HEXOKINASE from Microorganism

PREPARATION and SPECIFICATION Appearance White amorphous powder, lyophilized Activity GradeⅢ　150 U/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 ℃ for at least one year (Fig.1) Molecular weight approx. 82,000 (by gel filtration) Isoelectric point 4.1±0.1 Michaelis constants 2.3×10-4 M (D-Glucose), 7.7×10-5 M (ATP) Inhibitors Metal ions, p-chloromercuribenzoate, iodoacetamide, SDS, etc Optimum pH 8.0−9.0 (Fig.2) Optimum temperature 50 ℃ (Fig.3) pH Stability pH 4.0−9.0 (25 ℃, 20 hr) (Fig.4) Thermal stability below 45 ℃ (pH 7.0, 30 min) (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 in combination with glucose-6-phosphate dehydrogenase (=G-6-PDH,  G6D311 ,  G6D-321 ). ASSAY Principle The formation of NADH is measured at 340 nm by spectrophotometry. Unit definition One unit causes the formation of one micromole of NADH per minute under the conditions detailed below. Method Reagents A. Tris-HCl buffer, pH 8.0 50 mM, containing 13.3 mM MgCl 2 B. Glucose solution 0.67 M in Tris-HCl buffer solution (A) (The solution should be keep at room temperature for at least 1 hour before use) C. ATP solution 16.5 mM in Tris-HCl buffer solution (A) (Should be freshly prepared) D. NAD ＋  solution 6.8 mM in Tris-HCl buffer solution (A) (Should be freshly prepared) E. G-6-PDH solution 300 U/mL (Dilute with Tris-HCl buffer solution (A) and store on ice) F. Enzyme diluent Tris-HCl buffer solution (A) containing 0.1 % of bovine serum albumin Procedure 1.Prepare the following reaction mixture in a cuvette (d＝ 1.0cm) and equilibrate at 30℃ for approximately 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) Concentration in assay mixture Tris-HCl buffer 50 mM Glucose 0.11 M ATP 0.53 mM NAD ＋ 0.22 mM MgCl 2 13 mM BSA 3.2 μg/mL G-6-PDH approx.1.0 U/mL 2.Add 0.1ml of the enzyme solution *  and mix by gentle inversion. 3.Record the increase of optical density at 340 nm against water for 4 to 5 minutes with a spectrophotometer thermostated at 30 ℃ and calculate the ΔOD 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 that the enzyme diluent (F) is added instead of the enzyme solution. * Dissolve the enzyme preparation on ice-cold enzyme diluent (F) and dilute to 0.1−0.3 U/mL with the same buffer, immediately before the assay. Calculation Activity can be calculated by using the following formula : Volume activity (U/ml) = ΔOD/min (OD test−OD blank)×Vt×df 6.22×1.0×Vs ＝ ΔOD/min×5.0×df Weight activity (U/mg) ＝ (U/ml)×1/C Vt : Total volume (3.11 mL) Vs : Sample volume (0.1 mL) 1.0 : Light path length (cm) 6.22 : Millimolar extinction coefficient of NADH (cm 2 /micromole) df : Dilution factor C : Enzyme concentration in dissolution (c mg/mL) Table 1. Substrate specificity of Hexokinase [Pyruvate kinase-Lactate dehydrogenase system with 0.1 M Tris-HCl buffer, pH 7.5] Substrate(100mM) Relative activity(%) D-Glucose 100 D-Fructose 140 D-Mannose 52 2-Deoxy-D-glucose 91 Substrate(100mM) Relative activity(%) D-Galactose 0 D-Xylose 2 D-Glucosamine 58 Table 2. Effect of Various Chemicals on Hexokinase [The enzyme dissolved in 50mM K-phosphate buffer, pH 6.5 (5 U/mL) contg. 0.1 % bovine serum albumin was incubated with each chemical at 30 ℃ for 1hr.] Chemical Concn.(mM) Residual activity(%) None - 100 Metal salt AgNO 3 2.0 0 BaCl 2 2.0 99 CaCl 2 2.0 98 CdCl 2 2.0 85 CoCl 2 2.0 85 CuSO 4 2.0 25 FeCl 3 2.0 28 FeSO 4 2.0 80 HgCl 2 2.0 0 MgCl 2 2.0 98 MnCl 2 2.0 100 NiCl 2 2.0 100 Pb(OAc) 2 2.0 98 Zn(OAc) 2 2.0 98 ZnSO 4 2.0 99 NaF 20.0 101 NaN 3 20.0 102 Chemical Concn.(mM) Residual activity(%) PCMB 2.0 0 MIA 2.0 80 IAA 2.0 7 EDTA 5.0 103 (NH 4 ) 2 SO 4 20.0 104 Borate 20.0 102 o-Phenanthroline 2.0 101 α,α′-Dipyridyl 2.0 102 Urea 2.0 104 Guanidine 2.0 103 Hydroxylamine 2.0 104 Na-cholate 1.0 % 102 Triton X-100 1.0 % 105 Brij 35 1.0 % 0 SDS 0.1 % 25 Tween 20 0.1 % 101 Span 20 0.1 % 106 DAC 0.1 % 101 Ac, CH 3 CO; NEM, N-Ethylmaleimide; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; IAA, Iodoacetamide; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride. Fig.1. Stability (Powder form) (kept under dry conditions) Fig.2. pH-Activity 30 ℃ in the 50 mM buffer solution: pH 6.2-7.5, PIPES-NaOH: pH 7.5-9.0, Tris-HCI: pH 9.0-10.0, Glycine-NaOH Fig.3. Temperature activity (in 50 mM Tris-HCI buffer,pH 8.0) Fig.4. pH-Stability 25 ℃, 20 hr-treatment in the 0.1 M buffer solution: pH 4.0-8.0,Acetate-NaOH;pH 6.0-8.0, K-phosphate; pH 7.5-9.0,Tris-HCl;pH 9.0-10.5, Glycine-NaOH enzyme concn.: ca.10 U/mL Fig.5. Thermal stability 30 min-treatment with 50 mM K-phosphate buffer, pH 7.0, containing 0.1% bovine serum albumin enzyme concn.: ca.5 U/mL