Highly charged macromolecules

Analysis of ion atmospheres around highly charged macromolecules has traditionally been performed using numerical solutions to the nonlinear Poisson-Boltzmann (P-B) equation (Anderson and Record, 1980 Bai et al, 2007 Baker, 2004), in which the macromolecule is approximated as a collection of point charges embedded in a low dielectric cavity surrounded by a high-dielectric solvent. This approach utilizes the precise three-dimensional structure of the macromolecule (albeit in a static sense). We would not expect such a framework to capture subtleties, which are dependent on the partial dehydration of ions. 

Since DNA is a highly charged macromolecule surrounded by a layer of counter ions, it is more probable that the dielectric polarization of DNA arises from the polarization of the ion atmosphere. Various mechanisms of ionic polarization have been proposed. The theories of ionic polarization for a spherical particle suspension were reviewed and carefully discussed by Schwan (20, 21). Since DNA is a thin elongated molecule, those theories must be modified substantially. Various theories for ellipsoids are briefly reviewed here. 

In highly charged macromolecules Eq. (5.8.16) does not apply. It is then necessary to generalize these arguments to include hydration and deviations from spherical shape. However there are even more formidable complications to consider first. In aqueous solution, the macroion is surrounded by an ion atmosphere composed mainly of ions of opposite charge. This means that the local field —the field felt by the macroion— will be considerably different than the applied field. Corrections for this effect can be made if one uses the Debye-Huckel-Henry theory to calculate the properties of the ion atmosphere (see Chapters 9 and 13). An estimate (Tanford, 1961) gives... 

The thermodynamics of polyelectrolyte solutions is also a strong function of ionic strength and charge density. Very large osmotic second virial coefficients are observed for highly charged macromolecules. The Flory theory of these solutions in presented in Section 10.3. 

Plasmid DNA must penetrate the nuclear envelope to be transcribed for gene expression. Like a plasma membrane or endosomal membrane, the nuclear envelope constitutes an impenetrable barrier to highly charged macromolecules. Passive diffusion through the NPC is possible, but only for short DNAs of below 310 base pairs [182]. Without any assistance, only 1-2% of DNA can be transported to the nucleoplasm between the narrow gateway and the cytosolic digestion [183]. The disruption of the nuclear envelope during mitotic division is the only chance for free pDNA to enter the nucleoplasm. 

In summary, a practical realization of FEDs for the pure electrostatic detection of charged macromolecules by their intrinsic molecular charge, especially in high ionic-strength solutions such as physiological conditions, seems to be problematic. All the above discussed disturbing factors, together with a possible undesired adsorption or... 

At the same time, however, it must be concluded that the practical development of FEDs for a label-free detection of DNA and other charged macromolecules by their intrinsic molecular charge seems to be more complicated than originally expected. Although the discussed results are highly exciting, they are rather diverse and even sometimes inconsistent. Factors influencing the DNA immobilization and hybridization detection by FEDs are ... 

In the early 1980s, ionization techniques such as FAB, PD, and thermospray (TSP) made it possible to use MS in analysis of high-mass macromolecules since the production of gas phase ions from charged and polar compounds can be done without prior chemical derivatization. FAB is a soft ionization technique that performs well... 

In experiments where the ligand and macromolecule are charged, the equilibrium may be affected by the Donnan potential of the charged macromolecule, which is confined to one compartment. Donnan effects can be minimised by carrying out the dialysis in sufficiently high ionic strength media, such as in the presence of 0.1 M NaCl or KC1. 

While electrophoresis is mostly used for the separation of charged macromolecules, techniques are available for high-resolution separations, e.g. capillary electrophoresis, of small molecules such as amino acids, anions and catecholamines. 

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