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Results for “Isotope Labeling”

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PubMed2004

Isotopic labeling of recombinant proteins from the methylotrophic yeast Pichia pastoris.

The methylotrophic yeast Pichia pastoris is now an established expression system for the production of recombinant protein for nuclear magnetic resonance (NMR) studies. It is capable of expressing high levels of heterologous proteins and possesses the ability to perform many of the posttranslational modifications of higher eukaryotes. Here, we describe efficient methods for the production of uniformly 13C,15N-labeled proteins in shake flasks and of uniformly 13C,15N-labeled and 2H,13C,15N-labeled proteins in fermenters. We also provide details of two chromatographic procedures, cation exchange and concanavalin A lectin affinity, that have proved useful in purifying P. pastoris-expressed proteins for NMR studies.

Carbon Isotopes↗
PubMed1999

Simultaneous investigation of indinavir nonlinear pharmacokinetics and bioavailability in healthy volunteers using stable isotope labeling technique: study design and model-independent data analysis.

Indinavir follows nonlinear pharmacokinetics upon oral administration at clinical doses. A study employing the stable isotope administration technique in a three-treatment design was conducted to identify the source of the nonlinearity and to determine the dose-dependency of systemic bioavailability. In treatment A, 400 mg of unlabeled indinavir (D0) was coadministered orally with 16 mg of a hexadeutero analogue of indinavir (D6) intravenously. In treatment B, 800 mg of D0 po was coadministered with 16 mg of D6 intravenously. In treatment C, 16 mg of iv D6 was infused concurrently with 16 mg iv of D0. Plasma concentrations of D0 and D6 were determined by an LC/MS/MS assay method. Concentrations of indinavir in plasma increased greater than dose-proportionally over the 400- to 800-mg dose range. No meaningful kinetic isotope effects were found in treatment C. Plasma concentrations of D6 were dependent on the coadministered D0-indinavir dose and were lowest in treatment C, higher in treatment A, and highest in treatment B. The bioavailability of indinavir was high (60-65%) and comparable between the 400- and 800-mg doses. There was a significant contribution of nonlinear kinetics in the systemic circulation to the observed disproportional increase in plasma concentrations following oral dosing. The high bioavailability at clinically relevant doses suggests a high degree of saturation of first-pass metabolism. These results further demonstrate that the concomitant administration technique in combination with the LC/MS/MS method can provide a realistic and reliable means of elucidating important pharmacokinetic properties of drug candidates during product development.

Adult↗
PubMedDate not supplied

Direct measurement of nucleoside monophosphate delivery from a phosphoramidate pronucleotide by stable isotope labeling and LC-ESI(-)-MS/MS.

Amino acid phosphoramidates of nucleosides have been shown to be potent antiviral and anticancer agents with the potential to act as nucleoside monophosphate prodrugs. To access their ability to deliver 3'-azido-3'-deoxythymidine (AZT) 5'-monophosphate to cells, the decomposition pathway of an 18O-labeled AZT amino acid phosphoramidate was investigated by capillary reverse-phase high-performance liquid chromatography interfaced with negative ion electrospray ionization mass spectrometry (LC-ESI(-)-MS/MS). 18O-labeled L-AZT tryptophan phosphoramidate methyl ester ([18O]2) was synthesized with an 18O/16O relative ratio of 1.22 +/- 0.18. For CEM cells, a human T-lymphoblast leukemia cell line, incubated with [18O]2, values of 1.55 +/- 0.37, 0.34, and 0.13 were found for the 18O/16O relative ratio of intracellular AZT-MP for time intervals of 0.5, 4, and 20 h, respectively. The decrease in the level of labeled AZT-MP in CEM cells corresponded to a rapid increase in the amount of intracellular AZT presumably by dephosphorylation of AZT-MP. In contrast, for peripheral blood mononuclear cells (PBMCs), the 18O/16O relative ratio values of intracellular AZT-MP were 1.43, 1.06, and 0.61 for time intervals of 0.5, 4, and 20 h, respectively. Intracellular AZT in PBMCs was nearly undetectable for each time interval. Taken together, these results are consistent with the detection of direct P-N bond cleavage by CEM cells and PBMCs. However, AZT phosphoramidates are able to more effectively deliver AZT-MP to PBMCs than to CEM cells. Differential expression of 5'-nucleotidase in CEM cells relative to PBMCs is likely the reason for this discrepancy. Although applied to a phosphoramidate pronucleotide, the judicious use of 18O labeling and LC-MS is a general approach that could be applied to the investigation of the intracellular fate of other pronucleotides.

Amides↗
PubMed2000

IR spectroscopy of isotope-labeled helical peptides: probing the effect of N-acetylation on helix stability.

The effect of N-acetylation on the conformation of alanine-rich helical peptides is examined using isotope-edited Fourier transform infrared (FTIR) spectroscopy. A series of peptides with sequence AA(AAKAA)(3)AAY has been prepared; each peptide incorporates four (13)C-labeled alanines. These peptides have two amide I' bands in their FTIR spectra: one corresponding to the (12)C amino acids, and one assigned to the (13)C amino acids. The intensity and frequency of the (13)C amide I' band varies systematically with the position of the labels in the sequence and the presence or absence of an N-acetyl capping group. The intensity of the (13)C amide I' band correlates with helix stability at the labeled residues as predicted by thermodynamic models of the helix-coil transition. These results suggest that FTIR spectroscopy combined with specific isotope labeling can be used to dissect the conformation of helical peptides at the residue level.

Acetylation↗
PubMed2005

Assignments of the Pfr-Pr FTIR difference spectrum of cyanobacterial phytochrome Cph1 using 15N and 13C isotopically labeled phycocyanobilin chromophore.

The reversible red and far-red light-induced transitions of cyanobacterial phytochrome Cph1 from Synechocystis PCC 6803 were investigated by Fourier transform infrared (FTIR) difference spectroscopy. High-quality light-induced Pfr-Pr difference FTIR spectra were recorded for the 58 kDa N-terminal domain of Cph1 by repetitive photochemical cycling and signal averaging. The Pfr-Pr difference spectra in H(2)O and D(2)O were very similar to those previously reported for full-length 85 kDa Cph1.(1) Published assignments were extended by analysis of the effects of (13)C and (15)N isotope substitutions at selected sites in the phycocyanobilin chromophore and by (15)N global labeling of the protein. The Pfr-Pr difference spectra were dominated by an amide I peak/trough at 1653 cm(-1)(+)/1631 cm(-1)(-) and a smaller amide II band at 1554 cm(-1). Labeling effects allowed specific chromophore assignments for the C(1)=O (1736 cm(-1)(-)/1724 cm(-1)(+)) and C(19)=O (1704 cm(-1)(-)) carbonyl vibrations, C=C vibrations at 1589 cm(-1)(+), and bands at 1537(-), 1512(+), 1491(-), 1163(+), 1151(-), 1134(+), 1109(-), and 1072(-) cm(-1) that must involve chromophore C-N bonds. A variety of additional changes were insensitive to isotope labeling of the chromophore. Effects of (15)N labeling of the protein were used to tentatively assign some of these to specific amino acid changes. Those insensitive to (15)N labeling included a protonated aspartic or glutamic acid at 1734 cm(-1)(-)/1722 cm(-1)(+) and a cysteine at 2575 cm(-1)(+)/2557 cm(-1)(-). Bands sensitive to (15)N protein labeling at 1487 cm(-1)(+)/1502 cm(-1)(-) might arise from trytophan and bands at 1261 cm(-1)(+)/1244 cm(-1)(-) and 1107 cm(-1)(-)/1095 cm(-1)(+) might arise from a histidine environment or protonation change. These assignments are discussed in light of the 15Z-E photoisomerization model of phototransformation and the associated protein conformational changes.

Carbon Isotopes↗
PubMed1976

Simultaneous measurement of plasma levels of d-Propoxyphene and l-Propoxyphene using stable isotope labels and mass fragmentography.

When racemic propoxyphene was administered orally to dogs, plasma levels of d-propoxyphene were higher than those of l-propoxyphene. The half-life of d-propoxyphene was also longer than that of the l-isomer. Measurements were performed by labeling one isomer of a racemic mixture with deuterium and then measuring d- vs. l- ratios by mass fragmentography. An intravenous experiment was also performed and confirmed the preferential uptake of the levo isomer. To do these experiments successfully propoxyphene isomers were labeled on the benzyl methylene with deuterium atoms. In vivo studies demonstrated that this labeling position was sufficiently far removed from the point of metabolic attack in the molecule so that no interfering isotope effect existed. The methodology outlined in this paper will be of great value in the study of the disposition of the individual isomers of racemic drugs.

Animals↗
PubMed2003

Overexpression and purification of isotopically labeled Escherichia coli MutH for NMR studies.

MutH is one of the enzymes involved in the methyl directed -GATC-based DNA repair system. We report a significantly optimized protocol to prepare isotopically (15N and/or 13C) labeled MutH in minimal medium with high yields for NMR studies. Under the various conditions that we have standardized for the affinity purification of His(6) MutH, the yield of the purified MutH has been estimated to be 35-40 mg of protein from 1liter of M9 minimal media. The yield, thus, obtained by this method is significantly higher than those of previously reported methods. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy analysis revealed that the protein was pure and existed essentially in a monomeric form. Uniformly 15N-labeled protein, thus, produced has been characterized by recording a sensitivity enhanced 2D [15N]-[1H] HSQC spectrum. The dispersion seen in 15N-1H cross-peaks indicates the presence of a well-ordered structure for MutH and proper folding of the purified protein. The spectrum confirms further the existence of MutH as a monomer.

Amino Acid Sequence↗
PubMed2007

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for metabolic flux analyses using isotope-labeled ethanol.

We describe a novel method for the determination of the concentration and labeling degree of ethanol originating from 1-13C-labeling experiments. This method is suitable for high-throughput metabolic flux analysis because of the possible parallel sample preparation and fast final analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In a closed vial containing culture supernatant, ethanol is enzymatically oxidized to acetaldehyde. The acetaldehyde formed evaporates and is readily trapped in a second enclosed but open vial containing acidified 2,4-dinitrophenylhydrazine (DNPH). The 2,4-acetaldehyde dinitrophenylhydrazone (Ac-DNPH) that is formed is insoluble under these conditions. This leads to a constant conversion rate of the acetaldehyde produced from ethanol after 14 h minimum incubation time. MALDI-TOFMS was used to quantify the formed Ac-DNPH with [13C2]-ethanol as internal standard. The relative signal intensities of the unlabeled ethanol derivative as well as of [1-13C]-ethanol were linearly related to the ethanol concentration within a range of 1 to 50 mM with a limit of detection of 0.6 mM, a range which is sufficient for flux analysis in microtiter plate fermentation experiments. The method allows the estimation of the [1-13C]-ethanol originating from 1-13C-labeling experiments of Saccharomyces cerevisiae strains. In experiments where the expected flux range was exceeded, unlabeled ethanol was determined with a linear range from 30 to 500 mM. Ethanol quantification using this method was compared with enzymatic analysis and exhibited differences of less than 3.3% on average. Comparison of flux partitioning ratios between glycolysis and the pentose-phosphate pathway (PPP) based on MALDI-TOFMS and gas chromatography (GC)/MS methods showed good agreement, with differences for ethanol and alanine labeling of only 4.3%.

Carbon Isotopes↗