Activation of HPg by Sak. Human wtr-E¹-HPg (10 nmol/L) was activated by Sak (10 nmol/L) in the presence of the chromogenic substrate, S2251 (0.9 mmol/L). The HPm generated as a function of time was detected by the liberation of p-nitroanilide from the substrate and is depicted here as the absorbance at 405 nm. The curves illustrated are for wtr-E¹-HPg, wtr-E¹-HPg to which a small amount (0.5 nmol/L) of wtr-HPm was added at the 6-minute time point of the incubation of Sak and wtr-E¹-HPg, and for the mutants, r-[R⁵⁶¹A]E¹-HPg and r-[D⁶⁴⁶E]E¹-HPg. The reduced DodSO₄/PAGE gel insert shows the protein components present after 30 minutes of activation. The buffer was 10 mmol/L HEPES-NaOH, pH 7.4, at 37°C. The activation of wtr-E¹-HPg by lmw-uPA displayed in the gel insert was accomplished as in Fig 1.