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pH‐stat methodology in continuous monitoring of the kinetics of hydrolysis of phosphate esters catalysed by alkaline phosphatase from human placenta: II. Kinetic aspects
Author(s) -
Roig M. G.,
Serrano M. A.,
Bello J. F.,
Cachaza J. M.,
Kennedy J. F.
Publication year - 1991
Publication title -
polymer international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.592
H-Index - 105
eISSN - 1097-0126
pISSN - 0959-8103
DOI - 10.1002/pi.4990250203
Subject(s) - chromogenic , chemistry , alkaline phosphatase , kinetics , hydrolysis , phosphate , substrate (aquarium) , chromatography , enzyme , biochemistry , physics , oceanography , quantum mechanics , geology
The adaptation of the pH‐stat to continuous monitoring of the in‐vitro hydrolase activity of alkaline phosphatase in solution, an activity which until now has only been analysed by means of spectrophotometric methods in a continuous or static state, is described. To control the reliability of the method in the monitoring of these enzymic reactions, a series of kinetics of hydrolysis of chromogenic ( p ‐nitrophenyl and o ‐carboxyphenyl phosphates) and non‐chromogenic (ATP) substrates was carried out, comparing the results obtained via continuous spectrophotometric analysis of the corresponding phenol released or via a discontinuous technique by the phosphate produced with those results obtained via pH‐stat analysis of the H + released or utilized by the enzymic reaction of hydrolysis. It can be concluded that kinetic studies carried out on the pH‐stat of in‐vitro alkaline phosphatase activity offer results analogous to those obtained for the same system through classic spectrophotometric methods, offering as well notable advantages of speed and simplicity in the kinetic assays since it is always possible to monitor the enzymic activity continuously, with chromogenic or non‐chromogenic substrates. The pH‐stat methodology affords additional information on the kinetics of action of alkaline phosphatase from placenta acting on non‐chromogenic biological substrates. In this sense, kinetic studies with the pH‐stat on the enzymic hydrolysis of ATP were begun, determining their rate versus pH profile (optimum pH at 9.1) and proposing possible ‘non‐Michaelian’ type kinetic behaviour of the enzyme in solution as deduced from the graphical analysis of the data v ([ATP]).