Determination of biophysical parameters of polypeptide retro-inverso isomers and their analogues by capillary electrophoresis.
The relationship between the electrophoretic mobility, microobs, Stokes radius, rs, ionization state, and solution conformation of the all L-alpha-polypeptide, 1, the corresponding retro-all D-alpha-polypeptide, 2, and several truncated analogues, 3-5, has been investigated under low pH buffer conditions by high-performance capillary zonal electrophoresis (HPCZE) with coated capillaries. The results confirm that, under these conditions, the all L-alpha-polypeptide, 1, and its retro-inverso isomer, 2, exhibit nonidentical electrophoretic mobilities and thus different Stokes radii. At higher pH values, i.e., pH 5.0, the electrophoretic behavior of this retro-inverso isomer pair, however, converges. These results indicate that variations in the dipole characteristics of the polypeptide main chain and subtle differences introduced by the spatial constraints of the L-alpha-Pro-->D-alpha-Pro residue replacement lead to differences in the Stokes radii and electrophoretic mobilities of these polypeptides. Since the observed electrophoretic mobilities, microobs, reflect the mean of the mobilities of each charge species participating according to their Stokes radius or their intrinsic charge and mole fraction abundances, the results confirm that polypeptide retro-inverso isomers with unmodified amino and carboxy termini are resolvable. This outcome was achieved despite their notional topographical and conformational similarities as assessed from high-field proton nuclear magnetic resonance (1H NMR) spectroscopy and circular dichroism (CD) spectroscopy.
Hearn
M T
MT
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia. milton.hearn@med.monash.edu.au
Keah
H H
HH
Boysen
R I
RI
Messana
I
I
Misiti
F
F
Rossetti
D V
DV
Giardina
B
B
Castagnola
M
M
D000595:Amino Acid Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D019075:Electrophoresis, Capillary; D007202:Indicators and Reagents; D007536:Isomerism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*
D007202:Indicators and Reagents; D010455:Peptides