Predicting the elution behavior of proteins in affinity chromatography on non-porous particles.

Affinity chromatography on non-porous particles of microsize is particularly useful for the rapid analysis and micropreparative separation of proteins. The elution behavior of proteins in an affinity column packed with non-porous copolymerized particles of styrene, methyl methacrylate and glycidyl methacrylate was investigated both theoretically and experimentally, using the lysozyme-Cibacron Blue 3G-A affinity system. Equations used to predict the elution profiles, resulting from the elution by increasing the ionic strength (NaCl concentration) in the mobile phase, were obtained. The maximum adsorbate concentration, desorption rate constant and equilibrium constant under elution conditions were determined by matching experimental data with predicted elution profiles. Based on the parameters determined at a flow-rate of 0.5 ml/min and with 1 M NaCl in the elution buffer, the model equations could predict the elution profiles for other experimental runs, where different flow-rates and sodium chloride concentrations were used. Both the experimental and predicted results revealed that the affinity interaction kinetics are not significantly influenced by the flow-rate and, hence, the film mass transfer. To elute bound lysozyme from immobilized dye ligand, a higher value of the ionic strength leads to a faster elution and a sharper elution peak. The influence of elution conditions on the kinetic and thermodynamic parameters and, consequently, on the elution peak profiles was evaluated. The model equations can also predict the behavior of protein elution from an affinity column by changing the pH of the mobile phase, according to a previous study.     

A decade of high-resolution liquid chromatography of nucleic acids on styrene-divinylbenzene copolymers

The introduction of alkylated, nonporous poly-(styrene-divinylbenzene) microparticles in 1992 enabled the subsequent development of denaturing HPLC that has emerged as the most sensitive screening method for mutations to date. Denaturing HPLC has provided unprecedented insight into human origins and prehistoric migrations, accelerated the cloning of genes involved in mono- and polygenic traits, and facilitated the mutational analysis of more than a hundred candidate genes of human disease. A significant step toward increased sample-throughput and information content was accomplished by the recent introduction of monolithic poly(styrene-divinylbenzene) capillary columns. They have enabled the construction of capillary arrays amenable to multiplex analysis of fluorescent dye-labeled nucleic acids by laser-induced fluorescence detection. Hyphenation of denaturing HPLC with electrospray ionization mass spectrometry, on the other hand, has allowed the direct elucidation of the chemical nature of DNA variation and determination of phase of multiple alleles on a chromosome.     

Fractionation of DNA restriction fragments with ion-exchangers for high-performance liquid chromatography

The fractionation abilities of several ion-exchangers of the high-performance liquid chromatography type for two sets of DNA restriction fragments, ranging from 7 base pairs (bp) to about 650 bp and differing in their mean base composition, have been studied. The ion-exchangers tested comprise the RPC-5, the 5-PW DEAE and the Mono Q as polymer-based resins, and the Nucleogens 500 and 4000, both prepared from silica beads. The results indicate that all the ion-exchangers except the 5-PW DEAE perfectly separate fragment sizes up to about 90 bp, the 5-PW DEAE separating to 45 bp only. Above 200 bp only the Mono Q resin works in a satisfactory way provided that about 100 micrograms DNA mixture, containing less than 25 fragments within the given size range, is loaded per milliliter of packed resin. Appreciable base-pair specificities were detected for most of the resins which cause substantial retardations of the d(A + T)-rich fragments with respect to the eluting salt concentration. If the latter dominate in the DNA sample, acceptable results were only obtained with the Mono Q resin when the column was operated at elevated temperature.     

A new packing for separation of DNA restriction fragments by high performance liquid chromatography

TSK-GEL SW was found to be useful as a packing in high performance liquid chromatography for the separation of double-stranded DNA restriction fragments. DNA fragments smaller than 300 base pairs were separated as discrete peaks depending solely upon difference in chain length. The recovery of DNA fragments was higher than 90%.     

Effect of hairpin DNA on poly(phenylalanine) synthesis.

In a poly(U) dependent poly(Phe) synthesis, an unaminoacylated tRNA(phe) binds to a ribosome and inhibits poly(Phe) synthesis in excess tRNA(Phe). A small DNA fragment having a hairpin structure was added to this system and was found to prevent an unaminoacylated tRNA(phe) from binding to a ribosome and enhance the efficiency of poly(Phe) synthesis in the presence of excess tRNA(phe).     

New phase supports for liquid-liquid partition chromatography of biopolymers in aqueous poly(ethyleneglycol)-dextran systems. Synthesis and application for the fractionation of DNA restriction fragments

New phase supports for liquid-liquid partition chromatography, using aqueous poly(ethyleneglycol)-dextran systems have been developed by grafting linear polyacrylamide chains on to premanufactured chromatographic supports carrying primary or secondary aliphatic hydroxyl functions on their surface. Columns prepared from such supports have a higher binding capacity for the dextran-rich stationary phase and much higher performances than columns prepared from cellulose, the previously used phase support for this system. Test separations of DNA restriction fragments, ranging from 11 base pairs to 3829 base pairs, document a high resolution for DNA fragments larger than 200 base pairs.     

Separation and recovery of DNA fragments by electrophoresis through a thermoreversible hydrogel composed of poly(ethylene oxide) and poly(propylene oxide)

We have synthesized and characterized a thermoreversible hydrogel of multiplied block copolymers, composed of poly(ethylene oxide) and poly(propylene oxide), for DNA electrophoresis. The aqueous solution of block copolymers turned into a hydrogel upon heating at temperatures above 10-11 degrees C, whereas it reverted into a solution upon cooling. Linear double-stranded DNA molecules migrated through the gel matrices at a rate that was inversely proportional to the logarithm of the DNA length. The hydrogel is most effective for separating DNA fragments in the 10- to 2000-bp range. The resolving range lay in-between the effective ranges of polyacrylamide and agarose gel electrophoreses of DNA. The gel slices containing DNA fragments were liquefied by cooling on ice, and the DNA was precipitated with ethanol. No contaminants that inhibit enzymatic reactions were found in the DNA recovered from the hydrogel. Plasmid DNA recovered from the hydrogel was recircularized with T4 DNA ligase and yielded highly efficient Escherichia coli transformation. Therefore, thermoreversible gel electrophoresis will be a useful method for DNA separation and isolation in recombinant DNA technology.     

Optimization of urease immobilization onto non-porous HEMA incorporated poly(EGDMA) microbeads and estimation of kinetic parameters.

Jack bean urease (urea aminohydrolase, EC 3.5.1.5) was immobilized onto modified non-porous poly(ethylene glycol dimethacrylate/2-hydroxy ethylene methacrylate), (poly(EGDMA/HEMA)), microbeads prepared by suspension copolymerization for the potential use in hemoperfusion columns, not previously reported. The conditions of immobilization; enzyme concentration, medium pH, substrate and ethylene diamine tetra acetic acid (EDTA) presence in the immobilization medium in different concentrations, enzyme loading ratio, processing time and immobilization temperature were investigated for highest apparent activity. Immobilized enzyme retained 73% of its original activity for 75 days of repeated use with a deactivation constant kd = 3.72 x 10(-3) day(-1). A canned non-linear regression program was used to estimate the intrinsic kinetic parameters of immobilized enzyme with a low value of observable Thiele modulus (phi < 0.3) and these parameters were compared with those of free urease. The best-fit kinetic parameters of a Michaelis-Menten model were estimated as Vm = 3.318 x 10(-4) micromol/s mg bound enzyme protein, Km = 15.94 mM for immobilized, and Vm = 1.074 micromol NH3/s mg enzyme protein, Km = 14.49 mM for free urease. The drastic decrease in Vm value was attributed to steric effects, conformational changes in enzyme structure or denaturation of the enzyme during immobilization. Nevertheless, the change in Km value was insignificant for the unchanged affinity of the substrate with immobilization. For higher immobilized urease activity, smaller particle size and concentrated urease with higher specific activity could be used in the immobilization process.     

Reverse-phase high-performance liquid chromatography of hydrophobic proteins and fragments thereof

Reverse-phase high-performance liquid chromatography (HPLC) resolution and recovery of cytochrome P-450 and bovine rhodopsin, both integral membrane proteins, and large peptides derived from P-450 LM₂ were enhanced by utilizing ternary solvents. Surprisingly, most test materials eluted later in the gradient when using mixtures of acetonitrile and propanol in the mobile phase compared to using either solvent alone. Of the supports tested, the best recovery of hydrophobic cytochrome P-450 LM₄ was experienced on the less retentive CN-bonded phase. Two alternate solvents for HPLC of polypeptides are proposed: (1) 0.02–0.1 m hexafluoroacetone/NH₃, pH 7.2 for highly acidic peptides; and (2) 6 m formic acid/0.13 m trimethylamine, pH 1.5, vs 4 m formic acid/0.09 m trimethylamine in propanol for relatively insoluble peptides. Anomalous side reactions between formic acid and peptides can cause HPLC peak broadening, increased retention, and decreased resolution. These deleterious effects are thought to be due in part to formyl esterification of serine and threonine residues and appear to be reversible by aminoethanol treatment.     

Conformation of DNA in chromatin core particles containing poly(dAdT)-poly(dAdT) studied by 31 P NMR spectroscopy.

We have prepared semi-synthetic chromatin core particles from a complex of chicken erythrocyte inner histones (H2A, H2B, H3 and H4) with double-stranded poly(dAdT).poly(dAdT) and studied the conformation of the phosphodiester backbone using 31P NMR at 109.3 MHz. At 20 degrees C, the core particle spectrum is fit well by a single Lorenzian distribution with a line width of 110 Hz. This signal is significantly broader than that for the 145 base pair poly(dAdT).poly(dAdT) alone; the latter consists of two resonances, approximately equal in intensity, with average line width 41 Hz. Major changes in the spectrum ensue on heating the core particle preparation. In conjunction with other results (1) these data suggest four states for the core particle at increasing temperatures. Additionally, analysis of the spectrum of the unmelted core particle and its differences from protein-free DNA of the same length suggests that the conformation of the phosphodiester backbone and/or its interactions with histones along the length of the core particle DNA segment may not be uniform.     

Preparative fractionation of DNA restriction fragments by high pressure column chromatography on RPC-5.

High pressure liquid chromatography on the RPC-5 reversed-phase ion exchange system has been shown to have several potential applications as an initial high capacity step in the isolation of specific DNA restriction fragments. The fractionation of the Hinc II digest of lambda DNA, which contains 35 fragments with "flush ends" ranging in size from 3 x 10(6) to 7 x 10(4) daltons, has been used as a model system. Under certain conditions there are some restriction fragments whose elution relative to other fragments is different on RPC-5 chromatography than it is on gel electrophoresis. In some special circumstances it is possible to obtain satisfactory yields (60-70%) of a pure restriction fragment after a single passage through an RPC-5 column.     

Effect of temperature on the separation of DNA fragments by high-performance liquid chromatography and capillary electrophoresis: a comparative study

This study investigates the effect of experimental temperature on the separation of DNA fragments, 21–587 bp, by both high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The results show that the temperature plays an important role in the HPLC separation of DNA fragments. The optimum temperature was found to be between 40 and 50°C for HPLC, while 25°C was the optimum temperature for the CE separation. Also, although CE migration times became shorter, efficiency and resolution decreased with an increase in temperature from 25 to 50°C, but the separation was not significantly affected. Also, the optimum HPLC temperature might be different depending on the fragment sizes to be resolved.     

Fractionation of poly (dA-dT) by gel chromatography

A commercial sample of poly (dA-dT), a copolymer of 2′-deoxyribosyladenosine (dA) and 2′-deoxyribosylthymidine (dT) of perfectly alternating sequence, was fractionated by chromatography on Agarose gel. Paucidisperse fractions of different molecular size were obtained. The plot of log s⁰_20,w values shows a linear dependence on V_e. The buoyant densities of individual fractions do not differ over the molecular size range studied. On the other hand, the heat-induced hyperchromic effect was found to depend on molecular size below a certain limit, s⁰_20,w = 4.12 S.     

Large-scale purification of plasmid DNA by anion-exchange high-performance liquid chromatography.

Numerous methods have previously been reported for the final steps in the large-scale purification of plasmid DNA. Although gel permeation and reverse-phase high-performance liquid chromatography have been utilized for this procedure in the past, the limited capacity of these systems often necessitated multiple rounds of chromatography, especially with the high copy number plasmids commonly in use today. In this paper, the use of the high-capacity, high-resolution Protein-Pak DEAE 8HR column is presented for the large-scale isolation of highly purified plasmid DNA from crude E. coli cell lysates. Up to 5 mg of plasmid DNA have been purified in a single 50-minute chromatography run. The purified DNA demonstrated excellent biological activity as demonstrated by restriction endonuclease digestion, E. coli transformation and DNA-mediated gene transfection of eukaryotic cells.     

Preparation of DNA topoisomers by RP-18 high-performance liquid chromatography.

A method for the separation of superhelical DNA on the basis of superhelical density by reverse-phase HPLC on RP-18 columns is described. The technique can be used to prepare superhelical DNA in milligram amounts and narrow topoisomer distributions in 0.1 mg amounts. We show example separations of the plasmids pUC18 (2687 bp) and pi AN13 (895 bp). While the best separation for pUC18 yields topoisomer distributions of two or three major components, the small plasmid can be separated into single topoisomer fractions. The basis of the separation is probably an interaction of partially opened bases with the hydrophobic column matrix. This hypothesis is supported by the elution behavior of DNA fragments on this column: DNA fragments with sticky ends, even at a length of several hundred base pairs, elute at much higher methanol concentrations than blunt-ended fragments.     

The binding of poly(rA) and poly(rU) to denatured DNA. I. Model studies with homopolymers.

We have compared the properties of the poly(rA).oligo(dT) complex with those of the poly(rU).oligo(dA)n complex. Three main differences were found. First, poly(rA) and oligo(dT)n do not form a complex in concentrations of CsCl exceeding 2 M because the poly(rA) is insoluble in high salt. If the complex is made in low salt, it is destabilized if the CsCl concentration is raised. Complexes between poly(rU) and oligo(dA)n, on the other hand, can be formed in CsCl concentrations up to 6.6 M. Second, complexes between poly(rA) and oligo(dT)n are more rapidly destabilized with decreasing chain length than complexes between poly(rU) and oligo(dA)n. Third, the density of the complex between poly(rA) and poly(dT) in CsCl is slightly lower than that of poly(dT), whereas the density of the complex between poly(rU) and poly(dA) in CsCl is at least 300 g/cm3 higher than that of poly(dA). These results explain why denatured natural DNAs that bind poly(rU) in a CsCl gradient usually do not bind poly(rA).     

Separation of human Fab fragments on negative mode Ni(II)-TREN-agarose chromatography

We evaluated the feasibility of using immobilized metal-ion affinity chromatography (IMAC) with nickel ion complexed with Tris(2-aminoethyl)amine (TREN) immobilized on agarose gel for purification of human Fab fragments by negative chromatography. Efficient purification of Fab fragments from digested human IgG (immunoglobulin G) (106.4% purity) was accomplished in Tris-HCl buffer at pH 7.5 without NaCl (based on total protein concentration and radial immunodiffusion of human Fab). A technological application of Ni(II)-TREN-agarose using non transgenic soybean protein extract spiked with human Fab fragments as feedstream was also studied. Experiments using Tris-HCl at pH 7.0 as loading buffer allowed the adsorption of almost all of the soybean proteins. Sixty-six percent of the loaded human Fab fragments were recovered in the flowthrough and washing fractions with about 90% purity. These results demonstrate that Ni(II)-TREN-agarose is a potential adsorbent for recombinant Fab fragment purification.