Potentiometric and Colorimetric Methods for the Assay of 2′,3′‐cyclic Nucleotide 3′‐phosphohydrolase¹

A potentiometric titration method for the assay of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase is presented. Progress curves of the reaction were recorded automatically by pH‐stat. 2‐Mercaptoethanol was added to the reaction mixture to maintain a linear rate of reaction. The method is suitable for obtaining kinetic parameters and can be used for the rapid assay of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase in nervous tissues. An improved colorimetric method for estimation of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase activity at the optimum pH is described. This method employs the two‐step procedure in which decyclization by 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase and dephosphorylation by Escherichia coli alkaline phosphatase (EC 3.1.3.1) are carried out separately under the optimum conditions for each enzyme. The method is sensitive and most convenient for routine assays.