A comparison between electrokinetic capillary chromatography and absorption spectroscopy for the analysis of peptide‐micelle association by weak hydrophobic interactions

Abstract

Micellar electrokinetic capillary chromatography (MEKC) was compared to absorption spectroscopy to estimate equilibrium association constans (K(as)) for peptide-micelle systems involving three peptides (leucine-enkephalin, methionine-enkephalin and leucine-phenylalanine (LF)) and two surfactant micelles (sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB)). Buffer pH was chosen to minimize purely electrostatic interactions between peptides and micelles that could not be interrogated by absorption spectroscopy. Viscosity-corrected MEKC mobilities gave reasonably similar estimates of K(as) between the two methods for all three peptide-SDS micelle systems, with K(as) values ranging from 13.7 +/- 0.3 to 49 +/- 1 M(-1). For CTAB, estimates of K(as) for LF-CTAB micelle association were of the same order of magnitude as the SDS micelle by the two methods of estimation. On the other hand, enkephalin-CTAB micelle binding was about 10 times stronger (K(as) = 122 +/- 3 M(-1) to 311 +/- 9 M(-1)) than the enkephalin-SDS micelle binding. In addition, MEKC underestimated the K(as) values relative to spectroscopy by a factor of 2-3 for the enkephalin-CTAB system.

A pulsed field gradient NMR diffusion investigation of enkephalin peptide‐sodium dodecyl sulfate micelle association

“…These results confirm the hypothesis that Leu-enkephalin peptides bind to SDS micelles through a largely electrostatic interaction. 22 The PFG-NMR diffusion results for the cationic Leuenkephalin peptides also suggest that the identity of the positively charged amino acid does not play a large role in governing binding affinity. As noted above, the Leuenkephalin-Arg and Leu-enkephalinamide association constants were near equal.…”

“…From these results, the authors concluded that 'interaction of enkephalins with micelles is primarily hydrophilic or electrostatic in nature'. 22 Taken together, EKC and NMR analyses suggest that Leu-and Met-enkephalin interact with SDS micelles by a primarily electrostatic mechanism. The micelle bound Leu-enkephalin inserts its hydrophobic side chains into the micelle core, while Met-enkephalin does not.…”

“…18,19 These methods have been applied to solutions containing Leuenkephalin and both SDS and cetyltrimethylammonium bromide (CTAB) micelles. 22 At a pH of 8.1, the SDS micelle association constant of Leu-enkephalin was 49 š 1 M 1 , or approximately a factor of three less than the K eq value measured for Leu-enkephalin at pH 5.5 with PFG-NMR diffusion experiments. 22,23 This Leu-enkephalin association constant was also less than the corresponding value for the cationic CTAB micelles and the dipeptide Leu-Phe in both SDS and CTAB solutions.…”

“…22 At a pH of 8.1, the SDS micelle association constant of Leu-enkephalin was 49 š 1 M 1 , or approximately a factor of three less than the K eq value measured for Leu-enkephalin at pH 5.5 with PFG-NMR diffusion experiments. 22,23 This Leu-enkephalin association constant was also less than the corresponding value for the cationic CTAB micelles and the dipeptide Leu-Phe in both SDS and CTAB solutions. From these results, the authors concluded that 'interaction of enkephalins with micelles is primarily hydrophilic or electrostatic in nature'.…”

“…In their EKC study of enkephalin peptides, Hemond et al suggested that hydrophobic residues close to a protonated amino acid more readily insert into the micelle than those farther from the protonated residue. 22 In addition, Whitehead et al observed that a positively charged Lys adjacent to the hydrophobic residues in Substance P served as an 'anchor' that held the neuropeptide on the surface and allowed the hydrophobic side chains to insert. 11 Therefore, the presence of the positively charged Arg or Lys amino acids in the Leu-enkephalin peptides may enhance insertion of the Phe and Leu side chains into the core, thus increasing the hydrophobic interactions between the peptide and the SDS hydrocarbon chains and causing the cationic Leu-enkephalin peptides to have larger K eq values than their Met-enkephalin counterparts, where little to no insertion takes place.…”