HUMAN TRANSFER FACTOR: EXOGENOUS LABELLING, PURIFICATION, AND ROLE OF RIBONUCLEIC ACID SEGMENT

Abstract

This chapter presents data on the structures of both human transfer factor (TF) moieties derived from dialysates of leukocyte extracts (DLE) and on the radioactive labeling and subsequent further purification of human TF–H7. It describes experiments in which human TF–H5 and TF–H7 are purified from crude DLE by Sephadex G-25 chromatography, phenol extraction, ethanol precipitation, HPLC, cellulose TLC, and boronate affinity chromatography. In some cases, cellulose TLC was substituted for high-pressure reverse phase liquid chromatography and phenol extraction followed by ethanol precipitation was substituted for Sephadex G-25 chromatography. Bovine TF–C5 was similarly purified from material released by immune lymph node cells. Poiyethyleneimine cellulose (PEI) TLC was performed using ascending homochromatography at room temperature without prior wetting of the surface with water. The chapter presents degradation experiments with tobacco acid pyrophosphatase. This enzyme degrades pyrophosphate linkages such as those found in ATP or eucaryotic mRNA. As it is known that TAP has difficulty cleaving some pyrophoshate linkages, enough TAP is used to cleave the 5' cap linkage from 3μg of mRNA even though the physical amounts of each TP were below microgram levels. TF–H7 can be radiolabelled with 125I using the method of Prensky. This finding and the ability to study TF–H7 using PEI–TLC and acrylamide gel electrophoresis open up new frontiers for research on the structure of TF.