RESUMEN
Recombinant human erythropoietin (rHuEPO) is biologically functional when in a monomeric state; upon extensive heating, rHuEPO forms a dimer. The nature of this dimeric linkage was investigated after isolation of the dimer by gel filtration. The dimer fraction was subjected to tryptic digestion, and the peptides were separated by reversed-phase HPLC. SDS-PAGE, N-terminal sequencing, capillary electrophoresis and mass spectrometry (both liquid-chromatographic electrospray and matrix-assisted laser desorption ionization) were employed to compare the tryptic peptides from heat-treated rHuEPO and untreated rHuEPO. Results demonstrated that elevated heat broke the intramolecular disulfide bond between Cys-7 and Cys-161 and an intermolecular disulfide bond then formed from these residues, producing a covalently linked rHuEPO homodimer. Dimer formation was also mathematically modeled and shown to fit a simple equilibrium.
Asunto(s)
Eritropoyetina/química , Secuencia de Aminoácidos , Cromatografía en Gel , Dimerización , Electroforesis Capilar , Electroforesis en Gel de Poliacrilamida , Eritropoyetina/genética , Calor , Humanos , Cinética , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Conformación Proteica , Proteínas Recombinantes , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tripsina/metabolismoRESUMEN
The microheterogeneity of the carbohydrate structures on recombinant human erythropoietin (rHuEPO) expressed in Chinese hamster ovary cells has been evaluated by electrospray ionization (ESI) mass spectrometry (MS) of glycopeptide fragments. The microheterogeneity is largely associated with the presence or absence of terminal N-acetylneuraminic acid (Neu5Ac) residues, varying amounts of O-acetylation of the Neu5Ac residues, and the presence or absence of N-acetyllactosamine extensions. The N-linked carbohydrate structures were structurally diverse; 52 different N-linked oligosaccharide structures were identified. Consistent structural assignments could be made from data obtained using different proteolytic digests, ESI solvent systems (aqueous/methanol systems with acetic or formic acid), and on-line or off-line LC/MS analysis. All glycosylation sites exhibited some level of O-acetylation of Neu5Ac residues. Interestingly, glycosylation site asparagine-83 exhibits mono-O-acetyl and di-O-acetyl Neu5Ac residues, while the other sites, asparagine-24, asparagine-38, and serine-126, exhibit mainly mono-O-acetyl Neu5Ac derivatization. This difference in O-acetylation may be site specific or due to sample handling of labile structures. However, mild base treatment of rHuEPO with NaOH on ice removed the O-acetyl groups associated with a given carbohydrate structure, without adversely affecting the underlying oligosaccharide structure, resulting in a simplified mass spectra. Nuclear magnetic resonance spectroscopy of Neu5Ac residues released by neuraminidase treatment of total rHuEPO indicated that Neu5,9Ac2 residues were present. Additional resonances were also observed that were consistent with other Neu5Ac O-acetyl linkages; these O-acetyl resonances could be removed by mild base hydrolysis of rHuEPO.
Asunto(s)
Eritropoyetina/química , Secuencia de Aminoácidos , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Secuencia de Consenso , Cricetinae , Cricetulus , Eritropoyetina/genética , Femenino , Glicopéptidos/análisis , Humanos , Hidrólisis , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Datos de Secuencia Molecular , Ovario/citología , Ovario/metabolismo , Fragmentos de Péptidos/análisis , Proteínas Recombinantes/químicaRESUMEN
High-performance capillary electrophoresis (HPCE) has been employed to characterize the peptide map of recombinant human erythropoietin (rHuEPO) expressed from Chinese hamster ovary (CHO) cells. The methodology employs an ion pairing agent, 100 mM heptanesulfonic acid in 40 mM sodium phosphate buffer, pH 2.5, to increase peptide resolution, to decrease analyte wall interactions, and to evaluate glycopeptide microheterogeneity. The total tryptic map is segregated into two regions, nonglycosylated and glycosylated peptides. Reproducibility of the peptide map is excellent; the map results in baseline separation of 16 tryptic peptides and one doublet peak composed of two peptides (resolution 0.22). The map furthermore allows for the evaluation of the microheterogeneity associated with the three rHuEPO glycopeptides. At least 12 glycopeptide forms were separated in the initial peptide map. Peptides were identified by Edman sequencing, and the glycopeptides were further subjected to Dionex anion-exchange chromatography. To simplify the level of complexity associated with the glycopeptides, much of the characterization employed asialoglycopeptides and employed several endoproteolytic diagnosis. The relative percent distribution for each purified asialoglycopeptide was calculated to define the level of complexity and to tentatively assign a known structure to the HPCE peak. The level of structural complexity of the asialoglycopeptides appears to increase from the simplest O-linked form to the more complex N83, N38, and N24 glycosylation positions, respectively. HPCE evaluation of glycopeptide microheterogeneity appears to be simpler, faster, and just as sensitive as other more frequently employed methods for glycopeptide characterizations.
Asunto(s)
Eritropoyetina/química , Animales , Células CHO , Cricetinae , Electroforesis , Endopeptidasas , Glicopéptidos/química , Humanos , Hidrólisis , Mapeo Peptídico , Proteínas Recombinantes/químicaRESUMEN
College students (45 females and 12 males) estimated the frequency of pleasant and unpleasant English words. 28 subjects estimated how often they, personally, had seen the words (Personal condition), and 29 subjects estimated how often a "language expert" had encountered the words (Kucera-Francis condition). Byrne's Repression-Sensitization scores were obtained for all subjects. In both conditions repressors judged pleasant words to be more frequent, and sensitizers judged unpleasant words to be more frequent (p less than .01). The results may clarify some contradictions in the literature on frequency estimation and may help to explain the relationship between repression-sensitization and perceptual defense/vigilance.