Electrophoretic Behavior of the Papilloma Virus Protein. ∗

Abstract

In 1937 a specific heavy protein was isolated 1 by differential ultracentrifugation from infectious extracts of cottontail-rabbit papillomas. 2 Purified by several cycles of alternate low- and high-speed ultracentrifugation the material sediments in the analytical ultracentrifuge with the sharp boundary characteristic of a single molecular species and with a sedimentation constant of S₂₀° = 265 × 10^-13 cm sec^-1 dynes^-1. Studies on infectivity, 3 complement-fixing capacity, 4 and neutralization 5 with specific immune sera have demonstrated a high degree of uniformity in the biological behavior of the protein and its intimate relation to the papilloma virus. In the present work, further evidence of the homogeneity of the macromolecular protein is furnished by examination of its electrophoretic behavior.

The studies were made on about 150 mg of protein freshly prepared from 350 g of warts from many cottontail rabbits, extracted and purified by 3 ultracentrifugal cycles by the routine procedure previously described.¹ The purified product was dissolved in buffer solution consisting of 0.05 M sodium chloride and 0.05 M sodium veronal-sodium acetate to give a solution of 0.1 ionic strength and adjusted to the required pH with NaOH or HCl. Studies were made on dialyzed solutions at protein concentrations of 4.5 to 2.7 mg per cc. The instrument† employed was of the Tiselius 6 type. Movement of the protein boundary in the 11 cc cell under the influence of constant electric potential at 0°C and changes in boundary shape were recorded photographically by the refractive index method of Svensson. 7

The pH regions available for study of the intact protein are determined 8 by the limits of molecular stability in both acid and alkaline ranges and by insolubility near the isoelectric point. An example of the results obtained in the acid range of solubility is shown in Fig. 1 in the series of curves photographed after successive time intervals over a period of 5.5 hours at pH 3.78.