Natural human tumor necrosis factor beta (TNF./~) purified from supernatants of a human B-lymphoblastoid cell line was found to t~ heterogeneous in molecular mass, with seven components resolved by gel el~trophoresis. All components are N-glyeosylated at Asa~:; N-glycosylation does not contribute to heterogeneity, In addition, part of the molecules are O-glycosylated at Thr~; O-glycosylation is heterogeneous due to variable decoration with aeuraminie acid. The four lower molecular mass forms are derived from the full-length protein by trypsin-like prot¢olytic cleavage in the N~proximal region; these dipped molecules lack O-linked carbohydrates, Two allelic variants differing in amino acid position 26 (threonin~ a~paragine) were identified, modification. To clarify this situatio,~ and to identify the O-glycosylation site(s), we isolated natural TNF-p produced by the human B-lymphoblastoid cell line, RPMI-1788, and characterized the protein using HPLC methods, SDS-PAGE, amino acid sequence analysis and deglycosylating enzymes. MATERIALS AND METHODS Proch~ction and purification of natural TNF-~TNF-/ff was purified from supernatants of the human B-lymphoblastoid cell line. RPMI 1788, stimulated with mezerein by serial chromatography on controlled pore glass, Mono Q anion-exchange and concanavalin A columns, as described previously [16]. The resultin8 material was homogeneous as judged by gel l~rmeation HPLC and reverse-phase HPLC. Recombinant, E.colbderived TNF.fl wits kindly provided by G. Bodo and by Geaentech Inc., San Fr'aaciseo. SDS-PA OE, HPLC techniques, amino aeM sequence attafysis attd peptide mappOtgProtein samples were electrophora~¢d in the presence of 0.1% SDS on 15% acrylamide gels according to Laemmli [17], Reverse-phase HPLC was performed on a Bakerbond WP C18 column (4.6 x 250 ram, particle si~ 5 pna, pore diameter 300 A) at 30°C, usin/~ the rollowin~ solvents: solvent A, 0, I% trifluoroacetie acid in water;, solvent B, 0,1% trifluoroacetie acid in acetronitrile. The follow;rig program was used: 0-2 rain, 20% B; 2-26 rain, 20-68% B (linear gradient}; 26-36 rain, 68% B (flow rate 1 ml/min). Proteins were detected by their absorption at 214 and 280 rim. Gel permeation chromatography was performed on two Waters Protein-Pak 1 125 connected in series (7.8 ~< 300 mm each, particle size 10/.tin). using 0,:5 M Na2504, 0.02 M NaH.~PO~, pH 7.0, 0,04% Tween 20 in 25% propylene glycol (flow rate 0.5 ml/min). Proteins were detected by their absorption at 214 rim.For N-terminal amino acid sequencing, proteins v,,er~ dissolved in 70% formic acid and directly applied to the cartridge of an Applied Biosystems mod=i 477A puled dqui~-phas~ sequencer .... e s.que ....
An experimental and numerical investigation into the magnitude of longitudinal and transverse dispersion in a two-dimensional flow field over a particle Peclet number range of 50-8500 is reported. Numerical modelling using a Galerkin finite element method is used to test various models, notably those of Fried and combarnous and Koch and Brady. Dispersion at low Peclet numbers (<200) is found to be described adequately by either model, which at large Peclet, the degree of dispersion is significantly underestimated. An improved dispersion model for Peclet numbers greater than 200 is proposed. The transverse dispersion term and the choice of inlet boundary condition are found to have a negligible effect on the shape of the breakthrough curve.
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