Purification of Large "Troublesome" Polypeptides by RP-HPLC.

INTRODUCTIONThis protocol addresses the group of large polypeptides that have proved to be troublesome to handle because of their low solubility or the presence of secondary structural elements that favor supramolecular self-self assembly. Included in this group are polypeptides with amphipathic α-helical or ß-sheet structures with extensive runs of nonpolar (hydrophobic) amino acid side chains or proline-rich sequences. RP-HPLC provides one avenue to purify such troublesome examples provided certain steps and precautions are taken. (Related procedures are equally germane to polypeptides that have been lipidated or subjected to chemical modifications with nonpolar moieties, as well as to some core cyanogen bromide fragments of large proteins.) RP-HPLC methods not only enable concomitant desalting, removal of additives that aid dissolution of the polypeptide or protein, but also permit resolution and maintenance of a reasonably high concentration of the solutes, due to the presence of the (often) low-pH, aquo-organic solvent conditions.

A molecular recognition paradigm: promiscuity associated with the ligand-receptor interactions of the activin members of the TGF-beta superfamily.

The structure-function properties of the pleiotropic activins and their relationship to other members of the transforming growth factor-beta superfamily of proteins are described. In order to highlight the molecular promiscuity of these growth factors, emphasis has been placed on molecular features associated with the recognition by activin A and the bone morphogenic proteins of the corresponding extracellular domains of the ActRI and ActRII receptors. The available evidence suggests that the homodimeric activin A in its various functional roles has the propensity to fulfill key tasks in the regulation of mammalian cell behaviour, through coordination of numerous transcriptional and translational processes. Because of these profound effects, under physiologically normal conditions, activin A levels are closely controlled by a variety of binding partners, such as follistatin-288 and follistatin-315, alpha(2)-macroglobulin and other proteins. Moreover, the subunits of other members of the activin subfamily, such as activin B or activin C, are able to form heterodimers with the activin A subunit, thus providing a further avenue to positively or negatively control the physiological concentrations of activin A that are available for interaction with specific receptors and induction of cell signaling events. Based on data from X-ray crystallographic studies and homology modeling experiments, the molecular architecture of the ternary receptor-activin ligand complexes has been dissected, permitting rationalization in structural terms of the pattern of interactions that are the hallmark of this protein family.

Separation of structurally related synthetic peptides by capillary zone electrophoresis.

The separation of two different sets of synthetic peptides has been investigated by high-performance capillary zone electrophoresis utilising naked, fused silica capillaries. The effects of electrolyte pH, buffer concentration, capillary length and electric field strength on the separation efficiency and selectivity were systematically varied, with the highest resolution achieved with buffer electrolytes of low pH and relatively high ionic strength. Under optimised separation conditions utilising the "short end injection" separation approach with negative electric field polarity, a series of eight structurally-related synthetic peptides were baseline resolved within 4 min without addition of any modifier of the background electrolyte with separation efficiencies in the vicinity of 600000 theoretical plates/m. Further significant enhancement of separation efficiencies could be achieved by taking advantage of the "long end injection" approach with positive electric field polarity. The outcome of these experimental variations parallels the "sweeping" effect that has been observed in the capillary electrochromatographic and micellar electrokinetic separations of polar molecules and permits rapid resolution of peptides with focusing effects. In addition, small changes in the electrolyte buffer pH and concentration were found to have a significant impact on the selectivity of synthetic peptides of similar intrinsic charge. These observations indicate that multi-modal separation mechanisms operated under these conditions with the unmodified fused silica capillaries. This study, moreover, documents additional examples of peptide-specific multi-zoning behaviour in the high-performance capillary zone electrophoretic separation of synthetic peptides.