PARTIAL-PURIFICATION OF EPIDERMAL G2 CHALONE

Partial purification of epidermal G2 chalone from crude epidermal cell extracts from hairless mice is described. The procedure involved the sequential use of ammonium sulfate precipitation, affinity chromatography and gel filtration. Mitosis-inhibiting activity at each stage in the purification was tested in an in vitro assay system employing human epidermoid carcinoma cells in exponential growth phase and Colcemid for arresting mitoses. This assay system is more rapid and convenient than conventional in vivo systems. By the procedure described, 10,000-fold material purification of the active component was obtained. This corresponds to a 3000-fold purification measured by protein content, and a 300-fold increase in mitosis-inhibiting activity per unit weight. The active component is acidic, contains sugar residues and has gel chromatographic properties characteristic of a substance with a molecular weight of approximately 20,000. On SDS [sodium dodecylsulfate] polyacrylamide gel electrophoresis, 3 weak bands were found. The active component is resistant to trypsin and protease and stable between pH 6.0 and 8.5. It is easily inactivated at pH values below 6.0. At the present stage of purification, several components other than the active one remain and further purification steps must be eventually employed. Chalones play a possible role in carcinogenesis.