High performance liquid affinity chromatography (HPLAC) and its application to the separation of enzymes and antigens

Ohlson, Sten; Hansson, Lennart; Larsson, Per‐Olof; Mosbach, Klaus

doi:10.1016/0014-5793(78)80792-3

Cited by 195 publications

(51 citation statements)

References 12 publications

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“…Ohlson et al, (1978) introduced the concept of high performance affinity chromatography (HPAC), which combined the two chromatographic techniques of high performance liquid chromatography and affinity chromatograpy. Now, HPAC is a useful technique for the separation and purification of protein and enzyme.…”

Section: Introductionmentioning

confidence: 99%

Three novel high performance affinity chromatographic media for the separation of antithrombin III from human plasma

Zhao

Shangguan

et al. 2001

Biomedical Chromatography

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Novel monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) beads (PGMA) were employed as the support for high performance affinity chromatography. Heparin was covalently attached to PGMA beads by three different coupling methods. Heparin-PGMA-I was prepared by directly coupling amino-groups of heparin with PGMA. Heparin-PGMA-II and III were prepared by the coupling of heparin to amino-PGMA, which was obtained by amination of PGMA. Heparin-PGMA-II was prepared by coupling the carboxyl groups of heparin to amino-PGMA by using water-soluble carbodiimide as coupling reagent, and heparin-PGMA-III was prepared by the reductive amination of heparin and amino-PGMA with sodium cyanoborohydride. The heparin contents of heparin-PGMA-I, II and III were 1.6, 10.2 and 1.0 mg/g beads, respectively. Their affinity capacities for antithrombin III were investigated. Their binding activity to antithrombin III was not proportional to the content of heparin immobilized, and heparin-PGMA-I was the most efficient affinity medium for antithrombin III. The resultant affinity media presented minimal non-specific interaction with other proteins and can be used in a wide pH range. All the three heparin-PGMA beads were exploited for the separation of antithrombin III from human plasma. The purity of antithrombin III obtained was higher than 90%, which was confirmed by high performance size exclusion chromatography.

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Section: Introductionmentioning

confidence: 99%

Three novel high performance affinity chromatographic media for the separation of antithrombin III from human plasma

Zhao

Shangguan

et al. 2001

Biomedical Chromatography

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“…In this paper the enzyme amyloglucosidase has been immobilized to a modified silica surface via reductive amination [ 10,11]. Prior to immobilization of the protein, the diol groups on the silica surface were oxidized to aldehyde functions [ 12].…”

Section: Introductionmentioning

confidence: 99%

Use of immobilized amyloglucosidase as chiral selector in chromatography. Control of enantioselective retention and resolution in liquid chromatography

et al. 1999

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Key WordsColumn liquid chromatography Glucoamylase Enantioselective retention and resolution Effect of operating parameters Ionic strength SummarySeveral mobile phase parameters were investigated for controlling enantioselective retention and resolution on a chiral stationary phase made in-house. The chiral selector was the enzyme amyloglucosidase, which was immobilized onto a silica support via reductive amination. The influences of the mobile phase pH, concentration and type of uncharged organic modifier, ionic strength and column temperature on enantioselectivity were studied. The analysis time for resolving enantiomers could be adjusted with only a minor decrease in enantiosetectivity by using a high ionic strength mobile phase buffer. This indicated a retention mechanism involving ion-exchange interactions. It was further confirmed by the decreasing enantioselectivity of amines when using a mobile phase pH below the isoelectric point of the native protein. Interesting effects were observed when the organic modifier concentration was increased and also when the column temperature was raised. Both retention and enantioselectivity increased with increasing concentration of 2-propanol in the mobile phase. Examples are given where both enantioselectivity and retention increased with increasing column temperature. Thermodynamic studies were performed to calculate the entropy and enthalpy constants. The results showed that, depending on mobile phase composition, the enantioselective retention may be caused by differences in entropy or enthalpy.

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“…235,236 This method is based on weak ligand -ligate interactions at high concentrations of bound ligand on rigid particles approximately 10 µm in diameter. Use of monoclonal antibodies 237 or lectins as ligands 238 allows excellent chromatographic separation of oligosaccharides.…”

Section: Ion-exchange High-performance Liquidmentioning

confidence: 99%

Glycoprotein Analysis: General Methods

Debray¹,

Spik²

2000

Encyclopedia of Analytical Chemistry

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Glycosylation is a common co‐ and post‐translational modification of a protein which may have profound effects on protein structure and function. Many biologically interesting proteins are glycosylated at their asparagine, serine, and threonine residues. Glycosylation has now been recognized as being more ubiquitous and structurally varied than all other types of post‐translational modifications combined. The glycosylation pattern of a glycoprotein is not random but is dependent on cell type, physiological conditions and, in some cases, disease states. Carbohydrate chains or glycans attached to the peptide backbone are classified according to the nature of the linkage to the peptide and their structure; both of these parameters allow the definition of common cores. In addition to the extreme structural diversity and complexity of glycan structures, one feature of protein glycosylation is the presence at a single site of any one of a number of glycan structures. This property gives rise to an extremely heterogeneous population of glycoproteins, termed glycoforms. The complexity of the glycan structures, the multiple substitutions (microheterogeneity) at glycosylation sites, and the structural diversity associated with the protein backbone itself represent an enormous task for analytical structural studies. Indirect methods such as immunological approaches using antibodies or lectins provide some structural information concerning the nature of glycans. Prior to their structural study, glycans have to be released from the protein backbone by enzymatic or chemical methods. Different endoenzymes such as N ‐glycanase ® allow a complete deglycosylation of glycoproteins, providing a pool of oligosaccharides which may be further fractionated. Establishment of the site heterogeneity and assessment of the different glycoforms require isolation and analysis of the individual glycopeptides generated after hydrolysis with specific proteases. Fractionation of glycans or glycopeptides can be achieved by different high‐performance liquid chromatography (HPLC) or electrophoretic procedures. Lectins, due to their extreme specificities with regard to monosaccharide or oligosaccharide motifs, represent powerful tools for sequential glycan fractionation. Traditionally, a complete structural analysis of the glycan structures, including determination of the carbohydrate sequence and the sugar linkage, has been a tedious, multidisciplinary task. Characterization and determination of the relative amount of individual constituent monosaccharides is generally obtained by gas–liquid chromatography (GLC) after hydrolysis of the oligosaccharide chain. Information concerning the nature of linkages and branching points is furnished by the methylation procedure. Nevertheless, these chemical sequencing methods often necessitate milligrams of material. To progress from this situation, sensitive physicochemical or enzymatic methods have been developed. The nuclear magnetic resonance (NMR) study of oligosaccharides, even of the less sensitive of the physicochemical techniques, is extremely powerful because it allows determination in a mixture of glycans, the complete structure of individual glycans including nature of monosaccharides, sequence, type of linkages, anomericity and branching points. The recent advances in mass spectrometry (MS) techniques, mainly matrix‐assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI), have provided very sensitive means for the analysis of oligosaccharides and glycopeptides. The gain in sensitivity associated with a minimum number of sample manipulation steps allows work at the submicrogram level. The use of enzyme reagents is rapidly becoming popular in modern carbohydrate analysis because of their inherent selectivities for a sugar substrate and its linkage type. Exoglycosidases can be used in conjunction with other methods such as MALDI/MS or fluorophore‐assisted carbohydrate electrophoresis (FACE) separation. Complete structural information is now feasible for low‐microgram and submicrogram quantities of a glycoprotein; this quality of performance places the field of carbohydrate analysis closer to the sequencing methods available for other biomolecules, proteins or nucleic acids.

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High performance liquid affinity chromatography (HPLAC) and its application to the separation of enzymes and antigens

Cited by 195 publications

References 12 publications

Three novel high performance affinity chromatographic media for the separation of antithrombin III from human plasma

Three novel high performance affinity chromatographic media for the separation of antithrombin III from human plasma

Use of immobilized amyloglucosidase as chiral selector in chromatography. Control of enantioselective retention and resolution in liquid chromatography

Glycoprotein Analysis: General Methods

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