In response to our recent paper , in which we could not demonstrate prolyl oligopeptidase (POP) (prolyl endopeptidase: PREP; EC 220.127.116.11) in human plasma, Agustí-Cobos and Tenorio-Laranga  point out that their laboratory previously reported that POP activity in human plasma is not affected by EDTA (1.8 mg mL−1 EDTA), but is nearly 50% inhibited by 3.2 mg mL−1 citrate . They concluded that we did not detect POP activity in human plasma because our blood samples were treated with citrate.
Our recent paper reported that there is no POP activity in normal healthy human plasma, based on the data from two different substrates of the enzyme . One substrate, MEPLGRQLTSGP-AMC, is cleaved by POP (Km: 32 ± 3 μm; kcat: 2.3 ± 0.1 s−1) or fibroblast activation protein/antiplasmin-cleaving enzyme (FAP/APCE) (Km: 21 ± 2 μm; kcat: 1.2 ± 0.1 s−1), but not by dipeptidyl peptidase IV (DPPIV) [1,4]. The substrate cleavage activity in normal healthy plasma was not inhibited by a POP-specific inhibitor, acetyl-KLR-l-boroPro, and therefore, the oligopeptidase activity was totally attributed to FAP/APCE, also previously termed ZIP [5,6]. The specific POP inhibitor did block POP activity in POP-supplemented plasma . When either POP- supplemented or non-supplemented plasmas were treated with acetyl-Arg-(8-amino-3,6-dioxaoctanoic acid)-d-Ala-l-boroPro, which inhibits both POP and FAP/APCE activities, all oligopeptidase activity in both samples was completely abolished. These data indicate that human plasma does not contain POP activity. Using currently available substrates, MEPLGRQLTSGP-AMC , succinyl-Gly-Pro-AMC  or Z-Gly-Pro-AMC , we can assay the combined activity of both POP and FAP/APCE. By using any one of these substrates and a POP-specific inhibitor, POP activity is determined by subtracting remaining activity, that is FAP/APCE, from total cleavage activity.
To directly determine POP activity without interference of FAP/APCE activity, we designed and synthesized a POP-specific substrate, acetyl-KLRP-AMC, that is cleaved by POP with a Km of 30 ± 3 μm and kcat of 2.4 ± 0.1 s−1, but not by FAP/APCE or DPPIV . Using this POP-specific substrate, we developed an assay for POP activity in human plasma, which gave a sensitivity of 5–10 ng mL−1 and a lower limit of detection of 6.3 ± 0.6 ng mL−1; using this assay, we were not able to demonstrate POP activity in plasma samples from 24 healthy persons .
In response to the concern of Agustí-Cobos and Tenorio-Laranga , we directly compared the POP activities of EDTA-plasma with those of citrate-plasma, employing the same EDTA (1.8 mg mL−1) and citrate (3.2 mg mL−1) concentrations that their laboratory used . Using the POP-specific substrate, acetyl-KLRP-AMC, POP activities in both EDTA-plasma and citrated-plasma were measured in six healthy adults equally divided for gender, and in no instance did we detect POP activity in either EDTA or citrate-treated samples (Fig. 1A) – even after 18 h incubation (data not shown). Moreover, when plasma samples treated with EDTA or citrate were supplemented with human POP (final 50 ng mL−1 POP), each sample gave virtually the same level of POP activity, whether EDTA or citrate was used as anticoagulant (Fig. 1A). These data indicate that EDTA or citrate either do not affect, or affect equally, POP activity in human plasma.
We also analyzed samples of pools of six human plasmas, each treated with EDTA or citrate, and made to contain one of five different concentrations of human POP (25, 50, 100, 200, and 400 ng mL−1), (Fig. 1B); each sample was assayed in triplicate. We could not demonstrate that citrate-anticoagulated samples had lower POP activity when compared with EDTA-treated samples, and this was true for each of the five different POP concentrations. Figure 1A clearly shows that POP activity could not be detected in normal human plasma, whether anticoagulated with EDTA or citrate. Moreover, POP-supplemented EDTA or citrate anticoagulated plasma samples showed the same levels of POP activity (Fig. 1B). Hence, our conclusion remains the same, normal human plasma lacks POP activity.