Macromolecular Components of Untreated and of Formolized Normal Chick Embryo Tissue. ∗

Abstract

Macromolecular substances have been found in healthy tissues of both plants and animals. 1 In experiments associated with studies of the equine encephalomyelitis virus protein 2 and the non-infectious immunizing principle 3 of formalin-treated virus-diseased chick embryos, 4 we have obtained such substances from normal chick tissue and from the tissue treated with formalin.

Chick embryos incubated for 11 days were harvested, chilled, the heads removed and the bodies ground in a Ten Broeck grinder held in cracked ice. The resulting body mince was made to a 20% suspension and extracted as indicated. Large particles were removed with the angle centrifuge and the resulting extracts fractionated by alternate ultracentrifugal runs of 9,000 g for 5 minutes and 67,000 g for 1 to 1I/2 hours. The products have been studied by ultracentrifugal analysis.

The results observed when extraction was made and the pellets were dissolved with 0.005 M ammonium hydroxide-ammonium chloride buffer pH 8.2 are shown in Figures 1 and 2. After 2 ultra-centrifugal cycles, double or diffuse boundaries were present whereas only one of slight diffuseness was seen after the third cycle. Here s₂₀° = ca 70 × 10⁻¹³ cm sec⁻¹ dynes⁻¹. A similar material was obtained by extraction with glycerin in 50% concentration in the dilute buffer. When extraction was made in 0.9% sodium chloride solution alone or combined with glycerin and the pellets were dissolved in saline, indications of the same conponent were evident after the second cycle (Fig. 3) but further homogeneity was not attained by additional cycles (Fig. 4). The sharpest boundaries were obtained with material derived by extraction of tissue and solution of the pellets in distilled water (Fig. 9 and 10).