Effect of polyanions on the structure and stability of repifermin (keratinocyte growth factor-2).

The interaction of several of the fibroblast growth factors (FGFs) with polyanions is thought to be of physiological significance and has been exploited to create more stable pharmaceutical formulations of FGF-1 and -2. The extent of such phenomena throughout the 23-member FGF family is, however, unknown. In these studies, we examine the effect of several polyanions on the structure and stability of keratinocyte growth factor 2 (KGF-2, FGF-10), a candidate for use as a wound-healing agent. Employing a variety of methods sensitive to the protein's structure including circular dichroism (CD), intrinsic fluorescence, derivative near-UV absorption spectroscopy, bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5-disulfonic acid) fluorescence, differential scanning calorimetry (DSC), and dynamic light scattering (DLS), we find that a variety of polyanions (e.g., heparin, sucrose octasulfate (SOS), and inositol hexaphosphate (IHP)) stabilize KGF-2 by increasing the thermal-unfolding temperature by approximately 9-15 degrees C. Negatively charged liposomes produce a similar effect, arguing for relatively nonspecific interactions of polyanions with KGF-2. Unlike some other FGFs, no evidence for the presence of a molten globule state is found during thermal perturbation of this growth factor. The generality of this polyanion/protein interaction is discussed as well as its potential role in various cellular events such as protein folding and transport.

Comparative effects of pH and ionic strength on protein-protein interactions, unfolding, and aggregation for IgG1 antibodies.

Changes in protein-protein interactions, protein unfolding, and nonnative aggregation were assessed for a series of human IgG1 antibodies as a function of pH and solution ionic strength (I). Unfolding transitions were characterized with differential scanning calorimetry. Protein-protein interactions were characterized with the apparent second virial coefficient (A(2)) from light scattering. Aggregation pathways were assessed using size-exclusion chromatography and multi-angle laser light scattering, aggregation kinetics, and structural changes monitored by circular dichroism spectroscopy and thioflavine T (ThT) binding. Ionic strength had relatively minor qualitative effects on unfolding, while pH had large effects for all four antibodies. A(2) was sensitive to both pH and I, and indicated that electrostatic interactions and nonuniform surface-charge distributions were important near neutral pH. Depending on solution pH and I, distinct aggregation pathways were found for each antibody, and these shared similar patterns versus pH, I, and A(2). Main differences observed across different antibodies included thermal unfolding transitions in DSC and the effects of pH and I on aggregation kinetics and pathways. These correlated strongly with whether aggregates of a given antibody bound ThT, suggesting possible differences with respect to conformational changes and/or regions of the proteins that are structurally involved in stabilizing the aggregates.