Purification and physico-chemical characterization of rabbit tumor necrosis factor.

TLDR

Rabbit TNF was purified 2000‑fold by sequential salt precipitation, gel filtration, ion exchange, and lectin affinity chromatography, yielding a single species on SDS‑PAGE. TNF is an alpha‑globulin with pI 5.1, molecular weight 68 kDa (SDS‑PAGE), 55 kDa (gel filtration), 52 kDa (glycerol gradient), active over pH 6–10, heat stable up to 70 °C for 1 h, pronase sensitive but trypsin resistant, unaffected by neuraminidase or phospholipase C, still induces hemorrhagic necrosis in tumors, and its interferon titer drops from 3000 U in crude serum to <30 U after purification. The molecular weight was reported.

Abstract

Rabbit TNF has been purified 2000-fold by a series of salt precipitations, gel filtrations, ion exchange chromatography, and lectin affinity chromatography to a single species on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). TNF activity could be recovered from nondenaturing gel systems and has been shown to be an alpha-globulin with an isoelectric point of 5.1. The m.w. was estimated to be 68,000 d by SDS-PAGE, 55,000 by gel filtration, and 52,000 by glycerol gradient centrifugation. TNF activity was stable over the pH range of 6 to 10 and was relatively heat stable, not being inactivated at 70 degrees C for 1 hr. TNF activity was pronase sensitive, but relatively trypsin resistant. Neuraminidase and phospholipase C treatment did not destroy TNF activity. Partially purified TNF was still capable of eliciting hemorrhagic necrosis in susceptible tumors. Crude TNF serum had an interferon titer of 3000 U, whereas the partially purified sample had a titer of <30 U.