Figure 4.

A general description of the methodology to attain single turnover resolution measurements. (A) A method exploiting fluorescence property changes within each catalytic cycle of cofactors or covalently attached fluorophores; (B) Prefluorescent substrate analogues for prolonged observation of individual catalytic turnovers. The non-fluorescent substrate is converted by the enzyme to the highly fluorescent product, which subsequently diffuses away from the detection volume allowing recordings of individual catalytic cycles. Being limited only by substrate depletion this methods allows recordings of thousands of catalytic cycles for accurate statistical analysis; (C) A typical single molecule activity trace of lipase converting a prefluorescent substrate analogue, here CFDA, to highly fluorescent carboxy-fluorescein (red trace). Each spike corresponds to an individual catalytic cycle. The black trace corresponds to the background signal (Figure 4C reprinted with permission from [17]. Copyright (2012) American Chemical Society.)