CODE No.:B2P411

POLYNUCLEOTIDE PHOSPHORYLASE (PNPase)

[EC 2.7.7.8]
from Bacillus stearothermophilus

FOR DEPOLYMERIZATION REACTION

SPECIFICATION

State	:	Lyophilized
Specific activity	:	more than 2,000 U/mg protein
Contaminants	:	(as PNPase activity = 100%)
		Ribonuclease	<0.0001%

PROPERTIES

Molecular weight	:	300,000 - 340,000
Subunit molecular weight	:	ca. 85,000
Optimum pH	:	9.0 - 9.5	(Fig.1)
pH stability	:	9.0 - 11.0	(Fig.2)
Isoelectric point	:	4.0
Thermal stability	:	No detectable decrease in activity up to 55°C.	(Fig.3, 4)
Michaelis constants	:	(38 mM Tris-HCl buffer, pH 9.5, at 60°C)
		Poly A	0.27mM**
		KH2PO4	3.0mM
		**concentration of poly A was calculated as AMP concentration
Effectors	:	cations and anions	(Fig.5,6)

STORAGE

stable at -20°C for at least one year

APPLICATION

The enzyme is useful for the preparation of polyribonucleotide see below.

ASSAY

Principle

The change in absorbance is measured at 340 nm according to the following reactions.

Unit Definition

One unit of activity is defined as the amount of PNPase that forms 1 µmol of ADP per hour at 60°C by depolymerizing of Poly A.

Solutions
(Reaction I)

1. Buffer solution ; 100mM Tris-HCl, pH 9.5 ((1.212 g Tris + 0.074 g EDTA + 0.014 mL 2-mercaptoethanol + 0.610 g MgCl₂·6H₂O + 0.746 g KCl)/80mL distilled water, adjusted to pH 9.5 with 1 N-HCl and filled up to 100mL with distilled water)
2. KH₂PO₄ solution ; 65 mM (0.088 g KH₂PO₄/10mL distilled water)
3. polyadenylate (Poly A) solution ; (25 mg Poly A potassium salt/1 mL distilled water; ca. 35 mM based on AMP concentration)

(Reaction II)

4. Buffer solution ; 100mM Triethanolamine buffer, pH 7.6 ((9.300 g triethanolamine-HCl + 0.407 g MgCl₂·6H₂O + 0.373 g KCl)/400mL distilled water, adjusted to pH 7.6 with 1 N-NaOH and filled up to 500mL with distilled water)
5. NADH solution ; 13.1 mM (0.100 g NADH disodium salt·3H₂O/10mL distilled water)
6. Phosphoenolpyruvate (PEP) solution ; 56mM (0.150 g PEP MCA salt/10mL distilled water)
7. Pyruvate kinase (PK) ; (from rabbit muscle, Roche Diagnostics K.K., No. 128 155) crystalline suspension in 3.2 M (NH₄)₂SO₄ solution (10 mg/mL) approx. 200 U/mg at 25°C
8. Lactate dehydrogenase (LDH) ; (from hog muscle, Roche Diagnostics K.K., No. 127 221) 50% glycerol solution (25 mg/2.5 mL) approx. 550 U/mg at 25°C

Preparation of Enzyme Solution

Dissolve the lyophilized enzyme with distilled water and dilute to 1 to 5 U/mL with 50mM Tris-HCl buffer, pH 8.5.

Procedure
(Reaction I)

1. Prepare the following reaction mixture and pipette 0.55 mL of reaction mixture into a test tube.

   Solution I	2.50mL	Solution III	1.0mL
   Solution II	1.00mL	H2O	1.0mL

2. Add 0.10mL of enzyme solution and mix.
3. Incubate at 60°C for exactly 10 minutes.
4. After incubation, add 0.01 mL conc. HCl and mix.
5. Centrifuge at 10,000 rpm for 30 seconds.
   At the same time, repeat the Procedure 1 to 5 using distilled water in place of enzyme solution in Procedure 2 (as blank).

(Reaction II)

6. Prepare the following reaction mixture and pipette 2.50mL of the reaction mixture into a cuvette.

   Solution IV	24.18mL	Solution VII	0.12mL
   Solution V	0.40mL	Solution VIII	0.05mL
   Solution VI	0.25mL

7. Incubate at 30°C for about 3 minutes.
8. Add 0.10 mL of supernatant of Procedure 5 and mix.
9. Read absorbance at 340 nm (Abs·test).
   Repeat the Procedure using blank (Abs·blank).

Calculation

• d.f. ; dilution factor
• 6.22 ; millimolar extinction coefficient of NADH (cm²/µmol)
• *Protein concentration ; determined by the absorbance at 280nm (Abs280),where 1 Abs280 = 1 mg/mL

REFERENCE

1. Smith, J.C., and Eaton, M.A.W.; Nucleic Acids Research, 1, 1763 (1974)
2. Wood, J.N., and Hutchinson, D.W.; ibid., 3, 219 (1976)

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FOR POLYMERIZATION REACTION

PROPERTIES

Specific activity	:	more than 2,000 U/mg protein
Contaminants	:	(as PNPase activity = 100%)
		Ribonuclease	<0.0001%
Optimum pH	:	9.0 - 9.5
Michaelis constants	:	(79 mM Tris-HCl buffer, pH 9.5, at 60°C)
		ADP	2.2mM
Substrate specificity (25 mM)	:	(125 mM Tris-HCl buffer, pH 9.0, at 37°C)
		ADP	100%
		CDP	41%
		IDP	117%

ASSAY

Principle

The radioactivity of Poly A that obtained by polymerizing [C¹⁴] ADP is measured according to the following reaction.

Unit Definition

One unit of activity is defined as the amount of PNPase that polymerizes 1 µmol of ADP at 60°C per hour.

Solutions

1. Buffer solution ; 0.2M Tris-HCl, pH 9.5
2. ADP solution ; 100mM (0.507 g ADP disodium salt·2H₂O/(9.0mL distilled water + 1.0mL 1 N-NaOH))
3. [C¹⁴] ADP solution ; (0.05 mCi/2.5 mL, 43.3 mCi/mmol; New England Nuclear)
4. Mixed ADP solution ; (mix 9.5 volume of solution II and 5 volume of solution III)
5. EDTA solution ; 10mM (0.037 g EDTA disodium salt·2H₂O/10mL distilled water)
6. MgCl₂ solution ; 1 M (2.033 g MgCl₂·6H₂O/10mL distilled water)

Preparation of Enzyme Solution

referred to "Assay based on depolymerizing poly A"

Procedure

1. Prepare the following reaction mixture and pipette 150µL of the reaction mixture into a test tube.

   Solution I	590µL	Solution VI	80µL
   Solution IV	150µL	H2O	580µL
   Solution V	100µL

2. Preincubate at 60°C for about 5 minutes.
3. Start the reaction by adding 10 µL of the enzyme solution.
4. After exactly 4 minutes, stop the reaction by adding 0.3 mL of 10% trichloroacetic acid (TCA) solution and mixing. Allow to stand for about 10 minutes.
5. Filter the solution obtained in Procedure 4 through Whatman GF/C. Wash twice inside of the test tube with 5 mL of 1% TCA solution and filter the solution through the same filtering paper. Further, wash surface of the filter funnel with 5 mL of 1% TCA solution.
6. Air-dry the filter paper at room temperature.
7. Measure the counts of radioactivity with liquid scintillator. Prepare a calibration curve of dpm vs. ADP concentration previously.

Calculation

• A ; ADP concentration (µmol) per dpm
• d.f. ; dilution factor
• *Protein concentration ; determined by the absorbance at 280nm (Abs280),where 1 Abs280 = 1 mg/mL