Biological and physicochemical characterization of keyhole limpet hemocyanin-lnduced guinea pig lymphotoxin

Abstract

Biological and physicochemical properties of keyhole limpet hemocyanin (KLH)-induced guinea pig lymphotoxin (LT) were compared to those of LT induced by another antigen, ovalbumin, or by the mitogen, phytohemagglutinin (PHA). Mitogen- and antigen-induced LT had similar colony inhibitory activities toward guinea pig and rat tumorigenic cells but none toward nontumorigenic cells. Conversely, human nontumorigenic but not tumorigenic cells were inhibited by the three LT preparations. The molecular weight of each LT was 45,000 daltons on gel filtration chromatography but sucrose density gradient ultracentrifugation indicated greater heterogeneity. The KLH-, ovalbumin-, and PHA-induced LTs were resolved into multiple peaks upon preparative column isoelectric focusing (IEF) with IpH's in the range of 4.5 to 5.3. Analytic IEF of the KLH-induced LT revealed the presence of three distinct biologically active peaks with IpH at 4.77, 5.02, and 5.20. LT activity was stabilized during preparative IEF by inclusion of 0.1% polyethylene glycol (4000 molecular weight) in the ampholine gradient. This resulted in 90% recovery of LT activity with a concomitant removal of 96% of the KLH used as the antigenic stimulus for LT production. Fractionation of LT activity during diafiltration and IEF when exogenous proteins were included as protective agents altered the number and the isoelectric characteristics of the peaks obtained suggesting that LT was being bound by the added protein. Colony inhibitory (cytostatic) and 3H-release (cytolytic) LT activities copurified during fractionation. Thus, this study demonstrated that high yields (approximately 50% of the starting material) of LT, free of antigen or exogenous serum proteins, can be obtained by a combination of diafiltration and IEF performed in the presence of polyethylene glycol.