Phosphodiesterase I Assay

Method: The assay is essentially that of Razell and Khorana (1959) where the reaction velocity is determined by an increase in absorbance at 400 nm resulting from the hydrolysis of p-nitrophenyl thymidine-5'-phosphate. One unit hydrolyzes one micromole of p-nitrophenyl thymidine-5'-phosphate per minute at pH 8.9 and 25°C under the specified conditions.

Reagents

• 0.11 M Tris⋅HCl buffer, pH 8.9, with 0.11 M NaCl and 15 mM MgCl₂ (Tris⋅Salts buffer)
• 5 mM p-nitrophenyl thymidine-5'-phosphate. Note: The purity of commercial preparations varies somewhat and should be considered in preparing this reagent.

Enzyme

Dissolve at one mg/ml in Tris*Salts buffer to obtain a rate of 0.02-0.04 ΔA/minute.

Procedure

Set spectrophotometer at 400 nm and 25°C. Pipette into microcuvettes as follows:

Tris⋅Salts buffer	0.9 ml
5 mM p-nitrophenyl thymidine-5'-phosphate	0.1 ml

Incubate cuvettes in spectrophotometer for 3-5 minutes to reach temperature equilibrium and establish blank rate, if any. Add 10 microliters of diluted enzyme and record increase in A₄₀₀ for 3-5 minutes. The reaction remains linear until A₄₀₀ reaches about 1.2. Calculate ΔA₄₀₀/minute from initial linear portion of absorbance curve.

Calculation