HEXOKINASE - Toyobo

HXK-301
ASSAY
Principle:
hexokinase
D-GlucoseATP
Glucose-6-phosphateNAD
D-Glucose-6-phosphateADP
G-6-PDH
Glucono--lactone-6-phosphateNADHH
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
B. Glucose solution
C. ATP solution
D. NAD solution
E. G-6-PDH solution
F. Enzyme diluent
50mM, containing 13.3mM MgCl 2
0.67M in Tris-HCI buffer solution (A) (Should be prepared fresh)
16.5mM in Tris-HCl buffer solution (A) (Should be prepared fresh)
6.8mM in Tris-HCl buffer solution (A) (Should be prepared fresh)
300U/ml (Dilute with Tris-HCl buffer solution (A) and store on ice)
Tris-HCl buffer solution (A) contg. 0.1% of bovine serum albumin
Procedure
1. Prepare the following reagent mixture in a cuvette (d1.0cm)
Concentration in assay mixture
and equilibrate at 30 for about 5 minutes.
Tris-HCl buffer
50 mM
2.3 ml Tris-HCl buffer solution, pH 8.0 (A) Glucose
0.11 M
ATP
0.55 mM
0.50ml Glucose solution (B)
NAD
0.23 mM
0.10ml ATP solution (C)
MgCl 2
13 mM
0.10ml NAD solution (D) BSA
3.3g/ml
0.01ml G-6-PDH solution (E) G-6-PDH
ca.1.0 U/ml
2. Add 0.1ml of the enzyme solutionand mix by gentle inversion.
3. Record the increase in optical density at 340nm against water for 2 to 3 minutes in a spectrophotometer
thermostated at 30 and calculate theOD per minute from the initial linear portion of the curve (OD
test).
At the same time, measure the blank rate (OD blank) by using the same method as the test except that
the enzyme diluent (F) is added instead of the enzyme solution.
Dissolve the enzyme preparation in ice-cold enzyme diluent (F), dilute to 0.10.2U/ml with the same buffer
and store on ice.
Calculation
Activity can be calculated by using the following formula
OD/min (OD testOD blank)Vtdf
Volume activity (U/ml)
6.221.0Vs
Weight activity (U/mg)(U/ml)1/C
Vt Total volume (3.11ml)
Vs Sample volume (0.1ml)
6.22 Millimolar extinction coefficient of NADH (F/micromole)
1.0 Light path length (cm)
df Dilution factor
C Enzyme concentration in dissolution (c mg/ml)
OD/min5.0df
REFERENCES
1) I.T.Schulze et al.; J.Biol.Chem., 244, 2306 (1969).
2) E.M.Bessell et al.; Bilchem.J., 128, 199 (1972).
3) J.J.Schmidt et al.; Arch.Biochem.Blophys., 158, 458 (1973).
4) M.R.McDonals; Methods in Engymology, vol.1, p.269 Academic press, New York (1955).
5) A.Sole et al.; Biochem.Blophys.Acta, 30, 92 (1958).
6) W.H.Harrison and R.M.Gray; Arch.Biochem.Blophys., 151, 357 (1972).