Phosphodiesterase 1 (PDE1) Enzyme

Crotalus adamanteus venom (Eastern Diamondback rattlesnake)

Supplied as a lyophilized powder

Molecular Biology Grade

Lyophilized and reconstituted products are stable for 6 months after receipt at -20ºC. Reconstitute with sterile dH2O. Aliquot to avoid repeated freezing and thawing. Store at -20ºC. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

115kD

chromosome: Un

72.8 u/mg dry weight, 110 u/vial

1.68

0.75%

9.8-10.4

Reducing agents such as glutathione, cysteine and ascorbic acid. It is completely inhibited by 5mM EDTA while ATP, ADP and AMP are partial inhibitors.

The enzyme has an absolute requirement for Mg2+ indicates an optimum concentration of 15mM.

1 unit hydrolyzes 1 umole of p-nitrophenyl thymidine-5-phosphate per minute at 25ºC, pH 8.9.

Assay:

Method:
The assay is essentially that of Razell and Khorana (1959) where the reaction velocity is determined by an increase in absorbance at 400nm 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:
-0.11M Tris-HCl buffer, pH 8.9, with 0.11M sodium chloride 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 1 mg/ml in Tris*Salts buffer to obtain a rate of 0.02-0.04 ΔA/minute.

Protocol:
1. Set spectrophotometer at 400nm and 25ºC.
2. Pipette into microcuvettes as follows:
Tris*Salts buffer 0.9 ml; 5 mM p-nitrophenyl thymidine-5'-phosphate 0.1 ml.
3. Incubate cuvettes in spectrophotometer for 3-5 minutes to reach temperature equillibrium and establish blank rate, if any. Add 10ul 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:
Units/mg = ^ΔA400/min/_{16* x mg enzyme/ml reaction mixture}
*16 is the extinction coefficient of p-nitrophenol determined in the laboratory under these conditions.

Molecular Biology; MB-Enzymes; Phosphodiesterase