Determination of free phosphate release from AP substrate
Tris-HCl (50mmol L-1, pH6.0-8.0) buffer was used to
prepare 10mM of ATP, ADP, and AMP solution (ATP, Biotopped; ADP and AMP,
Shanghai source leaf biology) and also to dilute recIAP (1000U
ml-1) to 0.5, 5.0, 50.0U ml-1. 950μl
of recIAP and 50μl of ATP, ADP, and AMP solutions were mixed together
thoroughly, producing a final concentration of the solution is 0.5 mM,
and incubated at 37°C for 60 min. In the above measurement, recIAP that
was inactivated at 65 ° C for 60 minutes was used as a blank control. An
inorganic phosphorus test box (C006-1-1, Nanjing Jiancheng Biotechnology
Research Institute) was used to measure the content of free phosphate
(Pi) released through the recIAP’s effect on ATP, ADP, and AMP (0.5mM)
at different pHs, ranging between 6.5-8.0. The released Pi interacts
with molybdic acid to generate phosphomolybdic acid, which is then
reduced to molybdenum blue and exhibits a unique absorption spectrum
with a maximum peak at 660nm.