High-performance liquid chromatography of proteins by gel permeation chromatography

The ability of two high-performance liquid chromatography gel permeation columns to separate proteins was evaluated. These columns gave satisfactory molecular weight separations for some, but not all, proteins tested. These results indicate that there are limitations in confidence of molecular weight determinations made by this technique.     

Evaluation of radial chromatography versus axial chromatography, practical approach

Developments in packing and packing port design of radial columns in recent years have resulted in a claimed significant increase in performance of this process chromatography technology. In this first study, the main chromatographic parameters as efficiency, capacity factor, asymmetry and resolution were evaluated in a unique one-to-one comparison between a 120 ml bed-volume and 6 cm bed length radial chromatography mini-process column against a 50 mm diameter, 6 cm bed height and 120 ml bed-volume axial chromatography column. Radial chromatography showed an increase in efficiency by 31% in the number of plates per meter while the equilibration could be reduced by 0.4-0.5 column volumes. The asymmetry factor for bovine serum albumin in radial chromatography showed a reduction of 20% while the reduction of the asymmetry factor of the smaller protein ovotransferrin decreased even by 46% in comparison to the performance of the comparative axial chromatography column. Therefore in radial chromatography resolution improved up to 20%. The retention volume was similar in both cases. For radial chromatography, the decrease in "width at half height" at Height Equivalent of Theoretical Plates (HETP) measurements was 40% while the decrease of the over-all width of the peak was 27%. For adsorbed/desorbed proteins, the elution peak showed similar results: "width at half height" decreased to 45% while the over-all width of the peak decreased by 28%. The concentration of the non-retained protein in the flow-through (lysozyme), increased by 35% while the concentration of the eluted fraction (serum albumin bovine), increased with 40% in the radial chromatography columns. The better results obtained with the radial column were probably the consequence of the geometrical design of this device (larger inlet surface area and small outlet surface area which concentrate the eluted fraction).     

Improved performance of column chromatography by arginine: dye-affinity chromatography

Arginine has been effectively used in various column chromatographies for improving recovery and resolution, and suppressing aggregation. Here, we have tested the effectiveness of arginine as an eluent in dye-affinity column chromatography using Blue-Sepharose, which binds enzymes requiring adenyl-containing cofactors (e.g., NAD). A common eluent, NaCl, showed a broad elution peak with low recovery of lactate dehydrogenase, at most approximately 60% using 2M salt. The recovery decreased as the NaCl concentration was either decreased or increased; i.e., the recovery was maximum at 2M. On the contrary, addition of arginine to the eluent resulted in more than 80% recovery above 0.5M and the recovery was nearly independent of the arginine concentration. The elution peak was much sharper with arginine, leading to elution of more concentrated protein solution. Successful elution of proteins bound to the ATP-agarose resins by arginine was also described.     

Immunoaffinity chromatography

The basic procedure of immunoaffinity chromatography (IAC) is described. The insoluble support matrices available for IAC and their activation chemistries, including some of the most recently introduced, are reviewed. Means of selecting the most appropriate monoclonal antibody (MAb) are described, although an empirical approach is still required for the final choice of antibody. Precise methods of runing IAC columns are surveyed including the binding, washing, and elution stages, although no precise recommendations can be made particularly for the elution step since this is unique to a particular MAb and antigen. All IAC sorbents lose activity with time through a combination of MAb inactivation and ligand leakage. The relative importance of the two phenomena is discussed, and suggestions are made to minimize the problem along with an indication of the relative stabilities of a range of coupling chemistries. A sample of the proteins purified by IAC is given together with pointers to the future of the technique.     

Immunoaffinity chromatography

Immunoaffinity chromatography is a process in which the binding affinity of an antigen to a parent antibody is utilized as a basis of separation. Owing to the customized avidity and specificity, monoclonal antibodies (Mabs) have become indispensable for both protein characterization and purification. The immunosorbent performance is dependent on the support matrix upon which the antibody is immobilized and on the activation chemistry used couple the antibody to the matrix. This report details, protocols to immobilize Mabs on commercially available supports, and a method to compute immunosorbent efficiency.     

Countercurrent chromatography

Countercurrent chromatography is a newly developed versatile partition chromatography which totally eliminates the use of solid supports. The method utilizes the intriguing hydrodynamic behavior of two immiscible solvents in a rotating coiled tube. Various types of seal-free flow-through centrifuge schemes are introduced to facilitate a continuous elution process. The method yields high partition efficiencies comparable to liquid chromatography but without the complications arising from the use of solid supports. Countercurrent chromatography covers a wide spectrum of applicable samples ranging from small ions and molecules to macromolecules and even cell particles in both analytical and preparative scales.     

Paralog chromatography

As a mixed mode ligand, a small peptide can mimic an antibody's paratope (antigen recognition site). This report describes the construction of a representative set of paratope analogs, or "paralogs," which can be conjugated to a chromatographic sorbent to combine desirable characteristics of traditional high-performance liquid chromatography columns with the specificity of a moderate affinity antibody. The broad utility of this novel set of protein separatory reagents is illustrated on the complex mixture of proteins in a yeast lysate.     

Adsorption chromatography of proteins

A method for adsorption chromatography of proteins is proposed. A protein solution is passed through a cellulose column at a pH value corresponding to an isoelectric point of the protein. Depending on the charge of unwanted proteins, they either remain at the origin (if charges of protein and ion-exchanger are opposite) or are released from the column (if charges of protein and ion-exchanger coincide). Elution volume of the purified protein is higher than for the second group of unwanted proteins because movement of the uncharged protein of interest includes its adsorption on cellulose followed by subsequent desorption caused by the elution buffer. Problems of optimization of buffers and adsorbents are discussed. Applicability of the method of adsorption chromatography is illustrated using purification of horseradish peroxidase as an example.     

Affinity chromatography of transaminases

It is shown that glutamic-oxaloacetic transaminase from pig heart can be selectively and reversibly bound to a Sepharose column substituted with N′-alkyl derivatives of pyridoxamine 5′-phosphate. A simple procedure is described which includes formation of apotransaminase, its binding to the substituted Sepharose, and its elution under the same conditions that were used to prepare the apoenzyme. There is no binding of the transaminase to Sepharose substituted with unmodified pyridoxamine 5′-phosphate, indicating the importance of a certain “length” of the substituent for binding.     

Affinity chromatography of aminopeptidases

The recent data are generalized concerning a series of synthetic oligopeptides which are competitive inhibitors of aminopeptidases of animal, plant and microbic origin. A method for biospecific chromatography of these enzymes is developed, using as ligands such inhibitors as diazo derivatives of p-aminophenyl-, chloromethyl- and methylketones of L-amino acids and peptides, amino acids, aliphatic acid amides. It is established that the most effective inhibitors of aminopeptidases contain L-amino group in the uncharged form in the N-end position, hydrophobic lateral chain of L-configuration and a carbonyl group analogous to position of these groups in the substrate. Methods for synthesis of certain peptides are developed with respect to the above requirements. It is shown that peptides with a space-inaccessible peptide link and antibiotics are often used as ligands for affinity chromatography of aminopeptidases. At present a nonspecific (ion-exchange, hydrophobic) interaction of sorbent and aminopeptidases is observed, which necessitates to increase the specificity at the stage of the enzyme desorption in the further studies.     

Affinity chromatography of nucleases

The review concerns isolation and purification of nucleases by affinity chromatography. Different stationary ligands and the methods for their immobilization on supports are described, along with diverse eluents and various procedures for a nuclease detachment from the affinity sorbents. The data on the affinity chromatography application for measuring the dissociation constants of the enzyme complexes with either immobilized or soluble ligands are compiled.