Characterization of Prolidase Activity Using Capillary Electrophoresis with Tris(2,2'-bipyridyl)ruthenium(II) Electrochemiluminescence Detection and Application To Evaluate Collagen Degradation in Diabetes Mellitus

摘要

A new method for prolidase (PLD, EC 3.4.13.9) activity assay was developed based on the determination of proline produced from enzymatic reaction through capillary electrophoresis (CE) with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)_(3~(2+))] electrochemiluminescence detection (ECL). A detection limit of 12.2 fmol (S/N velence 3) for proline, corresponding to 1.22 X 10~(-8) units of prolidase catalyzing for 1min was achieved. PLD activity determined by CE-ECL method was in agreement with that obtained from the classical Chinard's one. CE-ECL showed its powerful resolving ability and selectivity as no sample pretreatment was needed and no interference existed. The clinical utility of this method was successfully demonstrated by its application to assay PLD activity in the serum of diabetic patients in order to evaluate collagen degradation in diabetes mellitus (DM). The results indicated that enhanced collagen degradation occurred in DM.