HEXOKINASE from Microorganism

HXK-311

PREPARATION and SPECIFICATION
Appearance:	White amorphous powder, lyophilized
Activity:	GradeⅢ 150U/mg-solid or more
Contaminants:	Phosphoglucose isomerase ≤1.0×10⁻¹% 6-Phosphogluconate dehydrogenase ≤1.0×10⁻²% Glucose-6-phosphate dehydrogenase ≤1.0×10⁻²% Myokinase ≤1.0×10⁻²% Glutathione reductase ≤ 5.0×10⁻¹%

PROPERTIES
Stability:	Stable at -20°C for at least One year (Fig.1)
Molecular weight :	approx. 82,000 (by gel filtration)
Isoelectric point :	4.1±0.1
Michaelis constants :	2.3×10⁻⁴M (D-Glucose), 7.7×10⁻⁵M (ATP)
Inhibitors :	Metal ions, p-chloromercuribenzoate, iodoacetamide, SDS, etc
Optimum pH :	8.0-9.0(Fig.2)
Optimum temperature :	50°C(Fig.3)
pH Stability :	pH 4.0-9.0 (25°C, 20hr)(Fig.4)
Thermal stability :	below 45°C (pH 7.0, 30min)(Fig.5)
Substrate specificity :	(Table 1)
Effect of various chemicals:	(Table 2)

APPLICATIONS

The enzyme is useful for enzymatic determination of glucose, adenosine-5'-triphosphate (ATP) and creatine phosphokinase when coupled with glucose-6-phosphate dehydrogenase (=G-6-PDH, G6D- 311, G6D-321).

ASSAY

Principle:

hexokinase

D-Glucose+ATP ► D-Glucose-6-phosphate+ADP

G-6-PDH

D-Glucose-6-phosphate+NAD⁺ ► Glucono-δ-lactone-6-phosphate+NADH+H⁺

The appearance of NADH is measured at 340nm by spectrophotometry.

Unit definition:

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

Method:

Reagents
A. Tris-HCl buffer, pH 8.0:	50mM, containing 13.3mM MgCl₂
B. Glucose solution:	0.67M in Tris-HCI buffer solution (A) (The solution Should be keep at room temperature at least for 1 hour before use)
C. ATP solution:	16.5mM in Tris-HCl buffer solution (A) (Should be prepared fresh)
D. NAD⁺ solution:	6.8mM in Tris-HCl buffer solution (A) (Should be prepared fresh)
E. G-6-PDH solution:	300U/ml (Dilute with Tris-HCl buffer solution (A) and store on ice)
F. Enzyme diluent:	Tris-HCl buffer solution (A) contg. 0.1% of bovine serum albumin

Procedure

Concentration in assay mixture
Tris-HCl buffer	50 mM
Glucose	0.11 M
ATP	0.53 mM
NAD⁺	0.22 mM
MgCl₂	13 mM
BSA	3.2µg/ml
G-6-PDH	ca.1.0 U/ml

1. Prepare the following reactin mixture in a cuvette (d=1.0cm) and equilibrate at 30°C for about 5 minutes.

2.30 ml Tris-HCl buffer solution (A)
0.50 ml Glucose solution (B)
0.10 ml ATP solution (C)
0.10 ml NAD⁺ solution (D)
0.01 ml G-6-PDH solution (E)

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

3. Record the increase of optical density at 340nm against water for 4 to 5 minutes in a spectrophotometer thermostated at 30°C and calculate theΔOD per minute from the initial portion of the curve (ΔOD test).
At the same time, measure the blank rate (ΔOD blank) by the same method as the test except the enzyme
diluent (F) is added instead of the enzyme solution.

* Dissolve the enzyme preparation on ice-cold enzyme diluent (F) and dilute to 0.1-0.3U/ml with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula :

ΔOD/min (OD test−OD blank ) ×Vt × df

Volume activity (U/ml) = =ΔOD/min×5.0×df

6.22×1.0×Vs

Weight activity (U/mg)=(U/ml)×1/C

Vt: : Total volume (3.11ml)
Vs: : Sample volume (0.1ml)
1.0: : Light path length (cm)
6.22: : Millimolar extinction coefficient of NADH (㎠/micromole)
df: : Dilution factor
C: : Enzyme concentration in dissolution (c mg/ml)

**Table 1. Substrate Specificity of Hexokinase**
[Pyruvate kinase-Lactate dehydrogenase system with 0.1M Tris-HCl buffer, pH 7.5]
Substrate(100mM)	Relative activity(%)	Substrate(100mM)	Relative activity(%)
D-Glucose	100	D-Galactose	0
D-Fructose	140	D-Xylose	2
D-Mannose	52	D-Glucosamine	58
2-Deoxy-D-glucose	91

**Table 2. Effect of Various Chemicals on Hexokinase**
[The enzyme dissolved in 50mM K-phosphate buffer, pH 6.5 (5U/ml) contg. 0.1% bovine serum albumin was incubated with each chemical at 30°C for 1hr]
Chemical	Concn.(mM)	Residual activity(%)	Chemical	Concn.(mM)	Residual activity(%)
None	−	100	PCMB	2.0	0
Metal salt			MIA	2.0	80
AgNO₃	2.0	0	IAA	2.0	7
BaCl₂	2.0	99	EDTA	5.0	103
CaCl₂	2.0	98	(NH₄)₂SO₄	20.0	104
CdCl₂	2.0	85	Borate	20.0	102
CoCl₂	2.0	85	o-Phenanthroline	2.0	101
CuSO₄	2.0	25	α,α′-Dipyridyl	2.0	102
FeCl₃	2.0	28	Urea	2.0	104
FeSO₄	2.0	80	Guanidine	2.0	103
HgCl₂	2.0	0	Hydroxylamine	2.0	104
MgCl₂	2.0	98	Na-cholate	1.0%	102
MnCl₂	2.0	100	Triton X-100	1.0%	105
NiCl₂	2.0	100	Brij 35	1.0%	0
Pb(OAc)₂	2.0	98	SDS	0.1%	25
Zn(OAc)₂	2.0	98	Tween 20	0.1%	101
ZnSO₄	2.0	99	Span 20	0.1%	106
NaF	20.0	101	DAC	0.1%	101
NaN₃	20.0	102

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

To get a quote, contact us at info@toyobousa.com, or INQUIRY.