Biochemical Purification of Antineoplastic Agents Obtained From Sturgeon Serum

Abstract

Discontinuous and continuous DEAE-Sephadex ion-exchange gradients were utilized to purify two sturgeon serum factors (A₂ and B₂) that possessed cytotoxic activity in vitro against human tumor cells but not against normal cells. After rabbit antisturgeon factor A₂ or B₂ was prepared, the antibodies were insolubilized. Sturgeon serum then was adsorbed to the insolubilized antibodies, and factors A₂ and B₂ were isolated in quantities large enough to conduct biologic studies. When the two factors purified either by ion-exchange chromatography or immunoadsorbent columns were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, factor A₂ consisted of one protein band with a molecular weight greater than 68,000 and an isoelectric point of approximately 6.5. Likewise, factor B₂ also consisted of one protein band with a molecular weight in excess of 68,000 and an isoelectric point of approximately 7.5. Both factors A₂ and B₂ were destroyed either by heating at 56°C for 30 minutes or by heating at 100°C for 2 minutes. Furthermore, the mechanism of tumor killing by the sturgeon factors appears to be by means of a soluble factor. When the kinetics of tumor killing was studied, the sturgeon factors were noncytotoxic after 4 or 8 hours of incubation and became cytotoxic after 24 hours of incubation.