Reaction of 4-methylumbelliferylguanidinobenzoate with proteases in human amniotic fluid.

An arginine esterase activity similar to that observed in plasma has been demonstrated in second trimester and term human amniotic fluid. Like plasma, the protease(s) hydrolyzed esters of arginine, were reactive towards 4-methylumbelliferylguanidinobenzoate (MUGB), a sensitive active site titrant of trypsin-like enzymes, and had a pI of 5.1--5.4. The pH optimum for proteolytic activity was 8.0. This protease activity was inhibited by soybean trypsin inhibitor (STI), benzamidine and (p-nitrophenyl)-p'-guanidinobenzoate (NPGB), and was insensitive to 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK) and p-hydroxymercuribenzoic acid (HMB). Upon gel filtration, two MUGB-reactive fractions were observed, one with an apparent molecular weight of 200,000 and the other, 100,000. Both fractions had arginine esterase activity and appeared to be sensitive to inhibition by STI and benzamidine. The mean MUGB titre value (nmoles of 4-methylumbelliferone released per ml amniotic fluid) for 300 mid-trimester amniotic fluids was 11.40 +/- 2.40 nmoles MU/ml. The mean specific activity was 2.36 +/- 0.41 nmoles MU/mg protein. Two amniotic fluids from pregnancies which delivered children with cystic fibrosis (CF) were analyzed in blind samples sent from other laboratories. The MU titre values obtained were 4.73 and 4.32 with specific activities of 1.24 and 1.30 respectively. A third was identified in our screening program of amniotic fluids obtained from amniocenteses done for the intrauterine detection of genetic abnormalities. The MU titre value was 5.52 nmoles/ml with a specific activity of 1.34. The specific activities of these fluids when compared to the controls were significantly different (p less than 0.001). The mean titre value for 23 term amniotic fluids samples was 8.14 +/- 1.69 nmoles MU/ml. The mean specific activity was 3.37 +/- 0.76 nmoles MU/mg protein. A term amniotic fluid obtained from a woman who delivered a baby with CF showed a markedly reduced level of MUGB reactivity (3.01 nmole/ml). The specific activity was 1.06 which was significantly different from the control term fluids. The MU titre values and specific activities of amniotic fluids obtained from abnormal pregnancies (such as those with neural tube defects, chromosomal abnormalities and polymorphisms, abortions and stillbirths) and fluids with elevated alphafetoprotein and maternal blood contaminants did not significantly vary from the mean control values (Table 3).