NIPRO ENZYMESPOLYNUCLEOTIDE PHOSPHORYLASE (PNPase3)[EC 2. 7. 7. 8]
from recombinant E. coli
RNAn+1 + Pi ↔ RNAn + Nucleoside diphosphate

FOR DEPOLYMERIZATION REACTION
SPECIFICATIONStateSpecific activity: Liquid: more than 2,000 U/mg protein

PROPERTIES
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Effectors: ca. 79,000: 9.0 - 9.5(Fig. 1)
: 8.0 - 11.0
(Fig. 2)
: No detectable decrease in activity up to 55 °C. (Fig. 3, 4): cations and anions(Fig. 5, 6)

STORAGE
at -20 °C
APPLICATIONThe enzyme is useful for the preparation of polyribonucleotide.

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reactions.
Poly An + Pi
ADP + PEPPNPase3
PKPyruvate + NADH + H+

Poly An-1 + ADP(I)

ATP + Pyruvate
LDH

Lactate + NAD+(II)

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)
Ⅰ Buffer solution ; 100 mM Tris-HCl, pH 9.5 ((1.212 g Tris + 0.074 g EDTA + 0.014 mL 2mercaptoethanol + 0.610 g MgCl2∙6H2O + 0.746 g KCl)/80 mL distilled water, adjusted to pH 9.5
with 1 N-HCI and filled up to 100 mL with distilled water)
Ⅱ KH2PO4 solution ; 65 mM (0.088 g KH2PO4/10 mL distilled water)
Ⅲ polyadenylate (Poly A) solution ; (25 mg PoIy A potassium salt/1 mL distilled water; ca. 35 mM
based on AMP concentration)(Reaction II)
Ⅳ Buffer solution ; 100 mM Triethanolamine buffer, pH 7.6 ((9.300 g triethanolamine-HCI + 0.407 g
MgCl2∙6H2O + 0.373 g KCl)/400 mL distilled water, adjusted to pH 7.6 with 1 N-NaOH and filled up
to 500 mL with distilled water)
Ⅴ NADH solution ; 13.1 mM (0.100 g NADH disodium salt∙3H2O/10 mL distilled water)
Ⅵ Phosphoenolpyruvate (PEP) solution ; 56 mM (0.150 g PEP MCA salt/10 mL distilled water)
Ⅶ 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
Ⅷ Lactate dehydrogenase (LDH) ; (from pig heart, Oriental Yeast Co. Ltd., Product Code: LDH-02)
ammonium sulfate suspension, approx. 5,000 U/mL at 25 °C
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 2 to 150 U/mL with 50 mM Tris-HClbuffer, pH 8.5.
Procedure(Reaction I)
1. Prepare the following reaction mixture and pipette 0.55 mL of reaction mixture into a test tube.
SolutionⅠ 2.50 mL
SolutionⅢ 1.00 mL
1.00 mL
SolutionⅡ 1.00 mL
H2O
2. Add 0.10 mL of enzyme solution and mix.
3. Incubate at 60 °C for exactly 10 minutes.
4. After incubation, add 0.01 mL conc. HCI 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 inProcedure 2 (as blank).
(Reaction II)
6. Prepare the following reaction mixture and pipette 2.50 mL of the reaction mixture into a cuvette.
SolutionⅣ 24.18 mL
SolutionⅦ 0.12 mLNIPRO ENZYMESSolutionⅤ 0.40 mL
SolutionⅧ
SolutionⅥ 0.25 mL
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
Volume activity (U/mL) = ((Abs•blank) - (Abs•test)) X

Specific activity (U/mg protein) =0.05 mL

2.60 х 0.65
6.22 X 0.10 х 0.10

X

Volume activity (U/mL)
Protein concentration (mg/mL)*

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

60
10X d.f.

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

100500456789101150012678910Fig. 1 pH profile

Fig. 2 pH stability

1112treated for 24 hr at 4 ℃ in thefollowing buffer solution (0.1 M);● Tris-HCl, ■ Glycine-KOH

100

100

Remaining activity (%)

Remaining activity (%)

5pH● Tris-HCl

5004pH3040506070

Temperature (℃)

Fig. 3 Thermal stability
treated for 15 min in 0.1 M
Tris-HCl buffer, pH 8.5

805000153045Time (min)Fig. 4 Thermal stability
treated in 50 mM Tris-HClbuffer, pH 8.5○ 55 ℃, □ 60 ℃, ● 65 ℃

60

NIPRO ENZYMES
150

Relative activity (%)

Relative activity (%)

150

100

500050

100

150

200

100

500050

100

150

200

Concentration (mM)Concentration (mM)

Fig. 5 Effect of various cations on theactivity of PNPase3 in the following Assay

Fig. 6 Effect of various anions on theactivity of PNPase3 in the following Assay

Measurement: 0.015 mL of each cation
solution, 0.010 mL of enzyme solution and0.055 mL of reaction mixture were mixed,
and reacted at 60 ℃. After 10 minutes, thequantity of ADP was determined.
○ NaCl, △ KCl, □ MgCl2

Measurement: 0.015 mL of each anion
solution, 0.010 mL of enzyme solution and0.055 mL of reaction mixture were mixed,
and reacted at 60 ℃. After 10 minutes, thequantity of ADP was determined.
○ NaCl, △ CH3COONa, □ Na2SO4, ●NaH2PO4