Real-time single-molecule kinetics of trypsin proteolysis.

Single-molecule enzymatic kinetics and enantioselectivity were monitored in real time by using total internal reflection fluorescence microscopy. The 300-kDa poly(L-lysine) (PLL) or poly(D-lysine) (PDL) was labeled with Alexa Fluor 532 and was covalently immobilized on a dithiobis(succinimidyl undecanoate) self-assembled monolayer (DSU SAM) prepared on a gold substrate. The PLL/PDL chains were more accessible to trypsin on DSU SAM than when they were immobilized on a bare glass substrate. Short-chain PDL was further used as a blocking agent to prevent readsorption of the hydrolyzed lysine fragments. Chain shortening due to enzymatic hydrolysis resulted in the reduction of the individual fluorescence intensities. A broad distribution was obtained when 100 single-molecule half-lives were analyzed. However, the detailed hydrolysis process involved also a long-lived component and an induction period that varied significantly among molecules. Charge and steric heterogeneity at the surface are responsible for these features. In contrast, standard Michaelis-Menten fitting of the decrease in molecule numbers with time masked out all such details.