The small-scale production of [U- 14C]acetylene from Ba 14CO 3: Application to labeling of ammonia monooxygenase in autotrophic nitrifying bacteria

Abstract

A small-scale method has been adapted from an established procedure for the generation of [U- 14C]acetylene from inexpensive and commonly available precursors. The method involves the fusing of Ba 14CO 3 with excess barium metal to produce Ba 14C 2. The BaC 2 is reacted with water to generate acetylene which is then selectively dissolved into dimethyl sulfoxide (DMSO). The results presented demonstrate the effect of Ba:BaCO 3 ratio on the concentrations of various gases released during the hydrolysis reaction and quantify the selectivity of the DMSO-trapping process for each gas. [U- 14C]-Acetylene generated by this method has been used to inactivate ammonia monooxygenase in three species of autotrophic nitrifying bacteria: Nitrosomonas europaea, Nitrosococcus oceanus, and Nitrosolobus multiformis. Our results demonstrate that acetylene inactivation of this enzyme in all three species results in the covalent incorporation of radioactive label into a polypeptide of apparent M r of 25,000–27,000, as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography.