Hormone and enzyme assays in pregnancy. V. A rapid method for measuring the placental cystine-aminopeptidase using 1-cystine-bis-1-cystine-bis-p-nitroanilide-nitroanilide as substrate.

A rapid and simple method for determination of the placental cystine-aminopeptidase (P-CAP) activity in plasma is presented. The enzyme-catalysed hydrolysis of the substrate, 1-cystine-bis-p-nitroanilide, is followed in a spectrophotometer by reading the absorbance of the product p-nitroaniline at 380 nm. The absorbance increases linearily with time after a lag-period of 30 seconds to 5 min. The reaction is followed for about 10 min and the increase in absorbtion per min is calculated from the linear part of the absorbtion curve. The test could also be performed by taking 2 readings at about 5 min intervals. In the studies of the enzyme kinetics a competitive inhibitor of the reaction was found in plasma. The amount of the inhibitory substances seemed to be nearly constant in the plasma samples studied from normal pregnant women as well as in plasma samples with high enzyme activity (twin-pregnancy) and low enzyme activity (severe pre-eclampsia). The normal enzyme activity pattern showed an increase from about 210 days of pregnancy and towards the term. A coefficient of correlation of plus 0.83 with the more time consuming method using 1-cystine-di-beta-naphtylamide as substrate was found. It was concluded that the present method could replace the method described by Babuna & Yenen (1966) at least from about 210 days of pregnancy and where a rapid answer is necessary.