzed oxidation. SOS competes with heparin and suramin for the aFGF polyanion binding site as measured by both fluorescence and light scattering based competitive binding assays. Front-face fluorescence measurements show that the native, folded form of aFGF binds to the insoluble aluminum salt of sucrose octasulfate (sucralfate). Moreover, sucralfate stabilizes aFGF against thermal and acidic pH-induced unfolding to the same extent as observed with SOS. Thus, due to their high charge density, SOS and sucralfate bind and stabilize aFGF via interaction with the aFGF polyanion binding site.

Nature of the interaction of growth factors with suramin.

Suramin inhibits the binding of a variety of growth factors to their cell surface receptors. The direct interaction of suramin with acidic fibroblast growth factor has been detected by the enhancement of the drug's fluorescence in the presence of the protein with the maximum effect occurring at a molar ratio of suramin to aFGF of 2:1. This interaction stabilizes aFGF to thermal denaturation and partially protects a free thiol in its polyanion binding site from oxidation. The binding of suramin to aFGF also induces aggregation of the growth factor to at least a hexameric state as detected by static and dynamic light scattering as well as by gel filtration studies. Both CD and amide I' FTIR spectra of aFGF in the presence and absence of suramin suggest that the drug may also be causing a small conformational change in the growth factor. Suramin produces an even greater aggregation of bFGF and PDGF but not of EGF or IGF-1. Evidence for a suramin-induced conformational change in IGF-1 but not EGF is found by CD, however. It is concluded that suramin binds to many growth factors and that this induces microaggregation and, in some cases, conformational changes. In the case of aFGF, suramin interacts at or near its heparin binding site. The relationship between these phenomena and the anti-growth factor activity of suramin remains to be clearly elucidated.