Selectivity due to conformational differences between helical and non-helical peptides in reversed-phase chromatography.
The reversed-phase retention behaviour of two series of peptides, one non-helical and the other alpha-helical, was studied under various linear AB gradients in order to determine the effect of peptide conformation on selectivity of the separation. The non-helical series, designated X1, with the sequence Ac-XLGAKGAGVG-amide, exhibited negligible alpha-helical content in a hydrophobic medium; whereas, the amphipathic alpha-helical series, designated AX9, with the sequence Ac-EAEKAAKEXEKAAKEAEK-amide, exhibited high alpha-helical content in a hydrophobic medium. We have shown that plots of log k vs. phi (where k is the median capacity factor and phi is the median volume fraction of organic solvent) are very similar for any one peptide conformation, i.e., peptides from either the non-helical or amphipathic alpha-helical series exhibit similar S (solute parameter) values and the b (gradient steepness parameter) values are also similar for 17 different amino acid substitutions within each series of peptides. If mixtures of peptides from the two different series are separated using either increasing or decreasing gradient rates, large increases in resolution occur due to selectivity, which may be attributed to the difference in the log k vs. phi plots for each series of peptides. In addition, by using a polymer of an X1 peptide, which is 20 residues in length, it has been shown that the molecular mass difference between the X1 and the AX9 series of peptides is not sufficient to account for the selectivity difference. The S value of a non-amphipathic alpha-helical peptide further suggested that the difference in selectivity between the two series of peptides was due to differences in conformation. We believe that the peptide mixtures presented here provide a good model for studying selectivity effects due to conformational differences between peptides, an important concern when attempting to develop rational approaches to the prediction and optimization of peptide separation protocols from primary sequence information alone.
Sereda
T J
TJ
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Mant
C T
CT
Hodges
R S
RS
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002942:Circular Dichroism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D010455:Peptides