Theory of chromatography of macromolecules with rigid structures on hydroxyapatite columns. I. Static part

A theory for the chromatography of rigid macromolecules on hydroxyapatite columns has been developed under the assumptions of instantaneous thermodynamic equilibrium between adsorbed phase and solution and of negligible longitudinal diffusion of macro-molecules. In the first or static part of this theory the adsorption isotherm of macro-molecules was studied as a function of phosphate concentration in the solvent.     

Heterogeneity of tropocollagen as investigated by chromatography on hydroxyapatite columns

The chromatography of native acid-soluble tropocollagen from calf skin on hydroxyapatite columns has been investigated. Resolution of a number of chromatographic peaks has been obtained by using shallow slopes of the eluting phosphate gradient. The results obtained suggest a heterogeneity of tropocollagen molecules which might be due to different distributions of absorbing sites on the molecules. The results obtained have also been used to study the mechanism of chromatography of proteins on hydroxyapatite and the resolving power of the columns.     

Investigations on the resolving power of hydroxyapatite columns

An investigation on the resolving power of hydroxyapatite columns is reported. The macromolecules chosen for this investigation have been five proteins endowed with a rigid structure and of different sizes: cytochrome c, lysozyme, β-lactoglobulin A, collagen, and T2 phage. The following points have been investigated in detail: (1) the dependence of the elution molarity upon column length and slope of the gradient; (2) the dependence of the elution molarity and the width of the protein peak upon the load and the presence of other chromatographic components; (3) the optimal conditions for the resolution of macromolecules having the same size or different sizes.     

Thermal elution chromatography of nucleic acids on hydroxyapatite.

Hydroxyapatite thermal elution chromatography was studied from an empirical standpoint. The dependence of elution temperature on elution buffer concentration was determined for various types of buffer, hydroxyapatite and nucleic acid. The results are analyzed in terms of the proper design and interpretation of thermal elution experiments. The potential for serious artifacts is demonstrated and the means by which they may be avoided is described. Various commercially available hydroxyapatites were tested in conjunction with various aqueous and partially non-aqueous buffer systems. Among the materials tested, potassium phosphate and Bio-Rad HTP were found to constitute the best buffer-hydroxyapatite system for most types of thermal elution study.     

Expanded bed chromatography of proteins in small-diameter columns. II. Methods development and scale up

The scaled down system developed in Part I of this series was further validated by using a 1-cm diameter column for method development studies for the separation of two model proteins, alcohol dehydrogenase and -glucosidase, from unclarified yeast homogenate by hydrophobic interaction expanded bed chromatography based on the STREAMLINE matrix. The efficacy of solids removal and establishment of optimal binding and separation condition by stepwise elution were investigated. Equilibration of the EBA column and loading at high salt strengths affected the subsequent recovery of the two target proteins. Although good resolution between the target proteins could be achieved, peak tailing was found to be a consistent problem. The optimised separation protocol was scaled up 25-fold to a column diameter of 5.0 cm. The results were in good agreement with the run conducted in the 1-cm column, indicating the potential of using the small columns as an viable approach for method scouting and development studies.     

Transport properties and hydrodynamic centers of rigid macromolecules with arbitrary shapes

A general formalism, which includes translation–rotation coupling, is proposed for calculating translational and rotational transport properties, as well as intrinsic viscosities, of rigid macromolecules with an arbitrary shape. This formalism is based on Brenner's theory of translational–rotational dynamics and on methods for the calculation of hydrodynamic properties that have been already presented, and can be regarded as a generalization of the one proposed by Nakajima and Wada. The calculated transport properties depend on the origin as predicted by Brenner's theory, but in a disagreement with him, the center of resistance and the center of diffusion do not coincide. As one can define several hydrodynamic centers, which in practice turn out to be located at different points, the influence of the choice of the center on the calculated transport properties is discussed. An analysis of the translation–rotation coupling effects in translational diffusion reveals that they arise exclusively from hydrodynamic interactions and are rather small in some cases of interest. Finally, we present a study of the rotational diffusion of rigid bent rods with a fixed length-to-diameter ratio. The diffusion coefficients obtained can be useful to estimate changes with respect to a straight rod.     

Hydrodynamic properties of rigid macromolecules composed of ellipsoidal and cylindrical subunits.

A procedure is devised for the calculation of hydrodynamic properties of rigid macromolecules composed subunits that are modeled as ellipsoids of revolution and cylinders. Owing to the axial symmetry of these shapes, smooth shell models can be constructured for the subunit structure. The bead shell model so constructed is employed for the calculation of the properties. A computer program, HYDROSUB, has been written implementing both the model building and the hydrodynamic calculation. A detailed example of the use of this methodology is presented for the case of the solution properties of the human antibody molecule immunoglobulin G3 (IgG3). Finally, hints are given on other uses and applications of the procedure.     

Cytosolic delivery of macromolecules. II. Mechanistic studies with pH-sensitive morpholine lipids

Drug carriers containing weak acids or bases can promote cytosolic delivery of macromolecules by exploiting the acidic pH of the endosome. We have prepared two pH-sensitive mono-stearoyl derivatives of morpholine, one with a (2-hydroxy) propylene (ML1) linker and the other, an ethylene (ML2) linker. The pK(a) values of lipids ML1 and ML2, when incorporated into liposomes, are 6.12 and 5.91, respectively. Both lipids disrupt human erythrocytes at pH equal to or below their pK(a) but show no such activity at pH 7.4. Confocal microscopy studies suggest partial endosome-to-cytosol transfer of fluorescent dextran (MW 10 kDa) encapsulated in liposomes that contained 20 mol% of morpholine lipids. Interestingly, co-incubation of morpholine lipids in free or micellar form (without liposomal incorporation) with dextran resulted in efficient cytosolic delivery. Upon acidification to the endosomal pH, liposomes containing ML1 revealed: (a). leakage of entrapped solute that is independent of solute size; (b). lack of liposomal collapse into micelles as evidenced by photon correlation spectroscopy and UV light scattering; and (c). minimal inter-bilayer interactions as shown in a fluorescence resonance energy transfer assay. These observations are consistent with progressive intravesicular reorganization of lipids into stable liposomes of smaller size, but of more homogeneous distribution, upon acidification. The results emphasize a need to manipulate liposomal formulations containing ML1 such that ML1 will promote catastrophic collapse of liposomes to mixed micelles upon exposure to acidic pH. It is only then that micelle-mediated permeabilization of the endosomal membrane will lead to efficient cytosolic delivery of macromolecules originally loaded in liposomes.     

Rapid purification of plasmid DNAs by hydroxyapatite chromatography.

A method is described for the rapid preparation of plasmid DNAs of molecular weight up to 14 X 10(6). This method involves the chromatography, at room temperature, of bacterial cleared lysates on hydroxyapatite in the presence of high concentrations of phosphate and urea. All detectable protein and RNA contamination of plasmid DNA is removed by this procedure and the conformation of the plasmid DNA is unaffected. Less than 0.5% chromosomal DNA is present in the purified preparation and even this can be removed if necessary by a simple extention of the procedure to include a heat-denaturation step. The method is extremely rapid and amenable to large-scale plasmid preparation; 5 mg ColE1 DNA have been purified within 40 min. The yield of plasmid DNA is similar to that obtained with the conventional dye-centrifugation technique, however the purity is greater.