Phosphodiesterase I Assay

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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

\frac{\text{Units}}{\text{mg}}\text{ = } \frac{\frac{\Delta \text{A}_{400}}{\text{min}}}{\text{16 x }\frac{\text{mg enzyme}}{\text{ml reaction mixture}}}\text{where 16 is the extinction coefficient of p-nitrophenol under these conditions}

Phosphodiesterase I Products

Description

Phosphodiesterase I

Source:

Crotalus adamanteus Venom

Purified by the method of Williams, Sung and Laskowski, JBC, 236, 1130 (1961). Further treated to inactivate contaminating 5'-nucleotidase activity according to Sulkowski and Laskowski: BBA, 240, 443 (1961). Lyophilized in vials.

Store at -20°C.

…

Ice Pack required

Activity

≥20 units per mg dry weight

Certificate of Analysis

Code

VPH

Product details

LS003926

100 un

$96.00

LS003928

Bulk

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

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Phosphodiesterase I - Assay

Phosphodiesterase I Assay

Phosphodiesterase I Products