Determination of biophysical parameters of polypeptide retro-inverso isomers and their analogues by capillary electrophoresis

The relationship between the electrophoretic mobility, mu(obs), Stokes radi us, r(s), ionization state, and solution conformation of the all L-alpha-po lypeptide, 1, the corresponding retro-all D-alpha-polypeptide, 2, and sever al truncated analogues, 3-5, has been investigated under low pH buffer cond itions by high-performance capillary zonal electrophoresis (HPCZE) with coa ted capillaries. The results confirm that, under these conditions, the all L-alpha-polypeptide, 1, and its retro-inverso isomer, 2, exhibit nonidentic al electrophoretic mobilities and thus different Stokes radii. At higher pH values, i.e., pH 5.0, the electrophoretic behavior of this retro-inverso i somer pair, however, converges. These results indicate that variations in t he dipole characteristics of the polypeptide main chain and subtle: differe nces introduced by the spatial constraints; of the L-alpha-Pro --> D-alpha- Pro residue replacement lead to:differences in the Stokes radii and electro phoretic mobilities bf these polypeptides, Since the observed electrophoret ic mobilities, mu(obs), reflect the mean of the mobilities of each charge s pecies participating according to their Stokes radius or their intrinsic ch arge and mole fraction abundances, the results confirm that polypeptide ret ro-inverso isomers with unmodified amino and carboxy termini are resolvable . This outcome was achieved despite their notional topographical and confor mational similarities as assessed from high-held proton nuclear magnetic re sonance (H-1 NMR) spectroscopy and circular dichroism (CD) spectroscopy.