CREATINE AMIDINOHYDROLASE from Actinobacillus sp.

PREPARATION and SPECIFICATION
Appearance:	White amorphous powder, lyophilized
Activity:	GradeⅡ 6.0U/mg-solid or more (containing approx. 50% of stabilizers)
Contaminants:	NADH oxidase ≤5.0×10⁻²% Catalase ≤2.0%
Stabilizers:	Sugars, EDTA

PROPERTIES
Stability:	Stable at -20℃ in at least one year(Fig.1,2)
Molecular weight:	approx. 100,000
Isoelectric point:	4.6±0.1
Michaelis constant:	1.9×10⁻² M (Creatine)
Structure:	2 subunits per enzyme molecule
Inhibitors:	Cu⁺⁺, Hg⁺⁺, Ag⁺
Optimum pH:	8.0(Fig.4)
Optimum temperature:	40℃(Fig.5)
pH Stability:	pH 5.5-9.0 (25℃, 16hr)(Fig.6)
Thermal stability:	below 50℃ (pH 7.5, 30min)(Fig.7)
Effect of various chemicals:	(Table 1)

APPLICATIONS

This enzyme is useful for enzymatic determination of creatine and creatinine when coupled with creatinine amidohydrolase (CNH-211, CNH-311) and sarcosine oxidase (SAO-351) in clinical analysis. ⁴⁾

CRH-211

ASSAY

Principle:

The appearance of yellow dye formed by condensation of urea and p-dimethylaminobenzaldehyde (DAB) (Ehrlich reaction) is measured at 435nm by spectrophotometry.

Unit definition:

One unit causes the formation of one micromole of yellow dye per minute under the conditions described
below.

Method:

Reagents
A. Creatine solution:	0.1M[1.49g creatine (Nacalai tesque)/100ml of 50mM phosphate buffer, pH 7.5] (Should be prepared fresh).
B. DAB solution:	Dissolve 2.0g of DAB in 100ml of dimethylsulfoxide and, to this solution, add 15ml of conc. HCl solution.
C. Enzyme diluent:	50mM Phosphate buffer, pH 7.5

Procedure

Concentration in assay mixture
Phosphate buffer	50mM
Creatine	90mM

1. Pipette 1.0ml of the substrate solution (A) into a test tube and equilibrate at 37℃ for about 5 minutes.

2. Add 0.1ml of the enzyme solution* and mix.

3. After exactly 10 minutes at 37℃, add 2.0ml of DAB solution (B) to stop the reaction.

4. Incubate at 25℃ for 20 minutes.

5. Measure the optical density at 435nm against water (OD test).
At the same time, prepare the blank by first mixing the substrate solution with 2.0ml of DAB solution after a 10 min-incubation at 37℃, followed by the addition of the enzyme solution, and carry out the same procedure as test (procedure 4 and 5)(OD blank).

* Dissolve the enzyme preparation in ice-cold enzyme diluent (C) and dilute to 2.0-3.0 U/ml with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula :

ΔOD (OD test−OD blank ) × Vt × df

Volume activity (U/ml) = =ΔOD×9.65×df

0.321×1.0× t ×Vs

Weight activity (U/mg) = (U/ml) × 1/C

Vt: : Total volume (3.1ml)
Vs: : Sample volume (0.1ml)
0.321: : Millimolar extinction coefficient of yellow dye(㎠/micromole)
1.0: : Light path length (cm)
t: : Reaction time (10 minutes)
df: : Dilution factor
C: : Enzyme concentration in dissolution (c mg/ml)

REFERENCES

D.Tsuru; Nucleic Acid and Amino Acids, 35, 31 (1977).
T.Yoshimoto, I.Oka and D.Tsuru; Arch.Biochem.Biophys., 177, 508 (1976).
T.Yoshimoto ,I.Oka and D.Tsuru; J.Biochem., 79, 1381 (1976).
D.Tsuru; Rinsho Kensa, 22, 1331 (1978).

**Table 1. Effect of Various Chemicals on Creatine amidinohydrolase**
[The enzyme dissolved in 50mM Tris-HCl buffer, pH 7.5 (80U/ml) was incubated at 25℃ for 30 minutes with each chemical.]
Chemical	Concn.(mM)	Residual activity(%)	Chemical	Concn.(mM)	Residual activity(%)
None	−	100	NaF	1.0	105
Metal salt	1.0		PCMB	0.33	3.3
CaCl₂		107	MIA	1.0	106
MnCl₂		109	IAA	1.0	103
MgCl₂		103	NaN₃	10	106
NiCl₂		107	o-Phenanthroline	1.0	108
CoCl₂		108	Hydroxylamine	1.0	105
Ba(OAc)₂		104	NEM	10	0.3
Cd(OAc)₂		88	Triton X-100	0.5%	94
FeCl₃		103	Brij 35	0.5%	103
FeSO₄		102	Tween 20	0.5%	100
HgCl₂		2.7	Span 20	0.5%	106
ZnSO₄		97	Na-cholate	0.5%	103
CuSO₄		11	SDS	0.25%	102
Pb(OAc)₂		108	DAC	0.5%	1.7
AgNO₃		2.5
EDTA	20	99
α,α′-Dipyridyl	1.0	100

Ac, CH₃CO; EDTA, Ethylenediaminetetraacetate; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; IAA,
lodoacetamide; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.

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