Semiautomated microtiter plate assay for monitoring peptidylprolyl cis/trans isomerase activity in normal and pathological human sera

Abstract

An UV/VIS spectrophotometric assay technique was developed that was able to routinely monitor peptidyl-prolyl cis/trans isomerase (PPIase) activity of biological fluids in 96-well microtiter plates. The assay, based on monitoring the cis-to-trans isomerization of succinyl-Phe-cisPro-Phe-4- nitroanilide as substrate in a chymotrypsin-coupled reaction, yields a throughput of 96 samples per 30 min. The assay's capacity was exemplified by dealing with the PPIase activity in several normal and pathological human sera. Reference values of 151 healthy subjects (83 females, 69 males, 17 to 60 years old) were found to possess significant sex-specific differences. PPIase activity factor K of the sera was significantly greater in males (5th, 50th, 95th percentiles: 17, 36, 55 K) than females (14, 30, 48 K). PPIase activities of sera from healthy donors (n = 151) were significantly higher (Mann-Whitney rank-sum test P <0.0001) than those of patients (n = 47). PPIase activity in serum samples stored at 4 °C was stable for at least 20 h.