Background: Prolidase is a metallo-exopeptidase hydrolyzing X-Pro and X-Hyp dipeptides. Its absence or reduced level is typical in prolidase deficiency (PD) patients, and altered prolidase activity was reported in various diseases. Therefore, standardized and accurate measurement of prolidase activity is essential for PD diagnosis, as well as to elucidate the pathophysiology of other disorders. Methods: Human recombinant prolidase was used to optimize a spectrophotometric enzyme activity assay. Kinetic parameters and Mn2+ affinity were evaluated. The method was validated on blood and fibroblasts from PD patients. Results: An activation step consisting in prolidase incubation with 1 mmol/l MnCl2 and 0.75 mmol/l reduced glutathione at 50 °C for 20 min was necessary to obtain the maximum activity and to accurately determine, for the recombinant enzyme, Vmax (489 U/mg), KM (5.4 mM) and Mn2+ affinity (54 mM−1). The method applied to PD diagnosis revealed an intra-assay CV=8% for blood and 9% for fibroblasts lysates. The interassay CV was 21% for blood and 20% for cell lysates. Conclusion: We optimized a faster spectrophotometric method to measure the activity when the enzyme is fully activated, this is crucial to allow a reliable evaluation of prolidase activity from different sources.
Improved prolidase activity assay allowed enzyme kinetic characterization and faster prolidase deficiency diagnosis
Besio R;Monzani E;Gioia R;Nicolis S;Rossi A;Casella L;Forlino A
2011-01-01
Abstract
Background: Prolidase is a metallo-exopeptidase hydrolyzing X-Pro and X-Hyp dipeptides. Its absence or reduced level is typical in prolidase deficiency (PD) patients, and altered prolidase activity was reported in various diseases. Therefore, standardized and accurate measurement of prolidase activity is essential for PD diagnosis, as well as to elucidate the pathophysiology of other disorders. Methods: Human recombinant prolidase was used to optimize a spectrophotometric enzyme activity assay. Kinetic parameters and Mn2+ affinity were evaluated. The method was validated on blood and fibroblasts from PD patients. Results: An activation step consisting in prolidase incubation with 1 mmol/l MnCl2 and 0.75 mmol/l reduced glutathione at 50 °C for 20 min was necessary to obtain the maximum activity and to accurately determine, for the recombinant enzyme, Vmax (489 U/mg), KM (5.4 mM) and Mn2+ affinity (54 mM−1). The method applied to PD diagnosis revealed an intra-assay CV=8% for blood and 9% for fibroblasts lysates. The interassay CV was 21% for blood and 20% for cell lysates. Conclusion: We optimized a faster spectrophotometric method to measure the activity when the enzyme is fully activated, this is crucial to allow a reliable evaluation of prolidase activity from different sources.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
