A continuous, coupled polarographic assay, which couples trehalose hydrolysis to OZ consumption using glucose oxidase (EC 1.1.3.4) and catalase (EC 1.11.1.6) as ancillary enzymes has been developed for the measurement of trehalase (o-a'-trehalose 1-D-glucohydrolase, EC 3.2.1.28) activity. With this procedure, 0, consumption was a linear function of time and the coupled reaction rate was directly proportional to the amount of protein assayed with both crude and partially purified enzyme preparations. The limits of sensitivity with this assay correspond to the production of 2.5 nmol of glucose/min. The validity of this assay was confirmed by comparative studies with a discontinuous calorimetric assay for the quantitation of glucose. In addition, the applicability of this assay was appraised by determining the K, of the enzyme for trehalose. The value obtained with the polarographic assay (i.e., 1.3 f 0.1 mM trehalose) showed excellent agreement with that obtained using a discontinuous calorimetric method (i.e., 1.2 mM trehalose). Thus the equivalence and applicability studies with the polarographic assay demonstrated that this procedure is a valid and sensitive method for the rapid quantitation of trehalase activity.
' Pbbreviations used: MES, 2-(N-Morpholino) ethanesulfonic acid.