Three stationary phases have been prepared for affinity liquid chromatography isolation and separation of porcine and human pepsin. The phases contain 3,5-diiodo-L-tyrosine (DIT) bound to the supports HEMA BIO VS, HEMA BIO E and EPOXY TOYOPEARL. These phases have been tested on a model sample of porcine pepsin A and applied to human pepsin. Fractions have been collected and the chymase activity determined in selected analyses. For affinity CE, capillaries have been prepared by modifying the wall with 3-aminopropyltriethoxysilane, followed either by direct binding of DIT, or by binding L-tyrosine that was subsequently iodated. The dissociation constant K(d) has been determined for the pepsin-DIT complex from the changes in the electrophoretic mobilities.
- MeSH
- Chromatography, Affinity methods instrumentation MeSH
- Diiodotyrosine chemistry MeSH
- Electrophoresis, Capillary methods instrumentation MeSH
- Research Support as Topic MeSH
- Humans MeSH
- Silicon Dioxide chemistry MeSH
- Pepsin A isolation & purification MeSH
- Swine MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
Carbonyl-reducing enzymes are important in both metabolism of endogenous substances and biotransformation of xenobiotics. Because sufficient amounts of native enzymes must be obtained to study their roles in metabolism, an efficient purification strategy is very important. Oracin (6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11H-indeno[1,2-c] isoquinoline) is a prospective anticancer drug and one of the xenobiotic substrates for carbonyl-reducing enzymes. A new purification strategy based on molecular recognition of carbonyl-reducing enzymes with oracin as a ligand is reported here. The type of covalent bond, ligand molecules orientation, and their distance from the backbone of the solid matrix for good stearic accessibility were taken into account during the designing of the carrier. The carriers based on magnetically active microparticles were tested by recombinant enzymes AKR1C3 and CBR1. The SiMAG-COOH magnetic microparticles with N-alkylated oracin and BAPA as spacer arm provide required parameters: proper selectivity and specificity enabling to isolate the target enzyme in sufficient quantity, purity, and activity.
- MeSH
- Alcohol Oxidoreductases isolation & purification MeSH
- Biological Assay MeSH
- Chromatography, Affinity MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Enzyme Assays methods MeSH
- Enzymes isolation & purification MeSH
- Ethanolamines chemistry MeSH
- Isoquinolines chemistry MeSH
- Ligands MeSH
- Magnetics * MeSH
- Microspheres MeSH
- Molecular Structure MeSH
- Antineoplastic Agents chemistry MeSH
- Schiff Bases chemistry MeSH
- Chromatography, High Pressure Liquid MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
... Methods for isolating RNA 15 -- Basic principles 15 -- Cell lysis and fractionation procedures 17 -- ... ... Isolation of total RNA by cell lysis 17 -- Isolation of RNA from cell fractions 19 -- Isolation of RNA ... ... 27 -- Isolation of total cellular RNA using the guanidinium-lithium chloride method 28 vi RNA ISOLATION ... ... -- Fractionation of RNA using magnetic beads 37 -- Isolation of newly synthesized RNA by affinity methods ... ... Isolation and analysis of ribonucleoproteins 167 -- Ribonucleoproteins (RNPs) 167 -- Isolation of ribonucleoproteins ...
xi, 196 stran : ilustrace, tabulky ; 24 cm
- MeSH
- Polymerase Chain Reaction MeSH
- Ribonucleoproteins MeSH
- RNA isolation & purification MeSH
- Sequence Analysis, RNA MeSH
- Publication type
- Monograph MeSH
- Conspectus
- Biochemie. Molekulární biologie. Biofyzika
- NML Fields
- biochemie
- molekulární biologie, molekulární medicína
Heterologous proteins are frequently purified by immobilized metal ion affinity chromatography (IMAC) based on their modification with a hexa-histidine affinity tag (His-tag). The terminal His-tag can, however, alter functional properties of the tagged protein. Numerous strategies for the tag removal have been developed including chemical treatment and insertion of protease target sequences in the protein sequence. Instead of using these approaches, we took an advantage of natural interaction of zinc finger domains with metal ions to purify functionally similar retroviral proteins from two different retroviruses. We found that these proteins exhibited significantly different affinities to the immobilized metal ions, despite that both contain the same type of zinc finger motif (i.e., CCHC). While zinc finger proteins may differ in biochemical properties, the multitude of IMAC platforms should allow relatively simple yet specific method for their isolation in native state.
- MeSH
- Chromatography, Affinity methods MeSH
- Escherichia coli genetics MeSH
- Gene Expression MeSH
- HIV-1 chemistry genetics MeSH
- Metals chemistry MeSH
- Mason-Pfizer monkey virus chemistry genetics MeSH
- Viral Proteins chemistry genetics isolation & purification MeSH
- Zinc analysis MeSH
- Zinc Fingers MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
