Rapid purification of two thermophilic proteinases using dye-ligand chromatography

Dye-ligand chromatography has been used successfully for the purification of extracellular thermostable proteinases from thermophilic Bacillus and Thermus cultures. Single step purification factors of up to 115-fold (for Thermus protease) and 2195-fold (for Bacillus protease) were obtained. Elution studies suggested that the mode of binding involved the enzyme active sites. The method was readily scaleable to 600 l volume.     

Expanded bed affinity chromatography of dehydrogenases from bakers' yeast using dye-ligand perfluoropolymer supports

Malate dehydrogenase (MDH) and glucose 6-phosphate dehydrogenase (G6PDH) have been partially purified from preparations of homogenized yeast cells using Procion Yellow H-E3G and Procion Red H-E7B, respectively, immobilized on solid perfluoropolymer supports in an expanded bed. A series of pilot experiments were carried out in small packed beds using clarified homogenate to determine the optimal elution conditions for both MDH and G6PDH. Selective elution of MDH using NADH was effective but the yields obtained were dependent on the concentration of NADH used. Selective elution was found to be most effective when a low concentration of NaCl (0.1 M) was present. MDH could be recovered in 84% yield with a purification factor of 94 when this strategy was adopted. In the case of G6PDH, specific elution using NADP(+) was successful in purifying G6PDH 178-fold in 96% yield. The dynamic capacity of both affinity supports was estimated by frontal analysis, in an expanded bed with unclarified homogenate, and corresponded to 17 U MDH/mL of settled Procion Yellow H-E3G perfluoropolymer support and 7.7 U H6PDH/mL of settled Procion Red H-E7B perfluoropolymer support. Expanded bed affinity chromatography of MDH resulted in an eluted fraction containing 89% of the applied activity with a purification factor of 113. Expanded bed affinity chromatography of G6PDH resulted in an eluted fraction containing 84% of the applied activity with a purification factor of 172. With both enzymes, the overall recovery of enzyme activity was greater than 94%, showing that the expanded bed approach to purification was nondenaturing. (c) 1995 John Wiley & Sons, Inc.     

Fractionation of plasma proteins using dye-ligand chromatography: elution profiles on immobilized green TSK-AF

The fractionation of human plasma by chromatography on immobilized Green TSK-AF was assessed by immunological analysis of the elution profiles of 27 different plasma proteins. A three-step procedure was used to elute proteins from the column. First a low-molarity buffer (30 mM sodium phosphate, pH 7.0, I = 0.053) was applied; then a linear salt gradient (0-1.0 M NaCl in the above buffer) was followed by an additional wash with four bed volumes of 1.0 M NaCl. Tightly bound proteins were finally stripped with 0.5 M NH4SCN. The elution profile of the proteins using this procedure appears to be very reproducible. Comparison with the profile obtained upon chromatography on Cibacron Blue 3GA [Gianazza, E. and Arnaud, P. (1982) Biochem. J. 201, 129-136] indicates significant differences between the binding properties of the two gels. These differences can be used to design a "tandem-chromatography" system which provides an efficient means for the separation of several plasma proteins.     

Separation of chymosin and pepsin in calf rennet by dye-ligand affinity chromatography

When calf rennet containing approximately 15% pepsin was applied to a Cibacron Blue agarose column at pH 5.5 in a low salt medium, pepsin passed through unadsorbed while chymosin was bound to the gel in the column. After washing the column, the bound chymosin was eluted with 1.7 M NaCl or 50% (v/v) aqueous ethylene glycol. The salt eluate was analyzed and found to contain greater than 97% pure chymosin. The fraction that passed through unadsorbed was found to contain greater than 96% pure pepsin. Thus a complete separation of chymosin and pepsin was effected by this technique without having to destroy either enzyme. Both enzymes are highly negatively charged at pH 5.5 but the separation does not arise from anion exchange since the gel functions as a cation exchanger. The separation appears to result from a combination of hydrophobic and electrostatic interactions of chymosin with Blue agarose. It is suggested that the enhanced affinity of chymosin to the Blue gel over pepsin may arise from topographically specified interaction between chymosin and the blue chromophore. Differential surface hydrophobicity may also play a key role, since in the presence of 0.7 M Na2SO4 the same behavior as at low ionic strength is observed.     

Purification of aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli by dye-ligand chromatography

Improved purification schemes are reported for the enzymes L-aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli. Dye-ligand chromatography on commercially available dye matrices are incorporated as key steps in these purifications. Red A-agarose has a high affinity for L-aspartase, which is then eluted as a homogeneous protein fraction with 1 mM L-aspartic acid. Green A-agarose shows a high binding affinity for the bifunctional enzyme aspartokinase-homoserine dehydrogenase I. Purification is accomplished by elution with NADP+, followed by formation of a ternary complex with NADP and cysteine, a good competitive inhibitor of the homoserine dehydrogenase activity, and rechromatography on Green A-agarose. The final specific activity of each purified enzyme equaled or exceeded previously reported values, the overall yield of enzymes obtained was significantly higher, and these improved purification schemes were found to be more amenable to being scaled up for the production of large quantities of purified enzyme.     

Homogeneous chicken heart mitochondrial creatine kinase purified by dye-ligand and transition-state analog-affinity chromatography

A method for the preparation of homogeneous mitochondrial creatine kinase from chicken heart is presented. The two-column procedure, which can be completed in 2 days, uses Procion red dye and transition-state analog-affinity chromatography. The transition-state analog-affinity chromatographic system utilizes an ADP-hexane-agarose column in conjunction with the transition-state analog complex originally developed by E. J. Milner-White and D. C. Watts (1971, Biochem, J. 122, 727-740) composed of KNO3, MgCl2, creatine, and ADP. The enzyme is a dimer composed of 2 Mr 43,000 subunits. The sequence of the first N-terminal 20 amino acids shows that the enzyme is different from the cytosolic isozymes but similar to human mitochondrial creatine kinase. The enzyme has an extinction coefficient of epsilon 280 nm = 2.22 +/- 0.10 ml X mg-1 X cm-1 and a maximum velocity of 200 IU/ml at pH 7.0. The kinetic constants for the chicken heart mitochondrial isozyme are comparable to values for the canine and human heart isozyme.     

Protein dye affinity chromatography using immobilized tetraiodofluorescein

The chromatographic behavior of a heterogeneous protein mixture and of a series of homogeneous proteins on the immobilized dye tetraiodofluorescein has been observed and analyzed. Less than 6%, of the millimolar concentration of dye immobilized to a porous agarose matrix is accessible to protein. The affinity of a protein for immobilized dye is dramatically increased by insertion of apolar spacer atoms between the dye and the matrix. Dye columns constructed with a 9-atom spacer can be used to advantage for the retention and competitive elution of proteins not found previously amenable to dye chromatography.     

Gram-scale purification of phosphorothioate oligonucleotides using ion-exchange displacement chromatography.

The purification of oligonucleotides by ion-exchange displacement chromatography is demonstrated on the gram-scale. Using a 50 mmD x 100 mmL (203 ml) column operated in the displacement mode, 1.2 g of a 24mer phosphorothioate oligonucleotide was purified. Product yield for this separation was 70% (780 mg) at a purity of 96.4% and the mass balance recovery of all oligonucleotide was 97.5%. The displacement purification of four additional phosphorothioate oligonucleotides ranging in length from 18 to 25 bases is also demonstrated on the semi-preparative (10-50 mg) scale. All of these oligonucleotides were purified using similar displacement conditions and typical results were 60% yield at 96% purity. The displacement portion of these separations required <15 min and total cycle time including equilibration, feed loading and regeneration can be performed in under 30 min. These results seem to indicate that displacement chromatography may be amenable to generalizations in separation protocol that would greatly reduce the effort required to obtain an optimized purification scheme for moderately long oligonucleotides.     

Affinity chromatography using enzymatically synthesized nucleotide-containing DNA binding polymers

A series of nucleotide-containing polyphenols has been synthesized by a simple, two-step enzymatic method. The binding properties of these synthetic polymers to complementary oligonucleotides have been evaluated using a commercially available oligo(dT)cellulose column. Complementary synthetic nucleosides were retained on this column to a greater extent than non-complementary synthetic nucleosides. These results suggest that the synthetic nucleosides prepared via this two-step enzymatic approach may have application as affinity matrices.     

Role of electrostatics in the binding of charged metallophthalocyanines to neutral and charged phospholipid membranes

Binding of the cationic tetra(tributylammoniomethyl)-substituted hydroxoaluminum phthalocyanine (AlPcN(4)) to bilayer lipid membranes was studied by fluorescence correlation spectroscopy (FCS) and intramembrane field compensation (IFC) methods. With neutral phosphatidylcholine membranes, AlPcN(4) appeared to bind more effectively than the negatively charged tetrasulfonated aluminum phthalocyanine (AlPcS(4)), which was attributed to the enhancement of the coordination interaction of aluminum with the phosphate moiety of phosphatidylcholine by the electric field created by positively charged groups of AlPcN(4). The inhibitory effect of fluoride ions on the membrane binding of both AlPcN(4) and AlPcS(4) supported the essential role of aluminum-phosphate coordination in the interaction of these phthalocyanines with phospholipids. The presence of negative or positive charges on the surface of lipid membranes modulated the binding of AlPcN(4) and AlPcS(4) in accord with the character (attraction or repulsion) of the electrostatic interaction, thus showing the significant contribution of the latter to the phthalocyanine adsorption on lipid bilayers. The data on the photodynamic activity of AlPcN(4) and AlPcS(4) as measured by sensitized photoinactivation of gramicidin channels in bilayer lipid membranes correlated well with the binding data obtained by FCS and IFC techniques. The reduced photodynamic activity of AlPcN(4) with neutral membranes violating this correlation was attributed to the concentration quenching of singlet excited states as proved by the data on the AlPcN(4) fluorescence quenching.     

Role of electrostatics in the binding of charged metallophthalocyanines to neutral and charged phospholipid membranes

Binding of the cationic tetra(tributylammoniomethyl)-substituted hydroxoaluminum phthalocyanine (AlPcN₄) to bilayer lipid membranes was studied by fluorescence correlation spectroscopy (FCS) and intramembrane field compensation (IFC) methods. With neutral phosphatidylcholine membranes, AlPcN₄ appeared to bind more effectively than the negatively charged tetrasulfonated aluminum phthalocyanine (AlPcS₄), which was attributed to the enhancement of the coordination interaction of aluminum with the phosphate moiety of phosphatidylcholine by the electric field created by positively charged groups of AlPcN₄. The inhibitory effect of fluoride ions on the membrane binding of both AlPcN₄ and AlPcS₄ supported the essential role of aluminum-phosphate coordination in the interaction of these phthalocyanines with phospholipids. The presence of negative or positive charges on the surface of lipid membranes modulated the binding of AlPcN₄ and AlPcS₄ in accord with the character (attraction or repulsion) of the electrostatic interaction, thus showing the significant contribution of the latter to the phthalocyanine adsorption on lipid bilayers. The data on the photodynamic activity of AlPcN₄ and AlPcS₄ as measured by sensitized photoinactivation of gramicidin channels in bilayer lipid membranes correlated well with the binding data obtained by FCS and IFC techniques. The reduced photodynamic activity of AlPcN₄ with neutral membranes violating this correlation was attributed to the concentration quenching of singlet excited states as proved by the data on the AlPcN₄ fluorescence quenching.     

Separation of plasmid DNA from protein and bacterial lipopolysaccharides using displacement chromatography

The preparation of plasmid DNA at large scale constitutes a pressing problem in bioseparation. This paper describes a first investigation of displacement chromatography as a means to separate plasmid DNA (4.7 kb) from E. coli lipopolysaccharides and protein (holo transferrin), respectively. Displacement chromatography has advantages in this regard, since the substance mixture is resolved into rectangular zones of the individual components rather than into peaks. Thus a higher total concentration can be maintained in the pooled product fractions. Hydroxyapatite (type I and II) and anion exchange stationary phases were included in the experiments. In addition to a conventional anion exchange column packed with porous particles, the recently introduced continuous bed UNOTM anion exchange column was investigated. No DNA purification was possible with either hydroxyapatite material. Conventional particle based columns in general were not suited to the separation of any two substances varying considerably in molecular mass, e.g. plasmid DNA and standard protein. Presumably, the direct competition for the binding sites, which is essential in displacement chromatography, was restricted by the size dependency of the accessible stationary phase surface area in this case. Better results were obtained with the continuous bed column, in which the adsorptive surface coincides with the walls of the flow through pores. As a result the accessible surface does not vary as much with the size of the interacting molecules as for the conventional stationary phase materials. Sharper transitions were also observed between substance zones recovered from the UNOTM column. The steric mass action model was used to aid method development in case of the anion exchange approach. While further research in obviously necessary, displacement chromatography on continuous bed columns has been shown to be capable of separating plasmid DNA from typical impurities. This revised version was published online in July 2006 with corrections to the Cover Date.     

Structures, energies and bonding in neutral and charged Li microclusters

Structural and chemical properties of charged and neutral Lithium microclusters are investigated for [Formula: see text]. A total of 18 quantum conformational spaces are randomly walked to produce candidate structures for local minima. Very rich potential energy surfaces are produced, with the largest structural complexity predicted for anionic clusters. Analysis of the electron charge distributions using the quantum theory of atoms in molecules (QTAIM) predicts major stabilizing roles of Non-nuclear attractors (NNAs) via NNA···Li interactions with virtually no direct Li···Li interactions, except in the least stable configurations. A transition in behavior for clusters containing more than seven nuclei is observed by using the recently introduced quantum topology to determine in a quantum mechanically consistent fashion the number of spatial dimensions each cluster has. We experiment with a novel scheme for extracting persistent structural motifs with increase in cluster size. The new structural motifs correlate well with the energetic stability, particularly in highlighting the least stable structures. Quantifying the degree of covalent character in Lithium bonding independently agrees with the observation in the transition in cluster behavior for lithium clusters containing more than seven nuclei. Good correlation with available experimental data is obtained for all properties reported in this work.     

Ion-exchange displacement chromatography of serum proteins, using carboxymethyldextrans as displacers

A system of displacers comprising carboxymethyldextrans with progressively higher content of carboxyl groups forms a displacement train in which absorbed proteins find positions according to their affinities for the adsorbent, an anion exchanger. Because little or no salt need be used, effluent fractions can be evaluated directly by gel electrophoresis. Application to the fractionation of serum proteins is demonstrated.     

Anion binding to neutral and positively charged lipid membranes

Aqueous anion binding to bilayer membranes consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated by using deuterium and phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. Only those anions that exhibit chaotropic properties showed significant binding to POPC membranes. A detailed investigation of thiocyanate binding to neutral POPC and to positively charged mixed POPC/dihexadecyldimethylammonium bromide (DHDMAB) (8:2 mol/mol) membranes revealed changes in the 2H NMR quadrupole splittings from POPC specifically deuteriated at either the alpha-segment or the beta-segment of the choline head group which were consistent with a progressive accumulation of excess negative charge at the membrane surface with increasing SCN- concentration. Both the 2H and 31P NMR spectra indicated the presence of fluid lipids in a bilayer configuration up to at least 1.0 M NaSCN with no indication of any phase separation of lipid domains. Calibration of the relationship between the change in the 2H NMR quadrupole splitting and the amount of SCN- binding provided thiocyanate binding isotherms. At a given SCN- concentration the positively charged membranes bound levels of SCN- 3 times that of the neutral membranes. The binding isotherms were analyzed by considering both the electrostatic and the chemical equilibrium contributions to SCN- binding. Electrostatic considerations were accounted for by using the Gouy-Chapman theory. For 100% POPC membranes as well as for mixed POPC/DHDMAB (8:2 mol/mol) membranes the thiocyanate binding up to concentrations of 100 mM was characterized by a partition equilibrium with an association constant of K approximately 1.4 +/- 0.3 M-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein refolding at high concentration using size-exclusion chromatography

A new method to improve refolding yields and to increase the concentration of refolded proteins in a single operation has been developed. The method uses size-exclusion chromatography matrices to perform buffer exchange, aggregate removal, and the folding reaction. The reduced diffusion of proteins in gel-filtration media has been shown to suppress the nonspecific interactions of partially folded molecules, thus reducing aggregation. Hen egg white lysozyme (HEWL) and bovine carbonic anhydrase (CAB) were successfully refolded from initial protein concentrations of up to 80 mg/mL using Sephacryl S-100 (HR). The aggregation reaction for lysozyme was reduced and was only detected at the highest protein concentration used. The average recovery of lysozyme was 63%, with an average specific activity of 104%. Carbonic anhydrase experiments also showed that aggregation was suppressed and the average protein recovery from the column was 56%, with a specific activity of 81%. This process enables refolding and the purification of active species to be achieved in a single step. (c) 1996 John Wiley & Sons, Inc.     

Isolation and fractionation of CHO chromosomes in aqueous two phase systems using charged polymers and base specific macroligands

Summary Chromosomes were isolated in a preparative scale by synchronisation of CHO cells with a double Thymidine block followed by an arrest in the metaphase by addition of Colcemid. Under proper cultivation conditions a mitotic index of 77% total cells could be routinely achieved. Bulk chromosome preparations free of nuclei and other subcellular particles have been obtained by low speed centrifugation followed by a 60 transfer countercurrent distribution using aqueous two phase systems composed of polyethylenglycol and dextran. The partition of CHO chromosomes previously purified in aqueous two phase systems were studied further to develop a protocol for the separation and isolation of individual chromosomes. Partition experiments with chromosomes changing the electrostatic phase potential by addition of charged PEG-derivatives suggest the existence of relatively highly charged chromosome groups. Most promising results with regard to separation were obtained using two PEG-derivatives, which interact specifically with the bases in DNA. For this affinity partitioning a GC- and AT-specific macroligand were employed. Comparing CCD's using each of these ligands information on the GC and AT content of exposed DNA in the chromosomes groups could be derived, demonstrating that specific sequences of DNA are accessible at the surface of metaphase chromosomes.     

Determination of barbiturates and some neutral drugs in serum using quartz glass capillary gas chromatography

Rapid methods for the glass capillary gas chromatographic determination of barbiturates and some neutral drugs are described. The analysis of barbiturates was performed using a nitrogen—phosphorus selective detector (NPD). The barbiturates were recovered from serum using charcoal adsorption followed by extraction with methylene chloride. The drugs were then alkylated by means of the Claisen carbonate method. Neutral drugs were extracted simultaneously with the barbiturates. The neutral drugs were determined underivatized with a flame ionization detector. In the underivatized form the barbiturates were not stable on the quartz column used. The selectivity of derivatization combined with an NPD was used to determine the barbiturates in the presence of neutral drugs with the aid of retention data.     

Purification of recombinant brain derived neurotrophic factor using reversed phase displacement chromatography

This work investigates the utility of RPLC displacement chromatography for the purification of recombinant brain derived neurotrophic factor (rHu-BDNF) from its variants and E. coli. protein (ECP) impurities. The closely associated variants (six in total) differ by one amino acid from the native BDNF and thus pose a challenging separation problem. Several operational parameters were investigated to study their effects on the yield of the displacement process. The results indicated that the concentration of trifluoroacetic acid (TFA) in the buffer was a key factor in achieving the desired purification. Displacement chromatography on an analytical scale column resulted in extremely high purity and yield in a single chromatographic step. The process was successfully scaled-up with respect to particle and column diameter. The production rate of a pilot scale RPLC displacement process was shown to be 23 times higher than the combined production rates of the current preparative ion exchange and hydrophobic interaction gradient elution steps that are used to remove variant and ECP impurities, respectively.     

Separation of neutral oligosaccharide alditols from human meconium using high-pH anion-exchange chromatography

Neutral reduced oligosaccharides are in general not sufficiently retained to achieve adequate separation and reproducible chromatography using high-pH anion-exchange chromatography. We describe a method to increase the retention using two columns in series. This method has been applied to the separation of oligosaccharides purified from human meconium glycoproteins, obtained as their alditols after alkaline-borohydride release of oligosaccharides. The neutral oligosaccharide alditols were significantly retained upon two CarboPac PA-100 columns, connected in series, and eluted in 80 mM sodium hydroxide between 4 and 10 min. Three sialylated alditols studied were substantially retained and could be eluted in a gradient of 0–500 mM sodium acetate—80 mM sodium hydroxide between 10 and 45 min. The elution patterns were based on their size, charge and linkage, such that oligosaccharide alditol isomers could be separated.