chapter  5
87 Pages

Conformational behaviour of polypeptides

With the development of high resolution biochromatography, reversed-phase highperformance liquid chromatography (RP-HPLC) has found wide analytical and laboratory scale preparative application in peptide and protein chemistry. The success of RP-HPLC procedures with porous n-alkylsilicas for the process scale purification of synthetic organic compounds and other substances of pharmaceutical interest, has been similarly matched over the past decade in the process manufacture of peptides and small polypeptides with molecular weights up to 7 000. Generally this success has not, however, been replicated with larger polypeptides (MW>10 000) and globular proteins, although numerous examples of the isolation of functional proteins at the micro-and ultramicro-preparative level by RP-HPLC procedures can be found in the scientific literature (Hearn, 1998a; Aguilar et al., 1996). Practitioners have often been discouraged from using RP-HPLC methods for the large scale purification of proteins because of their concerns about the combined influences of the acidic elution conditions that traditionally have been used with RP-HPLC procedures and the hydrophobicity per se of the nalkylsilica sorbents. In this context, under the influence of the perturbing low pH aquoorganic solvent conditions used during the RP-HPLC separation process, n-alkyl ligandor solvent-induced unfolding processes have frequently been implicated on an anecdotal basis of the loss of biological activity or the formation of multiple peak zones with samples of high compositional purity, as well as for the reduction in mass yields with proteins. Whilst these features may detract from the use of RP-HPLC as the technique of choice in large scale preparative purification protocols with proteins, these same characteristics provide a unique opportunity to study the folding/unfolding processes and conformational stability of polypeptides and proteins in aquo-organic solvents and hydrophobic environments. As a consequence, RP-HPLC can not only be used as a versatile separation technique par excellence for the high resolution separation and quantitation of complex mixtures of peptides and proteins, but this powerful separation method also can be employed to provide insight into the hierarchal structural features of these biosolutes. In particular, the interactive behaviour of polypeptides and proteins in hydrophobic environments associated with reversed phase chromatographic (RPC)

procedures provides an opportunity to examine the conformational properties and other secondary equilibria, which these biomolecules undergo at non-polar liquid-solid interfaces.