DIRECT MONITORING OF PROLIDASE ACTIVITY IN CULTURED SKIN FIBROBLASTS USING CAPILLARY ELECTROPHORESIS

Capillary electrophoresis (CE) was used as an alternative to current a nalysis schemes for detecting prolidase activity in erythrocytes and s kin fibroblast cultures because of its unique selectivity and high res olving power. Kinetic measurement of peptide bond hydrolysis was perfo rmed using porcine kidney prolidase on different substrates (Gly-Pro, Leu-Pro and Ala-Pro) and by following the disappearance of the peptide -substrate's peak. The K-m values obtained were in agreement with thos e previously reported. Interestingly, in the case of Phe-Pro as the su bstrate, simultaneous analysis of the product and parent peptide was p ossible, thus showing the superiority of the capillary electrophoresis (CE) assay with respect to the standard spectrophotometric method. Th e application of the CE technique to the characterization of prolidase activity in control and prolidase-deficient skin cultured fibroblasts was successful. Enzyme activity was easily calculated in all controls tested and the K-m values determined were slightly lower than those o btained with the colorimetric reaction, thus confirming our assumption that the CE assay shows higher specificity than the ninhydrin techniq ue. Our results demonstrate the feasibility of using CE as a simple an d reliable technique for determining prolidase activity.