Amphipathic chromatography

The main toxic pore forming component of P. marmoratus secretion, named pardaxin, was isolated by liquid column chromatography (5). Originally two toxic (5) polypeptides, Pardaxin I and II, were isolated. However, their primary sequences have been found to be identical (6) therefore, the two components most probably represent different aggregates of one polypeptide. This finding is in contrast to the secretion of P. pavonicuSj which contains three toxic polypeptides (8). Pardaxin is a single chain, acidic, amphipathic, hydrophobic polypeptide, composed of 33 amino acids and with a mass around 3500 daltons (5,6). The primary sequence is (6) NHj-Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Ile-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val-Gly-Ser-Ala-Leu-Ser-Ser-Ser-Gly-Gly-Gln-Glu-COOH. 

Zhou, N. E., Mant, C. T., and Hodges, R. S., Effect of preferred binding domains on peptide retention in reversed-phase chromatography amphipathic... 

Kaiser and colleagues (Fukushima et ai, 1980) showed that a 22-residue peptide can be synthesized entirely from Glu, Lys, and Leu arranged periodically in the typical class A distribution to form an am-phipathic a helix with equal polar and nonpolar faces. Indeed, as studied by quantitative ultrafiltration, gel-permeation chromatography, and circular dichroism, the peptide associated effectively with phospholipid and mimicked some of the physical and chemical properties of apoA-I (Fukushima a/., 1980 Kaiser and Kezdy, 1983, 1984 Nakagawa a/., 1985). Because of the presence of tandem repetitive amphipathic helical domains in apoA-I, a dimer of this 22-residue peptide was later synthesized and found to more closely mimic apoA-I than the monomer thus the study of peptide analogs supported the concept that 44-mers represent the minimal functional domain in apoA-I (Nakagawa et ai, 1985). 

Table I lists the available critical micelle concentrations of bile acids in water and in a salt solution. Accordingly, during chromatography the concentration of the bile acids should be kept below 0.01 M for the trihydroxy salts and below about 0.05 M for the dihydroxy derivatives. Further decreases in the concentration of the external solution at least may be required when working in the presence of salt or buffer and swelling amphipaths (monoglycerides, sterols). When more concentrated solutions are desired, the addition of alcohol to the aqueous solution of bile acids should be considered. The presence of simple and/or mixed micelles in the external solution during chromatography, however, may not necessarily be detrimental to the resolution of the acids, as the micelles would be expected to be in rapid equilibrium with the bile acids in the molecular solution. The actual rates of exchange of various bile acids between micelles and molecular solutions have not been determined.

In all modes of interactive chromatography, self-association of peptides and proteins can result in significant peak shape distortions, and RPC and HlC are no exception. Such behavior, often described in terms of isodemic interaction theory, is frequently a result of a specific molecular property of the peptide or protein, i.e., the propensity to form amphipathic helices or fibril-like structures or to selfassociate, and can lead to multiple peak zones for an otherwise compositionally homogeneous sample. Participation of such self-association processes can be assessed [127,128,338] by on-line light scattering (LALLS) spectroscopic procedures through an examination of the peak shape response as a function of sample concentration under specified RPC or HlC conditions at a defined pH and temperature. 

DISPLACEMENT WITH AMPHIPATHIC AGENTS is especially effective for removing adsorbed macromolecules from solid substrate, but it often is difficult to remove surfactants from the desorbed meicromolecules. Acetonitrile, which is widely used for the desorption step in reversed-phase liquid chromatography, works in the same way as surfactEmts, but higher concentrations are needed. However, concentrations higher than 30% are no longer... 

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