Determination of prolidase activity

The activity of prolidase was determined according to the method of Myara et al,26 which involves colorimetric determination of proline applying Chinard’s reagent. Protein concentration was measured by the method of Lowry et al.27

Briefly, the harvested cells were centrifuged at 2,000× g for 15 min. The supernatant was discarded and the cells were suspended in 1 mL of 50 mM HEPES, pH 7.8, and sonicated for 3×10 s at 0°C. The degraded cells were centrifuged at 12,000× g for 30 min at 4°C. To activate prolidase, the enzyme was incubated with Mn(II) in the following mixture: 100 µL of cell extract supernatant with 100 µL of 50 mM HEPES, pH 7.8, in the presence of MnCl₂ to achieve a final concentration of 1 mM in the mixture, and incubated for 24 h at 37°C. After this, the prolidase reaction was initiated by adding 100 µL of the activated mixture to 100 µL of 94 mM glycylproline (Gly-Pro) for a final concentration of 47 mM. The samples were then incubated for 1 h at 37°C. To terminate the reaction, 1 mL of 0.45 M trichloroacetic acid was added and the reaction mixture was centrifuged at 10,000× g for 15 min. The released proline was determined as follows: 0.5 mL of the reaction mixture was added to 2 mL of a 1:1 mixture of glacial acetic acid:Chinard’s reagent (25 g of ninhydrin dissolved at 70°C in 600 mL of glacial acetic acid and 400 mL of 6 M orthophosphoric acid) and incubated for 10 min at 90°C. The amount of released proline was determined by measurement of absorbance at 515 nm and reported as nanomoles per minute per milligram of protein.