The α-complementation reaction of β-galactosidase was studied at single-molecule resolution using arrays of femtoliter-sized wells. Single molecules of the complementation species were observed to be stable for long periods of time, demonstrating that the α-complementation reaction is irreversible. By directly counting the number of active molecules formed in the complementation reaction when different concentrations of enzyme acceptor (EA) and enzyme donor (ED) are used, we deduce that the EA:ED ratio in the complementation species is 4:1.

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The stochastic binding and release of two different inhibitors from a tetrameric enzyme is described at the single molecule level.