ABSTRACT
PURPOSE: To isolate and characterize a monomethioninesulfoxide variant of the commercially available therapeutic protein interferon alpha-2b. METHODS: The methionine (Met)-oxidized variant was isolated by reverse-phase high performance liquid chromatography and characterized by SDS-PAGE, peptide mapping and mass spectrometric analysis of the trypsin/V8-generated peptide fragments. The biological and immunological activities of the isolated variant were also evaluated. RESULTS: The rHuIFN alpha-2b variant was found to contain a Met sulfoxide residue at position 111 of the rHuIFN alpha-2b molecule. The far-UV CD spectra showed a slight loss of alpha-helical content and an increase in the beta-sheet contribution. The CD spectra indicate that both chromatographic conditions and Met oxidation contribute to the observed secondary structure changes. Both interferon alpha-2b main component and its methionine-oxidized variant showed different reactivity to monoclonal antibodies employed in immunoassays for the protein. CONCLUSIONS: A monomethioninesulfoxide rHuIFN alpha-2b variant was found to be present in the rHuIFN alpha-2b bulk drug substance in solution. The Met(111) residue was identified as Met sulfoxide by comparative tryptic/V8 mapping and mass spectrometric analysis. Nevertheless, the oxidation of the Met(111) residue did not seem to have a detectable effect on the biological activity of the molecule.
Subject(s)
Antiviral Agents/isolation & purification , Interferon-alpha/isolation & purification , Methionine/analogs & derivatives , Amino Acid Sequence , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Cells, Cultured , Chromatography, High Pressure Liquid , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Humans , Interferon alpha-2 , Interferon-alpha/chemistry , Interferon-alpha/pharmacology , Mass Spectrometry , Methionine/chemistry , Molecular Sequence Data , Peptide Mapping , Recombinant Proteins , Spectrophotometry, UltravioletABSTRACT
The analysis of lipids remains at the periphery of the biotechnology arena. Methods for analysis of lipids still center around the proven methods of thin layer chromatography, gas chromatography, gas chromatography-mass spectrometry and, more recently, high performance liquid chromatography. Advances have been made recently in understanding the biological role of lipid-conjugated molecules. Encapsulation of active substances in lipids is receiving more attention in order to achieve cellular penetration.
Subject(s)
Lipids/analysis , Humans , Lipids/chemistry , Lipids/physiology , Liposomes , Proteins/chemistrySubject(s)
Antibodies, Monoclonal , Animals , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/physiology , Antibodies, Monoclonal/therapeutic use , Antibody Specificity , Chromatography , Humans , Hybridomas/immunology , Immune Sera , MiceABSTRACT
We investigated the apparent requirement that Q beta replicase must add a nontemplated adenosine to the 3' end of newly synthesized RNA strands. We used abbreviated MDV-1 (+)-RNA templates that lacked either 62 or 63 nucleotides at their 5' end in Q beta replicase reactions. The MDV-1 (-)-RNA strands synthesized from these abbreviated (+)-strand templates were released from the replication complex, yet they did not possess a nontemplated 3'-terminal adenosine. These results imply that, despite observations that all naturally occurring RNAs synthesized by Q beta replicase possess a nontemplated 3'-adenosine, the addition of an extra adenosine is not an obligate step for the release of completed strands. Since the abbreviated templates lacked a normal 5' end, it is probable that a particular sequence at the 5' end of the template is required for terminal adenylation to occur.
Subject(s)
Coliphages/enzymology , Escherichia coli/enzymology , Q beta Replicase/metabolism , RNA Nucleotidyltransferases/metabolism , RNA, Bacterial/genetics , Base Sequence , Genetic Variation , Kinetics , Nucleic Acid Hybridization , Templates, GeneticSubject(s)
Mammary Tumor Virus, Mouse/analysis , Viral Proteins/analysis , Adsorption , Animals , Electrophoresis, Polyacrylamide Gel/methods , Female , Immunoglobulin G/isolation & purification , Mammary Neoplasms, Experimental/analysis , Mice , Radioimmunoassay/methods , Staphylococcus aureus/immunologyABSTRACT
Affinity-purified Maclura pomifera lectin (MPL) elutes from a gel filtration column as a single symmetrical peak with characteristics expected for a single protein of approximately 40 000 daltons. This material can be dissociated into two dissimilar polypeptide chains of approximately 10 000 daltons. Ion-exchange chromatography on DEAE-cellulose resolves affinity-purified MPL into five components. These proteins are structurally related and contain varying proportions of the two polypeptide chains. Two of these tetrameric lectins, each composed solely of one of these chains, display differences in mobility during discontinuous polyacrylamide gel electrophoresis and ion-exchange chromatography, but display no detectable differences in hemagglutination of human erythrocytes and interactions with carbohydrates.
Subject(s)
Lectins/isolation & purification , Plant Proteins/isolation & purification , Amino Acids/analysis , Chromatography, Affinity , Molecular Weight , Plant Lectins , Seeds/analysisABSTRACT
Maclura promifera seeds contain a protein which agglutinates human erythrocytes at concentrations as low as 4 ng/mL. This property is related to its ability to bind with high specificity various alpha-D-galactopyranosides. The agglutinin, which was pruified by affinity adsorption, exhibits one band on immunoelectrophoresis and displays one peak during ultracentrifugation, isoelectric focusing, and gel permeation chromatography. The active protein has a molecular weight of 40 000-43 000 and contains two dissimilar polypeptide chains of 12 000 and 10 000, respectively.
