Fig. 4. Enzyme activity in trypanosomal lysates to cleave Acetyl-SVLN-AMC. 1 mM
substrate was incubated with trypanosomal cell lysates (10% in citrate buffer,
pH 5.5) at 30°C. After incubation, the shifts of emission maxima were
measured. Lysate and buffer shifts were also measured at mock condition. For
determination of the pH optimum (b), samples were incubated in 50 mM citrate
buffer ranging from pH 2.5 to 6.5, and 50 mM Hepes buffer ranging from pH 5.5
to 9.5. The emission at 440 nm was measured on a spectrofluorimeter as
described in the Materials and Methods. (a) Addition of trypanosomal lysate
led to the release of AMC from the peptide substrate (
), while
substrate alone (
), lysate alone ([UNK]) or buffer alone (
) did
not. (b) Determination of the pH optimum. (c) Effect of hydrazine addition on
enzyme activity: liberation of AMC increased significantly after addition of
10 mM hydrazine () as compared with experiments without hydrazine
([UNK]). Again, substrate alone was stable. (d) Effect of pCMPSA addition on
enzyme activity: trypanosome lysates and substrate were incubated in the
presence ([UNK]) or absence () of 1 mM pCMPSA as described in the
Materials and Methods.
