(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-11REACH適合品

PHOSPHOLIPASE C [PLC]

from Bacillus cereus
(Phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3)

Preparation and Specification

Appearance: : White to brownish amorphous powder, lyophilized

Specific activity: : More than 30 U/mg solid

Properties

Molecular weight: : 20 kDa (Sephadex G–100)

Isoelectric point: : pH 7.0

Michaelis constants: : Phosphatidylcholine 2.0 × 10^-2M

Optimum pH: : 7.0–9.0Figure 1

pH stability: : 5.0–10.0 (in 0.1% BSA) Figure 2

Thermal stability: : Stable at 55℃ and below ( pH 7.5, 10 min) Figure3

Storage stability: : At least one year at －20℃Figure4

Effect of various chemicals: : See Table 1 and Table 2

Activator: : Ca²⁺, Sodium deoxycholate

Inhibitor: : Tris–HCl buffer

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of lecithin when coupled with alkaline phosphatase (T–08) and choline oxidase (T–05) .

	PLC
Lecithin + H₂O	→	Phosphorylcholine + Diglyceride
	ALP
Phosphorylcholine + H₂O	→	Choline + Pi
	COD
Choline + 2 O₂ + H₂O	→	Betaine + 2 H₂O₂
	POD
2 H₂O₂ + 4－AA + Phenol	→	Quinoneimine dye + 4 H₂O

Table 1. Effect of detergents on PLC activity

Detergent	Concentration (%)	Relative activity (%)
None	0	100
Triton X–100	0.1	43
Adekatol SO–120	0.1	63
Deoxycholate	0.1	248
Tween 20	0.1	16

Table 2. Effect of metal ions on PLC activity

Metal ion	Concentration (mM)	Relative activity (%)
None	0	100
NaCl	100	103
KCl	100	102
CaCl₂	10	128
MgCl₂	10	123
CoCl₂	10	33
MnCl₂	10	54
ZnCl₂	10	79
BaCl₂	10	91
EDTA	10	26

Fig.1 pH Optimum

〇: 3,3-Dimethylglutarate-NaOH buffer
●: Tris-HCI buffer
□: Collidine buffer
■: Diethanolamine-HCI buffer
△: Glycine-NaOH buffer

Fig.2 pH Stability

△: -BSA, 37℃, 60 min.
▲: -BSA, 37℃, 20 hr
〇: +BSA, 37℃, 60 min
●: +BSA, 37℃, 20 hr

Fig.3 Thermal Stability

pH 7.5, 10 min
〇: None
●: +BSA,10 min

Fig.4 Storage (lyophilized powder)

－20℃

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:

	PLC
Phosphatidyl choline+H₂O	→	1, 2–Diglyceride+Phosphoryl choline
	ALP
Phosphoryl choline+H₂O	→	Choline+Orthophosphate
	COD
Choline+2 O₂+H₂O	→	Betaine+2 H₂O₂
	POD
2 H₂O₂+Phenol+4–AA	→	Quinoneimine dye+4 H₂O

ALP: Alkaline phosphatase
COD: Choline oxidase

Unit definition

One unit is defined as the amount of enzyme which

liberates 1 μmole of phosphoryl choline per minute at 37℃ under the conditions specified in the assay procedure.

Reagents

Reaction mixture for the first reaction

0.2 M DMG–NaOH buffer pH 7.5	0.20 ml
10 mM Egg lecithin solution ¹⁾	0.20 ml
Distilled water	0.50 ml

DMG: 3, 3–Dimethylglutarate

1) :

10 mM Egg lecithin solution
Pour the entire contents of one ampoule of egg lecithin into a test tube and remove ether. Then dry contents under reduced air pressure with rotary evaporator. Accurately weigh 1 g of egg lecithin into a beaker and add 135 ml of 0.5% deoxycholate (DOC) ^※) solution to dried product and suspend in homogenizer.

※) :	0.5% DOC solution Suspend 250 mg of deoxycholic acid with 40 ml of distilled water, adjust pH to 8.0 with 4 N NaOH, and add distilled water to make a total of 50 ml.

Reagent mixture for the second reaction

15 mM 4–AA solution	0.10 ml
0.2% (W/V) Phenol	0.10 ml
50 mM Tris–HCl buffer, pH 8.0	0.50 ml
90 U/ml POD ²⁾	0.10 ml
30 U/ml COD ³⁾	0.10 ml
80 U/ml ALP ⁴⁾	0.10 ml

2) :	90 U/ml POD Dissolve 900 U (PPU) of POD with 10 ml of distilled water.
3) :	30 U/ml COD Dissolve 300 U of COD with 10 ml of 10 mM Tris–HCl buffer (containing 1% KCl) , pH 8.0.
4) :	80 U/ml ALP Dissolve 800 U of ALP with 10 ml of 10 mM Tris–HCl buffer pH 9.0.

Reaction stopper
1 M Tris–HCl buffer pH 8.0 containing 0.2% (W/V) sodium dodecyl sulfate (SDS)
Enzyme dilution buffer
10 mM DMG–NaOH buffer, pH 7.5 containing 0.1% (W/V) BSA
Reagents:
DMG: Tokyo Kasei Kogyo Co. Ltd. #D1322
Egg lecithin: Sigma Chemical Co. Type X VI –E #P–3556–1G
DOC: NACALAI TESQUE, INC. # 107–11
COD: Asahi Kasei Pharma Corporation # T–05
ALP: Asahi Kasei Pharma Corporation # T–08
4–AA: NACALAI TESQUE, INC. Special grade # 01907–52
POD : Sigma Chemical Co. Type II #P–8250

Enzyme solution

Weigh about 20 mg of test sample exactly and add enzyme dilution buffer to make a total of 20 ml. Dilute it with enzyme dilution buffer as required.

Procedure

Pipette 0.90 ml of reagent mixture for the first reaction into a small test tube and preincubate at 37℃.
After 5 min, add exactly 100 μl of enzyme sample and mix to start the first reaction at 37℃.

※ In the case of a test blank, add 100 μl of enzyme

　 dilution buffer in place of enzyme solution.
After 10 min, add 1.0 ml of reaction stopper and mix.
On stopping the first reaction, add 1.0 ml of the reaction　mixture for the second reaction immediately to start　the second reaction at 37℃.
After 20 min, measure the absorbance at 500 nm.

Absorbance sample : As

blank : Ab

△A = (As−Ab) ≦ 0.30 Abs

Calculation

Activity (U/mg) = {(△A/10)/(12.0)} × 3.00/0.10 × 1/x

12.0 :	millimolar extinction coefficient of quinoneimine dye at 500 nm (cm²/ μmole)
10 :	reaction time (min)
3.0 :	final volume (ml)
0.10 :	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 (Figure 4)

References

Zwaal, R. F., Roelofsen, B., Comfurius, P.S. and Van
Deenen , L. L. M (. 1971) Biochim. Biophys. Acta, 233, 474.
Otnaess, A. B., Prydz, H., Bjoerklid, E. and Berre, A. (1972) Eur. J. Biochem., 27, 238.

PLC 活性測定法 (Japanese)

試薬液

第一反応試薬混合液

0.2M DMG–NaOH 緩衝液 pH7.5	0.20 ml
10mM 卵黄レシチン溶液¹⁾	0.20 ml
精製水	0.50 ml

1) ：

10mM 卵黄レシチン溶液
卵黄レシチン1 g をビーカーに量りとり、0.5%DOC 溶液※) 135ml を加えてホモジナイザーにより分散懸濁する。

※) :	0.5%DOC 溶液デオキシコール酸250mg を精製水40mlに懸濁して4N NaOH を加えpH8.0 位にして溶解した後、精製水で全容50ml とする。

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
30U/ml COD 溶液²⁾	0.10 ml
90U/ml POD 溶液³⁾	0.10 ml
80U/ml ALP 溶液⁴⁾	0.10 ml
50mM トリス–HCl 緩衝液pH8.0	0.50 ml

2) :	30U/ml COD 溶液 COD 300 単位 (U) を1% KCl を含む10mM トリス–HCl 緩衝液pH8.0 10ml で溶解する。
3) :	90U/ml POD 溶液 POD 900 単位 (PPU) を精製水10ml 溶解する。
4) :	80U/ml ALP 溶液 ALP 800 単位 (U) を10mM トリス–HCl 緩衝液pH9.0 10ml で溶解する。

反応停止液
0.2% (W/V) ドデシル硫酸ナトリウム (SDS) を含む1M トリス–HCl 緩衝液pH8.0
酵素溶解希釈用液
0.1% (W/V) BSA を含む10mM　DMG–NaOH 緩衝液pH7.5
試薬
DMG (3,3–ジメチルグルタル酸) ：
東京化成製　#D1322
卵黄レシチン (L–α–フォスファチジルコリン) ：
シグマ製Type X VI–E #P–3556–1G
DOC (デオキシコール酸) ：ナカライテスク製
#107–11
COD (コリン酸化酵素) ：旭化成ファーマ製　#T–05
ALP (アルカリフォスファターゼ) ：
旭化成ファーマ製　#T–08
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0.90ml 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100 μl を正確に加えて混和し、37℃で第一反応を開始する。

※ 盲検は酵素試料液の代わりに酵素溶解希釈用液100μl を加える。
10 分経過後、反応停止液1.0ml を加えて混和後、直ちに第二反応試薬混合液1.0ml を加えて混和し、37℃で第二反応を開始する。
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) ≦ 0.30 Abs

計算

活性 (U/mg) = {(ΔA/10)/(12.0)} × 3.00/0.10 × 1/x

12.0 :	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)
3.00 :	反応総液量 (ml)
0.10 :	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)

Absorbance sample :	As
blank :	Ab

PHOSPHOLIPASE C [PLC] (T-11)