Cytofluorometric quantification of the activity and reaction kinetics of acid phosphatase

This paper describes how fluorogenic substrates derived from naphthol AS can be used for the microscopic demonstration and cytofluorometric quantification of the activity and reaction kinetics of acid phosphatase in single living cells. A special study has been made of acid naphthol AS-BI phosphatase. However, the method can be extended to other hydrolytic enzymes. The method is sensitive and accurate because: quantification of very low enzyme activity is possible; the reaction kinetics can be evaluated with a good degree of precision inasmuch as the initial reaction velocity is derived over short times; there is an absence of distributional error; and the errors due to extra-cellular diffusion of the hydrolysed substrate, to photodecomposition, and to autofluorescence can be contained within very narrow limits. The procedures for determining enzyme activity and reaction kinetics, and the instrumental characteristics and devices required for carrying out these measurements, are described. Some possible applications are indicated.