Publisher Summary Reversed phase high performance liquid chromatography (RP-HPLC) now plays a cri... more Publisher Summary Reversed phase high performance liquid chromatography (RP-HPLC) now plays a critical role in the analysis and purification of peptides and proteins from natural and synthetic sources. The extraordinary popularity of RP-HPLC for polypeptide analysis can be attributed to a number of factors hi9ghlighted in this chapter. Some of the potential of RP-HPLC in the purification of polypeptides and proteins is demonstrated by the selected samples. Despite the current wide usage of RP-HPLC techniques in protein sequencing studies and many other purification areas involving high resolution polypeptide separations, the selection of a particular chemically-bonded stationary phase and mobile phase composition has frequently been based on empirical criteria. Not uncommonly, such arbitrary selections may not adequately address the issues of optimal chromatographic resolution, band shape or solute recovery. This chapter documents recent theoretical developments which characterize the RP-HPLC of polypeptides and proteins, and describes recent advances in the evolution of fully mechanistic models which describe in physicochemical terms the nature of the interaction between biological macromolecules and biological and synthetic surfaces.
The chromatographic properties of 16 thyromimetic iodoamino acids and related compounds on microp... more The chromatographic properties of 16 thyromimetic iodoamino acids and related compounds on microparticulate non-polar stationary phases have been examined and conditions determined which allow optimised resolution with analysis time ca. 60 minutes. These compounds elute in order of increasing hydrophobicity which correlates with the progressive increase in the number of iodogroups present in the tyrosine or thyronine aromatic nucleus. The reverse isomers, e.g. rT3, have consistently greater k' values than their corresponding analogues, e.g. T3. Conditions for the direct application of the rapid HPLC analyses of the iodoamino acids in biological or pharmaceutical samples have been examined.
Previously only one of the six disulphide bonds within the β-subunit of bovine thyrotropin (bTSHβ... more Previously only one of the six disulphide bonds within the β-subunit of bovine thyrotropin (bTSHβ) has been unequivocally assigned. In the present investigation, the fluorescent alkylating reagent 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulphonic acid has been employed as part of a double-alkylation strategy to allow the relative reactivities and the location of the six disulphide bonds of bTSHβ, after selective reduction, to be assigned by using reversed-phase HPLC peptide mapping techniques and associated methods of structural analysis. The most reactive disulphide bond was Cys88–Cys95; the second most reactive group of disulphide bonds involved the half-cystine residues Cys16, Cys19, Cys67 and Cys105 with the experimental results consistent with the assignment of disulphide bonds to Cys16–Cys67 and Cys19–Cys105. The least reactive group of half-cystine residues consisted of Cys2, Cys27, Cys31, Cys52, Cys83 and Cys85. The isolation, by high-performance ion-exchange chromatogr...
INTRODUCTIONThe RP-HPLC technique can be used to "desalt" peptide or protein samples de... more INTRODUCTIONThe RP-HPLC technique can be used to "desalt" peptide or protein samples derived from extraction procedures, from chemical reactions such as reductive alkylation in the presence of urea or guanidine hydrochloride, citraconylation, iodination, or cyanogen bromide cleavage, or recovered from other chromatographic separation. The peptide or protein solution is injected onto a small RP-HPLC column. An aqueous buffer is used to elute the salts while the peptides or proteins are concentrated at the top of the column. After elution of the salts, monitored by UV detection, the peptides or proteins are eluted with H(2)O-acetonitrile or H(2)O-isopropanol mobile phases.
Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.], 2001
The eight basic modes of HPLC currently in use for peptide and protein analysis and purification ... more The eight basic modes of HPLC currently in use for peptide and protein analysis and purification are presented in this overview. They include: size-exclusion chromatography (HP-SEC), ion-exchange chromatography (HP-IEX), normal phase chromatography (HP-NPC), hydrophobic interaction chromatography (HP-HIC), reversed-phase chromatography (RP-HPLC), hydrophilic interaction chromatography (HP-HILIC), immobilized metal ion affinity chromatography (HP-IMAC), and biospecific/biomimetic affinity chromatography (HP-BAC). In addition, some subsets of these chromatographic modes, e.g., mixed mode chromatography (HP-MMC), charge transfer chromatography (HP-CTC), or ligand-exchange chromatography (HP-LEC) are described. The unit includes overviews of the system requirements and planning strategies for set-up of the systems.
In this study, the advantages of carrying out the analysis of peptides and tryptic digests of pro... more In this study, the advantages of carrying out the analysis of peptides and tryptic digests of proteins under gradient elution conditions at pH 6.5 by reversed-phase liquid chromatography (RP-HPLC) and in-line electrospray ionisation mass spectrometry (ESI-MS) are documented. For these RP separations, a double endcapped, bidentate anchored n-octadecyl wide pore silica adsorbent was employed in a capillary column format. Compared to the corresponding analysis of the same peptides and protein tryptic digests using low pH elution conditions for their RP-HPLC separation, this alternative approach provides improved selectivity and more efficient separation of these analytes, thus allowing a more sensitive identification of proteins at different abundance levels, i.e. more tryptic peptides from the same protein could be confidently identified, enabling higher sequence coverage of the protein to be obtained. This approach was further evaluated with very complex tryptic digests derived from a human plasma protein sample using an online two-dimensional (2D) strong cation-exchange (SCX)-RP-HPLC-ESI-MS/MS system. Again, at pH 6.5, with mobile phases of different compositions, improved chromatographic selectivities were obtained, concomitant with more sensitive on-line electrospray ionisation tandem
INTRODUCTIONThis protocol addresses the group of large polypeptides that have proved to be troubl... more INTRODUCTIONThis protocol addresses the group of large polypeptides that have proved to be troublesome to handle because of their low solubility or the presence of secondary structural elements that favor supramolecular self-self assembly. Included in this group are polypeptides with amphipathic α-helical or β-sheet structures with extensive runs of nonpolar (hydrophobic) amino acid side chains or proline-rich sequences. RP-HPLC provides one avenue to purify such troublesome examples provided certain steps and precautions are taken. (Related procedures are equally germane to polypeptides that have been lipidated or subjected to chemical modifications with nonpolar moieties, as well as to some core cyanogen bromide fragments of large proteins.) RP-HPLC methods not only enable concomitant desalting, removal of additives that aid dissolution of the polypeptide or protein, but also permit resolution and maintenance of a reasonably high concentration of the solutes, due to the presence o...
The dynamic behaviour of a series of closely related cytochrome c molecules has been investigated... more The dynamic behaviour of a series of closely related cytochrome c molecules has been investigated in reversed-phase high-performance liquid chromatography. In particular, the influence of temperature and gradient time on the experimental bandwidths of equine, tuna, canine and bovine cytochrome c and equine and tuna apocytochrome c has been determined with a C,, and a C, sorbent. The observed bandbroadening changes are discussed in comparison to the corresponding retention behaviour of the cytochrome c molecules reported in the preceding paper [l]. The results demonstrate that bandwidth measurements can be used as an indicator of the conformational status of a protein under a particular set of chromatographic conditions.
The retention behaviour of equine, tuna, canine and bovine cytochrome c and the corresponding equ... more The retention behaviour of equine, tuna, canine and bovine cytochrome c and the corresponding equine and tuna apocytochrome c has been investigated using reversed-phase gradient elution chromatographic procedures. A range of operating temperatures between 5 and 85°C were utilised to monitor the influence of protein unfolding on the corresponding retention parameters. Chromatographic measurements were obtained with both an n-octadecyl (C,,) and n-butyl (C,) sorbent in order to investigate the role of ligand structure on protein retention. The results demonstrated significant differences in the S and log k, values between all cytochrome c proteins despite the very high degree of sequence homology. These observations suggest that specific amino acid substitutions are directly involved in the chromatographic contact region of the cytochrome c molecules. In addition, the prosthetic haem moiety was also shown to provide a significant structural role in terms of relative protein stability under reversed-phase chromatographic conditions. Overall, the results of the present study further demonstrate the utility of interactive modes of chromatography to provide information on physicochemical aspects of protein-surface interactions.
Recombinant human growth hormone (r-hGH) expressed in Escherichia coli, was 70-80% purified by a ... more Recombinant human growth hormone (r-hGH) expressed in Escherichia coli, was 70-80% purified by a combination of ion-exchange chromatography and metal ion affinity chromatography. For the last purification step, a multicompartment electrolyzer was used, containing three compartments delimited by isoelectric membranes and two additional anodic and cathodic chambers. The central compartment was situated between two membranes having isoelectric points (pI) of 5.08 (anodic) and of 5.16 (cathodic), i.e. equidistant from the pI value of hGH (pI 5.12). r-hGH was isoelectric between these two membranes and could not leave the central chamber, while more acidic and more cathodic impurities collected in the two lateral chambers under the influence of the electric field. The r-hGH, thus purified, exhibited a single band by isoelectric focusing (IEF) in immobilized pH gradients (IPG) and gave recoveries greater than 90%. The problem of isoelectric precipitation in a practically ion-free environment was alleviated by focusing in 30% glycerol added with 1% neutral detergent (Nonidet-P40). The latter was eliminated by passage through a Q-Sepharose column after collecting the pI 5.12 band from the electrolyzer. Also the pre-hormone (pre-hGH) can be purified in a similar manner (30% glycerol, 1% Nonidet P-40) between two membranes having pIs 4.77 (anodic) and 4.87 (cathodic) (pre-hGH pI 4.82). This paper demonstrates the possibility of purifying by a focusing process also poorly soluble proteins at the pI.
Abstract In recent publications we have described the analysis of peptides and proteins by revers... more Abstract In recent publications we have described the analysis of peptides and proteins by reverse-phase high-pressure liquid chromatography (1–3). These studies demonstrated that the addition of 0.1% phosphoric acid to the mobile phase allowed the rapid and reproducible analysis of peptidic compounds. In addition phosphoric acid allows uv detection to be used at wavelengths down to 190 nm, thus permitting high sensitivity detection of underivatized samples. It is the purpose of the report to show that this chromatographic system allows the facile analysis of a variety of amino acids. The high sensitivity of the method is demonstrated by the analysis of 0.1 ng of tyrosine.
Two different series of polyethylenimine (PEI) block copolymers grafted with linear poly(ethylene... more Two different series of polyethylenimine (PEI) block copolymers grafted with linear poly(ethylene glycol) (PEG) were investigated as delivery systems for oligodeoxynucleotides (ODN) and ribozymes. The resulting interpolyelectrolyte complexes were characterized with respect to their physicochemical properties, protection efficiency against enzymatic degradation, complement activation, and biological activity under in vitro conditions. The effect of PEG molecular weight and the graft density of PEG blocks on complex characteristics was studied with two different series of block copolymers. The resulting ODN complexes were characterized by photon correlation spectroscopy (PCS) and laser Doppler anemometry (LDA) to determine complex size and zeta potential. Electrophoresis was performed to study the protective effects of the different block copolymers against enzymatic degradation of ODN. Intact ODN was quantified via densitometric analysis. Ribozymes, a particularly unstable type of oligonucleotides, were used to examine the influence of block copolymer structure on biological activity. The stabilization of ribozymes was also characterized in a cell culture model. Within the first series of block copolymers, the grafted PEG chains (5 kDa) had marginal influence on the complex size. Two grafted PEG chains were sufficient to achieve a neutral zeta potential. Within the second series, size and zeta potential increased with an increasing number of PEG chains. A high number of short PEG chains resulted in a decrease in complex size to values comparable to that of the homopolymer PEI 25 kDa and a neutral zeta potential, indicating a complete shielding of the charges. Complement activation decreased with an increasing number of short PEG 550 Da chains. Ribozyme complexes with PEG-PEI block copolymers achieved a 50% down-regulation of the target mRNA. This effect demonstrated an efficient stabilization and biological activity of the ribozyme, which was comparable to that of PEI 25 kDa. PEGylated PEI block copolymers represent a promising new class of drug delivery systems for ODN and ribozymes with increased biocompatibility and physical stability.
Abstract High-performance ion-exchange liquid chromatography was utilized for the purification of... more Abstract High-performance ion-exchange liquid chromatography was utilized for the purification of the acidic isozyme of adenylosuccinate synthetase from rat liver. Initial steps in the purification included ammonium sulfate fractionation and DEAE-cellulose and agarose-GTP affinity columns. The final steps were done on a SynChropak AX-300 anion-exchange support. The enzyme was purified 3000-fold with an overall yield of 10%. The enzyme preparation exhibited only one protein band on gel electrophoresis.
Synthesis of Novel Derivatives of Benzoxazole in Bis-Ionic Liquid [BDBDIm]Br. -The title ionic li... more Synthesis of Novel Derivatives of Benzoxazole in Bis-Ionic Liquid [BDBDIm]Br. -The title ionic liquid (IMB) is an effective catalyst for the synthesis of benzoxazoles from 2-hydroxybenzoic acid and 2-aminophenol derivatives by a solvent-free and simple method under mild conditions. -(NIKPASSAND*, M.;
Publisher Summary Reversed phase high performance liquid chromatography (RP-HPLC) now plays a cri... more Publisher Summary Reversed phase high performance liquid chromatography (RP-HPLC) now plays a critical role in the analysis and purification of peptides and proteins from natural and synthetic sources. The extraordinary popularity of RP-HPLC for polypeptide analysis can be attributed to a number of factors hi9ghlighted in this chapter. Some of the potential of RP-HPLC in the purification of polypeptides and proteins is demonstrated by the selected samples. Despite the current wide usage of RP-HPLC techniques in protein sequencing studies and many other purification areas involving high resolution polypeptide separations, the selection of a particular chemically-bonded stationary phase and mobile phase composition has frequently been based on empirical criteria. Not uncommonly, such arbitrary selections may not adequately address the issues of optimal chromatographic resolution, band shape or solute recovery. This chapter documents recent theoretical developments which characterize the RP-HPLC of polypeptides and proteins, and describes recent advances in the evolution of fully mechanistic models which describe in physicochemical terms the nature of the interaction between biological macromolecules and biological and synthetic surfaces.
The chromatographic properties of 16 thyromimetic iodoamino acids and related compounds on microp... more The chromatographic properties of 16 thyromimetic iodoamino acids and related compounds on microparticulate non-polar stationary phases have been examined and conditions determined which allow optimised resolution with analysis time ca. 60 minutes. These compounds elute in order of increasing hydrophobicity which correlates with the progressive increase in the number of iodogroups present in the tyrosine or thyronine aromatic nucleus. The reverse isomers, e.g. rT3, have consistently greater k' values than their corresponding analogues, e.g. T3. Conditions for the direct application of the rapid HPLC analyses of the iodoamino acids in biological or pharmaceutical samples have been examined.
Previously only one of the six disulphide bonds within the β-subunit of bovine thyrotropin (bTSHβ... more Previously only one of the six disulphide bonds within the β-subunit of bovine thyrotropin (bTSHβ) has been unequivocally assigned. In the present investigation, the fluorescent alkylating reagent 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulphonic acid has been employed as part of a double-alkylation strategy to allow the relative reactivities and the location of the six disulphide bonds of bTSHβ, after selective reduction, to be assigned by using reversed-phase HPLC peptide mapping techniques and associated methods of structural analysis. The most reactive disulphide bond was Cys88–Cys95; the second most reactive group of disulphide bonds involved the half-cystine residues Cys16, Cys19, Cys67 and Cys105 with the experimental results consistent with the assignment of disulphide bonds to Cys16–Cys67 and Cys19–Cys105. The least reactive group of half-cystine residues consisted of Cys2, Cys27, Cys31, Cys52, Cys83 and Cys85. The isolation, by high-performance ion-exchange chromatogr...
INTRODUCTIONThe RP-HPLC technique can be used to "desalt" peptide or protein samples de... more INTRODUCTIONThe RP-HPLC technique can be used to "desalt" peptide or protein samples derived from extraction procedures, from chemical reactions such as reductive alkylation in the presence of urea or guanidine hydrochloride, citraconylation, iodination, or cyanogen bromide cleavage, or recovered from other chromatographic separation. The peptide or protein solution is injected onto a small RP-HPLC column. An aqueous buffer is used to elute the salts while the peptides or proteins are concentrated at the top of the column. After elution of the salts, monitored by UV detection, the peptides or proteins are eluted with H(2)O-acetonitrile or H(2)O-isopropanol mobile phases.
Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.], 2001
The eight basic modes of HPLC currently in use for peptide and protein analysis and purification ... more The eight basic modes of HPLC currently in use for peptide and protein analysis and purification are presented in this overview. They include: size-exclusion chromatography (HP-SEC), ion-exchange chromatography (HP-IEX), normal phase chromatography (HP-NPC), hydrophobic interaction chromatography (HP-HIC), reversed-phase chromatography (RP-HPLC), hydrophilic interaction chromatography (HP-HILIC), immobilized metal ion affinity chromatography (HP-IMAC), and biospecific/biomimetic affinity chromatography (HP-BAC). In addition, some subsets of these chromatographic modes, e.g., mixed mode chromatography (HP-MMC), charge transfer chromatography (HP-CTC), or ligand-exchange chromatography (HP-LEC) are described. The unit includes overviews of the system requirements and planning strategies for set-up of the systems.
In this study, the advantages of carrying out the analysis of peptides and tryptic digests of pro... more In this study, the advantages of carrying out the analysis of peptides and tryptic digests of proteins under gradient elution conditions at pH 6.5 by reversed-phase liquid chromatography (RP-HPLC) and in-line electrospray ionisation mass spectrometry (ESI-MS) are documented. For these RP separations, a double endcapped, bidentate anchored n-octadecyl wide pore silica adsorbent was employed in a capillary column format. Compared to the corresponding analysis of the same peptides and protein tryptic digests using low pH elution conditions for their RP-HPLC separation, this alternative approach provides improved selectivity and more efficient separation of these analytes, thus allowing a more sensitive identification of proteins at different abundance levels, i.e. more tryptic peptides from the same protein could be confidently identified, enabling higher sequence coverage of the protein to be obtained. This approach was further evaluated with very complex tryptic digests derived from a human plasma protein sample using an online two-dimensional (2D) strong cation-exchange (SCX)-RP-HPLC-ESI-MS/MS system. Again, at pH 6.5, with mobile phases of different compositions, improved chromatographic selectivities were obtained, concomitant with more sensitive on-line electrospray ionisation tandem
INTRODUCTIONThis protocol addresses the group of large polypeptides that have proved to be troubl... more INTRODUCTIONThis protocol addresses the group of large polypeptides that have proved to be troublesome to handle because of their low solubility or the presence of secondary structural elements that favor supramolecular self-self assembly. Included in this group are polypeptides with amphipathic α-helical or β-sheet structures with extensive runs of nonpolar (hydrophobic) amino acid side chains or proline-rich sequences. RP-HPLC provides one avenue to purify such troublesome examples provided certain steps and precautions are taken. (Related procedures are equally germane to polypeptides that have been lipidated or subjected to chemical modifications with nonpolar moieties, as well as to some core cyanogen bromide fragments of large proteins.) RP-HPLC methods not only enable concomitant desalting, removal of additives that aid dissolution of the polypeptide or protein, but also permit resolution and maintenance of a reasonably high concentration of the solutes, due to the presence o...
The dynamic behaviour of a series of closely related cytochrome c molecules has been investigated... more The dynamic behaviour of a series of closely related cytochrome c molecules has been investigated in reversed-phase high-performance liquid chromatography. In particular, the influence of temperature and gradient time on the experimental bandwidths of equine, tuna, canine and bovine cytochrome c and equine and tuna apocytochrome c has been determined with a C,, and a C, sorbent. The observed bandbroadening changes are discussed in comparison to the corresponding retention behaviour of the cytochrome c molecules reported in the preceding paper [l]. The results demonstrate that bandwidth measurements can be used as an indicator of the conformational status of a protein under a particular set of chromatographic conditions.
The retention behaviour of equine, tuna, canine and bovine cytochrome c and the corresponding equ... more The retention behaviour of equine, tuna, canine and bovine cytochrome c and the corresponding equine and tuna apocytochrome c has been investigated using reversed-phase gradient elution chromatographic procedures. A range of operating temperatures between 5 and 85°C were utilised to monitor the influence of protein unfolding on the corresponding retention parameters. Chromatographic measurements were obtained with both an n-octadecyl (C,,) and n-butyl (C,) sorbent in order to investigate the role of ligand structure on protein retention. The results demonstrated significant differences in the S and log k, values between all cytochrome c proteins despite the very high degree of sequence homology. These observations suggest that specific amino acid substitutions are directly involved in the chromatographic contact region of the cytochrome c molecules. In addition, the prosthetic haem moiety was also shown to provide a significant structural role in terms of relative protein stability under reversed-phase chromatographic conditions. Overall, the results of the present study further demonstrate the utility of interactive modes of chromatography to provide information on physicochemical aspects of protein-surface interactions.
Recombinant human growth hormone (r-hGH) expressed in Escherichia coli, was 70-80% purified by a ... more Recombinant human growth hormone (r-hGH) expressed in Escherichia coli, was 70-80% purified by a combination of ion-exchange chromatography and metal ion affinity chromatography. For the last purification step, a multicompartment electrolyzer was used, containing three compartments delimited by isoelectric membranes and two additional anodic and cathodic chambers. The central compartment was situated between two membranes having isoelectric points (pI) of 5.08 (anodic) and of 5.16 (cathodic), i.e. equidistant from the pI value of hGH (pI 5.12). r-hGH was isoelectric between these two membranes and could not leave the central chamber, while more acidic and more cathodic impurities collected in the two lateral chambers under the influence of the electric field. The r-hGH, thus purified, exhibited a single band by isoelectric focusing (IEF) in immobilized pH gradients (IPG) and gave recoveries greater than 90%. The problem of isoelectric precipitation in a practically ion-free environment was alleviated by focusing in 30% glycerol added with 1% neutral detergent (Nonidet-P40). The latter was eliminated by passage through a Q-Sepharose column after collecting the pI 5.12 band from the electrolyzer. Also the pre-hormone (pre-hGH) can be purified in a similar manner (30% glycerol, 1% Nonidet P-40) between two membranes having pIs 4.77 (anodic) and 4.87 (cathodic) (pre-hGH pI 4.82). This paper demonstrates the possibility of purifying by a focusing process also poorly soluble proteins at the pI.
Abstract In recent publications we have described the analysis of peptides and proteins by revers... more Abstract In recent publications we have described the analysis of peptides and proteins by reverse-phase high-pressure liquid chromatography (1–3). These studies demonstrated that the addition of 0.1% phosphoric acid to the mobile phase allowed the rapid and reproducible analysis of peptidic compounds. In addition phosphoric acid allows uv detection to be used at wavelengths down to 190 nm, thus permitting high sensitivity detection of underivatized samples. It is the purpose of the report to show that this chromatographic system allows the facile analysis of a variety of amino acids. The high sensitivity of the method is demonstrated by the analysis of 0.1 ng of tyrosine.
Two different series of polyethylenimine (PEI) block copolymers grafted with linear poly(ethylene... more Two different series of polyethylenimine (PEI) block copolymers grafted with linear poly(ethylene glycol) (PEG) were investigated as delivery systems for oligodeoxynucleotides (ODN) and ribozymes. The resulting interpolyelectrolyte complexes were characterized with respect to their physicochemical properties, protection efficiency against enzymatic degradation, complement activation, and biological activity under in vitro conditions. The effect of PEG molecular weight and the graft density of PEG blocks on complex characteristics was studied with two different series of block copolymers. The resulting ODN complexes were characterized by photon correlation spectroscopy (PCS) and laser Doppler anemometry (LDA) to determine complex size and zeta potential. Electrophoresis was performed to study the protective effects of the different block copolymers against enzymatic degradation of ODN. Intact ODN was quantified via densitometric analysis. Ribozymes, a particularly unstable type of oligonucleotides, were used to examine the influence of block copolymer structure on biological activity. The stabilization of ribozymes was also characterized in a cell culture model. Within the first series of block copolymers, the grafted PEG chains (5 kDa) had marginal influence on the complex size. Two grafted PEG chains were sufficient to achieve a neutral zeta potential. Within the second series, size and zeta potential increased with an increasing number of PEG chains. A high number of short PEG chains resulted in a decrease in complex size to values comparable to that of the homopolymer PEI 25 kDa and a neutral zeta potential, indicating a complete shielding of the charges. Complement activation decreased with an increasing number of short PEG 550 Da chains. Ribozyme complexes with PEG-PEI block copolymers achieved a 50% down-regulation of the target mRNA. This effect demonstrated an efficient stabilization and biological activity of the ribozyme, which was comparable to that of PEI 25 kDa. PEGylated PEI block copolymers represent a promising new class of drug delivery systems for ODN and ribozymes with increased biocompatibility and physical stability.
Abstract High-performance ion-exchange liquid chromatography was utilized for the purification of... more Abstract High-performance ion-exchange liquid chromatography was utilized for the purification of the acidic isozyme of adenylosuccinate synthetase from rat liver. Initial steps in the purification included ammonium sulfate fractionation and DEAE-cellulose and agarose-GTP affinity columns. The final steps were done on a SynChropak AX-300 anion-exchange support. The enzyme was purified 3000-fold with an overall yield of 10%. The enzyme preparation exhibited only one protein band on gel electrophoresis.
Synthesis of Novel Derivatives of Benzoxazole in Bis-Ionic Liquid [BDBDIm]Br. -The title ionic li... more Synthesis of Novel Derivatives of Benzoxazole in Bis-Ionic Liquid [BDBDIm]Br. -The title ionic liquid (IMB) is an effective catalyst for the synthesis of benzoxazoles from 2-hydroxybenzoic acid and 2-aminophenol derivatives by a solvent-free and simple method under mild conditions. -(NIKPASSAND*, M.;
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