(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-222REACH適合品

PHOSPHOLIPASE D [PLDⅡ]

from Streptomyces chromofuscus
(Phosphatidylcholine phosphatidohydrolase: EC 3.1.4.4)

Preparation and Specification

Appearance: : Pale grayish to grayish or brownish to light purple lyophilizate

Specific activity: : More than 30 U/mg solid

Contaminants: :

Catalase: Less than 0.6 % (U/U)

Glucose oxidase: Less than 0.02 % (U/U)

Properties

Substrate specificity: : See Table 1

Molecular weight: : 58 kDa (SDS-PAGE)

Isoelectric point: : pH 5.9 (estimated from amino acid sequence)

Michaelis constants: : 1,2-Dioleoyl-sn-glycero-3-phosphocholine　9.3 × 10⁻⁴ M

Optimum pH: : 6.7–7.1Figure 1

pH stability: : 5.3–9.7Figure 2

Thermal stability: : Stable at 60℃ and below (pH 8.0, 10 min) Figure3

Storage stability: : At least one year at −20℃

Effect of metal ions: : See Table 2

Activator: : Ca²⁺

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of phospholipids when coupled with choline oxidase (T–05) .

	PLDⅡ
Phosphatidylcholine + H₂O	→	Choline + Phosphatidic acid
	COD
Choline + 2 O₂ + H₂O	→	Betaine + 2 H₂O₂
	POD
2 H₂O₂ + 4-AA + Phenol	→	Quinoneimine dye + 4 H₂O

Table 1. Substrate specificity

Substrate	Specific activity (%)
1,2-Dioleoyl-sn-glycero-3-phosphocholine	100
2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine	98
L-α-Phosphatidylcholine	95
L-α-Lysophosphatidylcholine	99
1-Oleoyl-sn-glycero-3-phosphocholine	99
L-α-phosphatidylethanolamine	14
Sphingomyelin	26

Table 2. Effect of metal ions (Activators)

Divalent cation (1 mM)	Relative activity (%)
None	2
Ca2⁺	100
Mg2⁺	2
Mn2⁺	0
Ba2⁺	1

Fig.1 pH Optimum

〇: 3,3-Dimethylglutarate-NaOH buffer
□: Tris-HCI buffer
●: Glycine-NaOH buffer

Fig.2 pH Stability

37℃, 60 min.
10 mM buffer containing 0.1%
TritonX-100 and 0.05% BSA
〇: 3,3-Dimethylglutarate-NaOH buffer
□: Tris-HCI buffer
●: Glycine-NaOH buffer

Fig.3 Thermal Stability

pH 8.0, 10 min.
10 mM Tris-HCI buffer containing
0.1% TritonX-100 and 0.05% BSA

Assay

Principle

The assay is based on the increase in absorbance at 500 nm as the formation of quinoneimine dye proceeds in the following reactions:

	PLD Ⅱ
Lecithin +H₂O	→	Phosphatidic acid+Choline
	COD
Choline +2 O₂+H₂O	→	Betaine+2H₂O₂
	POD
2 H₂O₂+4–AA+Phenol	→	Quinoneimine dye+4 H₂O

COD: Choline oxidase

Unit definition

One unit is defined as the amount of enzyme which hydrolyzes 1 μmole of phosphatidylcholine to phosphatidic acid and choline per minute at 37℃ under the conditions specified in the assay procedure.

Reagents

Reaction mixture for the first reaction

0.1 M Tris–HCl buffer pH 8.0
0.20 ml

0.1 M CaCl₂ solution
0.05 ml

25 mM substrate solution ¹⁾ 0.10 ml

Distilled water
0.15 ml

1) : 25 mM substrate solution
Dissolve 88.5 mg of 1,2-Dioleoyl-sn-glycero-3-phosphocholine with 4.5 ml of 5 % (W/V) Triton X–100 solution.

Reaction mixture for the second reaction

15 mM 4–AA solution	0.10 ml
0.2 % (W/V) Phenol solution	0.10 ml
60 mM EDTA pH 8.0	0.10 ml
50 mM Tris–HCl buffer pH 8.0	2.00 ml
90 U/ml POD solution ²⁾	0.10 ml
30 U/ml COD solution ³⁾	0.10 ml

EDTA: Ethylenediaminetetraacetic acid

2) :	90 U/ml POD solution Dissolve 900 U (PPU) of POD with 10 ml of distilled water.
3) :	30 U/ml COD solution Dissolve 300 U of COD with 10 ml of 10 mM Tris–HCl buffer pH 8.0.

Enzyme dilution buffer
10 mM Tris–HCl buffer (pH 8.0) containing 0.05%
(W/V) BSA and 0.1% (W/V) Triton X–100
Reagents
Triton X–100: The Dow Chemical Company
1,2-Dioleoyl-sn-glycero-3-phosphocholine:
Sigma Chemical Co. #P–6354
EDTA (2 Na・2H₂O) : KISHIDA CHEMICAL Co., Ltd.
#060–29133

COD: Asahi Kasei Pharma Corporation #T–05
BSA: Millipore Fraction V pH 5.2 #81–053
4–AA : NACALAI TESQUE, INC. Special grade #01907–52
POD: Sigma Chemical Co. Type Ⅱ #P–8250

Enzyme solution

Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20 ml. Dilute it with enzyme dilution buffer to adjust the concentration as required.

Procedure

Pipette accurately 0.50 ml of reaction mixture for the first reaction into a small test tube and preincubate at 37℃.
After 5 min, add 50 μl of enzyme solution and mix to start the reaction at 37℃.
At 10 min after starting the reaction, add 2.50 ml of reaction mixture to the second reaction and mix to start the second reaction.

※ In the case of a blank test, add 50 μl of enzyme dilution buffer solution at this time.
At 20 min after starting the reaction, measure the absorbance at 500 nm.

Absorbance sample : As

blank : Ab

△ A = (As－Ab) ≦ 0.60 Abs

Calculation

Activity (U/mg of powder) = {(△A/10)/(12.2)} × 3.05/0.05 × 1/x

12.2 :	millimolar extinction coefficient of quinoneimine dye (cm²/ μmole)
10 :	reaction time (min)
3.05 :	final volume (ml)
0.05 :	volume of enzyme solution (ml)
X :	concentration of the sample in enzyme solution ( mg/ml)

Storage

Storage at －20℃ in the presence of a desiccant is recommended. Enzyme activity will be retained for at least one year under this condition.

References

Imamura, S. and Horiuchi, Y. (1979) J. Biochem., 85, 75–95.

PLDⅡ 活性測定法 (Japanese)

試薬液

第一反応試薬混合液

0.1M トリス－HCl 緩衝液pH8.0
0.20 ml

0.1M 塩化カルシウム溶液
0.05 ml

25mM 基質溶液¹⁾ 0.10 ml

精製水 0.15 ml

1) ： 25mM 基質溶液
1 , 2 -ジオレオイルsn- グリセロ- 3 - ホスホコリン88.5mg を5％ (W/V) トリトンX–100 溶液4.5ml で溶解する。

第二反応試薬混合液

15mM 4–AA 溶液	0.10 ml
0.2％ (W/V) フェノール液	0.10 ml
60mM EDTA 溶液pH8.0	0.10 ml
50mM トリス－HCl 緩衝液pH8.0	2.00 ml
90U/ml POD 溶液²⁾	0.10 ml
30U/ml COD 溶液³⁾	0.10 ml

2) :	90U/ml POD 溶液 POD 900 単位 (PPU) を精製水10ml で溶解する。
3) :	30U/ml COD 溶液 COD 300 単位 (U) を10mM トリス－HCl 緩衝液pH8.0 10ml で溶解する。

酵素溶解希釈用液
0.05％ (W/V) BSA と0.1％ (W/V) トリトンX–100 を含む10mM トリス–HCl 緩衝液pH8.0
試薬
トリトンX–100：Dow Chemical 製
1 , 2 -ジオレオイルs n -グリセロ- 3 -ホスホコリン：
シグマ製 #P–6354

EDTA (エチレンジアミン四酢酸・2Na・2H₂O) ：
キシダ化学製 #060–29133
COD (コリン酸化酵素) ：旭化成ファーマ製　#T–05
BSA: Millipore 製　Fraction V pH5.2 #81–053
4–AA：ナカライテスク製　特級　#01907–52
POD：シグマ製　Type Ⅱ　#P–8250

酵素試料液

検品約20mg を精密に量り、酵素溶解希釈用液で全容20ml とする。
その液を酵素溶解希釈用液で適宜希釈する。

測定操作法

小試験管に第一反応試薬混合液0.50ml を正確に分注し、37℃で予備加温する。
5 分経過後、酵素試料液50 μl を正確に加えて混和し、37℃で第一反応を開始する。
10 分経過後、第二反応試薬混合液2.50ml を加えて混和し、37℃で第二反応を開始する。

※ 盲検はこの時点で酵素溶解希釈用液50 μl を加える。
20 分経過後、500nm における吸光度を測定する。求められた吸光度を試料液はAs、盲検液はAb とする。
ΔA = (As－Ab) ≦ 0.60Abs

計算

活性 (U/mg) = {(ΔA/10)/(12.2)} × 3.05/0.05 × 1/x

12.2 :	キノンイミン色素の500nm におけるミリモル分子吸光係数 (cm² / μmole)
10 :	反応時間 (min)
3.05 :	反応総液量 (ml)
0.05 :	反応に供した酵素試料液量 (ml)
X :	酵素試料液中の検品濃度 (mg/ml)

0.1 M Tris–HCl buffer pH 8.0	0.20 ml
0.1 M CaCl₂ solution	0.05 ml
25 mM substrate solution ¹⁾	0.10 ml

0.1M トリス－HCl 緩衝液pH8.0	0.20 ml
0.1M 塩化カルシウム溶液	0.05 ml
25mM 基質溶液¹⁾	0.10 ml
精製水	0.15 ml

Absorbance sample :	As
blank :	Ab

PHOSPHOLIPASE D [PLDⅡ] (T-222)