Direct measurement of alkaline phosphatase kinetics on bioactive paper

Abstract: The reaction kinetics of alkaline phosphatase (ALP) adsorbed on paper was quantified by image analysis using a colorimetric technique under conditions of excess enzyme. A classical reaction kinetics approach integrated with image processing technique was used to analyse the coloured product formation from the enzyme substrate reaction against time. The order of reaction and rate constant of enzyme activities on paper surface were calculated. ALP was either physisorbed directly on paper or adsorbed on paper treated with a monolayer of a high molecular weight cationic polyacrylamide (CPAM), an anionic polyacrylic acid (PAA) or a high molecular weight polyethylene oxide (PEO). The reaction rate of enzymatic paper follows a first order reaction with respect to the concentration of enzyme–substrate complex. ALP immobilized on paper has a reaction rate 2 to 3 orders of magnitude lower than the free ALP enzyme in buffer solution. This might be due to a critical loss of enzyme flexibility upon adsorption. No increase in reaction rate was achieved by immobilizing ALP on paper treated with a high molecular weight cationic polyacrylamide (CPAM) or a highly charged anionic polyacrylic acid oligomer (PAA), which suggests the enzyme orientation was not significantly affected. However, the reaction rate for ALP on PEO treated paper was lower than that on CPAM or PAA treated papers. That is probably due to some PEO-enzyme interaction affecting the tertiary structure of the ALP enzyme. [Copyright &y& Elsevier]