Identity of trypanosome growth factors in serum II. Active globulin components

This paper describes the identification of the active fractions of rat serum that support the growth and survival of Trypanosoma lewisi in the heterologous mouse host. The relationship between dose of serum and the size of trypanosome challenge was first established. There was reciprocity between the amount of serum and the inoculum, so that at high serum dosages smaller inocula were sufficient for either a viable transfer back to the rat or for a microscopically demonstrable parasitemia in the mouse. At high inocula (> 6 × 10⁷) organisms were detected on transfer back to the rat after 5 days. A problem arising in the fractionation of rat serum was the toxicity of certain preparations to the mouse. Since this effect was also dose dependent it was minimized by adjusting the inoculum size knowing the relationship between protein dose and inocula. Rat serum was then fractionated to determine which components retained the ability to promote T. lewisi parasitemias in the mouse. The proteins were active while the crystalloids were ineffective after their separation by ultrafiltration, dialysis, and Sephadex G-25. Precipitation of the globulins with (NH₄)₂SO₄ and Na₂SO₄ yielded β and γ globulins which supported gross parasitemias in the mouse and albumin which did not. Further fractionation of the globulins by DEAE and Sephadex G-200 column chromatography, as well as ultracentrifugation, pointed to activity in the γ₂ globulins and macroglobulins.