A general method of tritium labeling utilizing microwave discharge activation of tritium gas. Methodology and application to biological compounds.

A method of general tritium labeling is described which is applicable to a broad range of organic compounds of biological interest, including amino acids, peptides, and proteins. The method is rapid, inexpensive, and nondestructive, thereby making the purification of labeled material relatively uncomplicated. The reaction apparatus consists of a closed system in which tritium gas at a pressure of 4 mm Hg or less can be cycled by an internal pump. The labeling reaction(s) is initiated by microwave discharge activation of the tritium gas and the sample, cooled by liquid nitrogen, remains outside the region of the high ultraviolet and electron flux from the tritium plasma. The evidence suggests that tritium-hydrogen exchange occurs as a result of bombardment of the labeled substrate with tritium atoms. Studies of the chemistry of the labeling reaction with aromatic hydrocarbons, of the analytical requirements for radiochemical purity, of the characteristic specific activities noted for several classes of compounds, and of the application of the method to the preparation of tritium-labeled peptides are presented. Tritiumlabeled adrenocorticotropin (ACTH) with a specific activity of 15 Ci per mmole and full biological activity has been prepared by this method. This represents a lOOO-fold increase in the specific activity previously obtained by Wilzbach labeling of ACTH.