Interaction of calf intestinal alkaline phosphatase with 8-anilinonaphthalene-1-sulphonate

Calf intestinal alkaline phosphatase is inhibited by 8-anilinonaphthalene-1-sulphonate (ANS). The inhibition is uncompetitive but non-linear. Hill plots of the inhibition data have slopes of 1.4-1.8 suggestive of positive cooperativity. Fluorescence titration revealed that 2 molecules of ANS bind per molecule of enzyme with no evidence of cooperativity. The Kd for ANS obtained by fluorescence was 1.8 X 10(-6) mol/l but the approximate Ki for inhibition was 1 X 10(-3) mol/l. Thus, the fluorescence and kinetic experiments appear to monitor different events.