Partial purification of human leukocytic pyrogen.

Abstract

Human leukocytes stimulated in vitro release leukocytic pyrogen (LP), a protein which is the mediator of fever. In order to study human leukocytic pyrogen, we attempted to purify this molecule from the large quantity and variety of proteins which are present in leukocyte supernates. Human peripheral leukocytes were stimulated in vitro by phagocytosis of killed staphylococci, and several methods were used to isolate the pyrogen protein. First, using isoelectric focusing, it was found that crude leukocyte supernates contained two molecular species of LP which were separable by precipitation in cold alcohol. Isoelectric focusing, although used for confirmation of the molecular homogeneity of LP, could not be employed as a preparative purification technique. Following alcohol precipitation, human LP was chromatographed on ion-exchange materials at various pH with modest recovery of initial activity but marked increase in specific activity. Gel-filtration was also employed and yielded partially purified LP. When alcohol precipitation was combined with ion exchange at alkaline pH and followed by gel-filtration, resulting LP preparations contained 5 or 6 contaminating proteins. These results demonstrate that human LP can be partially purified from the large quantity and variety of proteins present in crude leukocyte supernates and during purification procedures, the pyrogen did not change in either molecular weight or isoelectric point. This work provides reliable techniques for initial purification of human LP.