Reversible subunit dissociation of tumor necrosis factor during hydrophobic interaction chromatography.

Hydrophobic interaction chromatography (HIC) of recombinant tumor necrosis factor (TNF) results in reversible dissociation of the quaternary protein structure yielding separation of trimer and monomer peaks. Gel electrophoresis, size exclusion, fluorescence polarization and rechromatography were used to identify the trimeric and monomeric species. Relative amounts of these peaks varied as a function of temperature and column contact time. When TNF was chromatographed in the presence of partially proteolyzed [14 kilodalton (kD)] TNF, two additional peaks, identified as the 14-kD monomer and heterotrimer of TNF and the 14-kD fragment, appeared. Rechromatography of this heterotrimer produced TNF monomer and 14-kD peaks establishing that the multiple peak pattern in HIC was due to quaternary dissociation. Incubating TNF in denaturants prior to non-denaturing size-exclusion chromatography resulted in apparent protein unfolding. However, free, undenatured monomer was not observed. We conclude that TNF is most likely a trimer, which is tightly held together by hydrophobic forces, and that the tertiary structure of the monomer is stabilized through this subunit association. The hydrophobicity of the sorbent surface mediates reversible dissociation of the trimers to monomers through hydrophobic stabilization of the monomeric tertiary structure. After elution, the TNF monomers reassociate to form the native TNF trimer.

High-sensitivity phenylthiohydantoin amino acid analysis using conventional and microbore chromatography.

Reversed-phase microbore high-performance liquid chromatography was investigated for high-sensitivity analysis of phenylthiohydantoin (PTH) amino acids. A mixed nitrile alkylsilane bonded phase was developed and ternary gradient elution conditions were devised for resolution of the common PTH amino acids. Elution conditions were developed with a conventional 150 X 4.6 mm I.D. column and transferred to a 150 X 1 mm I.D. microbore column. The performance of these columns was evaluated in terms of PTH amino acid resolution, enhanced sample detectability, and retention time precision. For this work a general purpose high-performance liquid chromatograph was modified to reduce extra column band broadening and a preformed gradient elution technique was developed to achieve rapid analysis times at microbore flow-rates. The microbore high-performance liquid chromatographic system is useful for high-sensitivity analysis of PTH amino acids in micro-sequencing applications.

The effect of occlusion on carbon dioxide emission from human skin.

The effect of occlusion on the carbon dioxide (CO2) emission rate (CDER) of human skin was determined. Occlusive plastic tape elevated the CDER 4.5 times (90nl/cm2/hr) over the normal CDER (20nl/cm2/hr). This increase was noted within a 3-hour period. Non-occlusive paper tape had no effect on CDER. Quantitation of the amount of CO2 under plastic tape revealed that CO2 was present at a concentration of 8-10%. Removal of the plastic tape after 24 hours allowed the CDER to return to approximately normal values within 2 hours. The mechanism by which occlusive plastic tape mediates this dramatic effect on CDER as well as the significance of elevated CO2 concentrations under occlusion are discussed.

Skin barrier properties in the newborn. Transepidermal water loss and carbon dioxide emission rates.

Relative permeabilities of newborn and adult skin to small molecules were determined by measuring emission of carbon dioxide and water vapor through skin. Transepidermal water loss (TEWL) measured on the forearm of 22 term infants was lower than that observed in 30 control adults (0.31 +/- 0.09 vs. 0.39 +/- 0.15 mg/cm2/h). There was no significant difference between newborn carbon dioxide emission rates (CDER) and adults CDER. When environmental temperature was raised from 74 to 80 derees F, large dramatic increases in adult TEWL were observed in contrast to small increases in newborns. This phenomenon suggests diminished eccrine activity in the newborn. No correlation of TEWL or CDER with respect to birth weight, sex, age or gestational age was observed. Similar permeability to carbon dioxide in neonates and adults and a decreased TEWL in neonates compared to adults suggest that even at birth the skin functions as an efficient barrier to these molecules, penetration rates, in adults, of other polar compounds should be similar to those in neonates.