Non-porous Agarose Beads Research Articles

Real-time kinetic analysis of limited proteolysis by ion-exchange chromatography using compressed, non-porous agarose beads.

In biotechnology there is a great need for methods that can be used for kinetic studies of ongoing processes, such as limited proteolysis. Ion-exchange chromatography on a nonporous, compressed agarose matrix was used in the study presented in this paper to monitor papain cleavage of fungal cellulases. This non-porous matrix allows completion of a chromatographic analysis, with baseline resolution, within 30-60 s. Utilizing this very fast chromatographic method one can monitor a proteolysis 'continuously' by repeated chromatographic analysis of the incubation mixture and, if desired, terminate it at the optimal moment. Fractions were also collected for identification by activity assays and analytical SDS-PAGE.

High-performance liquid chromatography of proteins on deformed nonporous agarose beads: immuno-affinity chromatography, exemplified with human growth hormone as ligand and a combination of ethylene glycol and salt for desorption of the antibodies

An affinity chromatography column packed with nonporous agarose beads derivatized with human growth hormone via carbonyldiimidazol was used for the purification of antibodies against human growth hormone from antiserum. Desorption with 1 M sodium chloride in 60% ethylene glycol at pH 9.8 gave 100% total recovery of the antibodies, as measured by radioimmunoassay. The adsorption/desorption process is discussed in terms of hydrophobic and electrostatic interaction (these interactions may be involved in the bond between antibody and antigen in a cooperative fashion). The binding capacity of the column was estimated at about 50 micrograms of antibodies per gram sedimented agarose beads.

General methods to render macroporous stationary phases nonporous and deformable, exemplified with agarose and silica beads and their use in high-performance ion-exchange and hydrophobic-interaction chromatography of proteins

New general methods for the preparation of nonporous beads from macroporous beads are described. One method is based on filling the inner volume of the beads with a solution of a monomer which binds to the matrix at the same time as it is allowed to polymerize. The method is illustrated with agarose and silica as matrices and glycidol as monomer. In an alternative, but principally similar method, we first attached allyglycidyl ether to agarose via the epoxide groups and then allowed acrylamide to react with the immobilized allyl groups during polymerization. In an analogous way, nonporous silica beads were prepared by coupling γ-methacryloxypropyltrimethoxy silane to the macroporous beads followed by polymerization of acrylamide or N-methylolacrylamide on the immobilized methacryl groups. The latter monomer has the advantage of giving a polymer rich in OH groups, which can be used for crosslinking or/and attachment of different ligands (the glycidol polymers have the same advantage). The nonporous agarose beads have chromatographic properties similar to those of the previously described nonporous agarose beads prepared by shrinkage and subsequent crosslinking. For instance, the beds are compressible, which favors the resolution: compressed beds of large beads give the same or higher resolution than do beds of small beads. Another similarity is that the resolution is independent of flow rate or is even enhanced upon an increase in flow rate, maybe in part owing to the generation of a flow pattern which transports the solute from one bead to another faster than does diffusion. These similarities are demonstrated by anion-exchange and hydrophobic-interaction chromatography of proteins. Even the nonporous silica beads are somewhat deformable owing to the relatively thick polymer coating and share with the nonporous agarose beads the attractive relation between resolution and flow rate. In addition, in comparison with naked silica beads they exhibit very little protein adsorption and are more pH stable. A compressed bed of nonporous, coated 30–45 μm silica beads gave in an HIC experiment a resolution comparable to that obtained with a bed of noncompressed, nonporous 1.5-μm silica beads, which give a very high resolution, as shown by Unger and coworkers [5].

High-Performance Liquid Chromatography of Proteins on Deformed Nonporous Agarose Beads. Affinity Chromatography of Dehydrogenases Based on Cibacron Blue-Derivatized Agarose

Nonporous agarose beads, prepared by shrinkage and cross-linking in organic solvents, were derivatized with Cibacron Blue F3G-A. A compressed bed of these beads was used for purification of dehydrogenases (glucose-6-phosphate dehydrogenase, lactate dehydrogenase and alcohol dehydrogenase). The chromatographic conditions for the purification of glucose-6-phosphate dehydrogenase were optimized by varying the pH of the buffer; the concentrations of eluting agents, i.e. NADP (specific elution) and sodium chloride (nonspecific elution); flow rate; residence time of the protein on the column bed; and protein load. Specific elution with NADP (2 mM in 0.025 M Tris-HCl, pH 8.0) gave the highest recovery (140%) and highest purification factor (200-fold) of the enzyme. The ability of the compressed bed of nonporous agarose beads to tolerate high flow rates was essential, since the recovery of the enzyme activity increased with an increase in flow rate.

High-performance liquid chromatography of proteins on deformed non-porous agarose beads: Fast boronate affinity chromatography of haemoglobin at neutral pH

Aminophenylboronic acid was attached to epoxy-activated non-porous agarose beads with diameters of 12–15 μm and this boronate gel column was used for the fractionation of glycosylated from non-glycosylated haemoglobin. By varying the experimental conditions it was shown that the ratio between the second peak (glycosylated haemoglobin) and the first peak (non-glycosylated haemoglobin) was virtually independent of pH in the range 7–8, ionic strength, flow-rate and sample load (up to at least 160 μl ofhaemolysate on a 0.7-ml column). It is therefore not necessary to control thoroughly these parameters in order to obtain reproducible results, which is a great advantage in fast routine analyses of glycosylated haemoglobin. At a flow-rate of 4.0 ml/min an analysis was finished within 2 min on a 2.5 cm X 0.6 cm I.D. column. The total time of an analysis was also short because a sample from a droplet of blood could be applied directly onto the column after haemolysis for 1 min without removal of cell debris by time-consuming centrifugation. The experiments were performed at a pH close to the isoelectric point of haemoglobin, because at this pH haemoglobin has a negligible net charge and will therefore not interact with the charged groups of the ligands, the matrix and proteins adsorbed in previous runs.

High-performance adsorption chromatography of proteins on deformed non-porous agarose beads coated with insoluble metal compounds : I. Coating: Ferric oxyhydroxide with stoichiometrically bound phosphate

A new support for adsorption chromatography of proteins based on crosslinked agarose gels containing ferric (hydr)oxide is described. Two methods have been used for immobilization of the ferric (hydr)oxide on agarose beads; namely,entrapment of preformed ferric oxide particles in connection with the preparation of macroporous agarose beads and precipitation of ferric oxyhydroxide on and in preformed non-porous agarose beads. In the former method many of the adsorption sites of the ferric oxide may become sterically blocked, resulting in chromatographic properties which are similar to those of cation exchangers. Therefore, attention was focused on the second method. Upon using compressed beds of non-porous agarose beads the resolution became independent of the flow-rate, even when the bead diameter was relatively large. When ferric oxyhydroxide agarose is equilibrated with phosphate buffer, some phosphate becomes stoichiometrically and irreversibly bound to the matrix. The separation mechanism of this ferric oxyhydroxide phosphate agarose is discussed. It differs from those of ion-exchange, hydrophobic-interaction, and molecular-sieve chromatography. One characteristic is that phosphate buffers are the most efficient eluting agents. Chromatography on ferric oxyhydroxide phosphate-agarose is accordingly a good complement to these three conventional methods. Some chromatographic properties of the adsorbents are described (resolution as a function of gradient time, flow-rate and protein load; and the pH dependence of the adsorption). A successful separation of a model mixture of six proteins was achieved. Purification of a commercial preparation of β-glucosidase is also presented.

High-performance adsorption chromatography of proteins on deformed non-porous agarose beads coated with insoluble metal compounds : II. Coating: aluminium and zirconium (hydr)oxide with stoichiometrically bound phosphate

Two novel supports for adsorption chromatography of biomolecules, based on deformed non-porous cross-linked agarose beads coated with aluminium or zirconium (hydr)oxide, were prepared. Some fundamental chromatographic properties of these bed materials are discussed, including the adsorption mechanism and high-resolution separations of mixtures of proteins, nucleotides and coenzymes. Both aluminium and zirconium (hydr)oxide agarose irreversibly and stoichiometrically bind phosphate ions, thus some of the chromatographic properties resemble those of calcium phosphate and hydroxyapatite. Fractionation of a β-glucosidase preparation into two components possessing enzymatic activity was achieved on the aluminium (hydr)oxide phosphate agarose, whereas chromatography on zirconium (hydr)oxide phosphate agarose removed several inactive contaminants but gave only one peak associated with enzyme activity. This finding indicates that the adsorption mechanisms of the two adsorbents are not identical, although other experiments show that there are several analogies. For instance, model proteins chromatographed on these two supports were eluted in the same order, although the relative retention times differed, i.e., the chromatograms did not have the same appearance. Compressed beds of cross-linked non-porous agarose beads, including those coated with aluminium and zirconium (hydr)oxide, have the unique feature that the resolution is roughly independent of flow-rate and that there is no need for small beads of narrow size distribution.

High-performance liquid chromatography on continuous polymer beds

Current research in this laboratory is directed toward the investigation of various possible means to increase performance in electrophoresis and chromatography. These methods are studied in parallel because the separation mechanisms are analogouP, which means that solutions of methodological problems in electrophoresis are often applicable to analogous problems in chromatography and vice versa. In the chromatographic field we give an high priority to practical solutions (with the aid of theoretical considerations) of the following fundamental questions: (1) Is it possible to design chromatographic beds such that the resolution is independent of or even increases with an increase in the flow-rate and bead size? Classical chromatographic theory says no. However, the experiments described in refs. 4-6 and forthcoming papers show that compressed beds of non-porous agarose beads have the desired unique relationship between the resolution, flow-rate and bead-size. (2) Is it possible to design a chromatographic bed by bulk polymerization directly in the chromatographic tube? It has been taken for granted that a chromatographic bed must be built up of granulated particles, preferably in spherical form. Even when the spheres are monodisperse the packing is never perfect. The theoretical maximum resolution can therefore never be attained. Further disadvantages of packed beds are the time-consuming and expensive steps required for preparation of the beads, the sieving of the beads to select the desired size (if not monodisperse in the preparation) and the packing of the column with the beads, A continuous gel plug with channels sufficiently large to permit an hydrodynamic flow might be the ideal chromatographic column. One could then expect the zones to be almost as sharp as those obtained in agarose or polyacrylamide gel electrophoresis. Unfortunately, the latter continuous gels cannot be used for chromatography, since they collapse when pressure is applied, i.e., water cannot be pressed through them. However, more than 20 years ago we prepared a polyacrylamide gel (cross-linked in a special way) directly in a glass tube and on this gel plug separated monomers and dimers of albumin by molecular-sieve chromatography. The flow-rate was relatively low, which limited its usefulness. We have now resumed these experiments and at the same time have tried to improve the mechanical properties of the gel plug so that it will withstand higher pressures and thereby permit higher flow-rates. The polymerization technique is still under development and will be published else-

High-performance liquid chromatography of proteins on compressed, non-porous agarose beads : I. Hydrophobic-interaction chromatography

Macroporous agarose beads were converted into non-porous beads by shrinkage and cross-linking in organic solvents. These beads could be used for high-performance hydrophobic-interaction chromatography without derivatization with non-polar ligands, because the 1,4-butanediol diglycidyl ether, used as cross-linker, gives relatively hydrophobic bridges. The resolution for compressed columns packed with these beads was determined as a function of gradient time at constant flow-rate, flow-rate at constant gradient volume and flow-rate at constant gradient time and as a function of loading capacity. Interestingly, the resolution is virtually independent of flow-rate at constant gradient volume even when the column is packed with relatively large beads (diameter 30 μm). The beads have the advantage of being stable up to pH 14.

High-performance liquid chromatography of proteins on compressed, non-porous agarose beads : II. Anion-exchange chromatography

Macroporous agarose beads were rendered impermeable to proteins by shrinkage and cross-linking in organic solvents. The chromatographic properties of compressed beds of these non-porous beads derivatized for high-performance ion-exchange chromatography were studied, e.g., the resolution as a function of gradient time, flow-rate (at constant gradient volume) and loading capacity. The columns permit high flow-rates and the resolution is about the same at low and high flow-rates. The beads are stable up to pH 14.