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Hexokinase (HK)

(ATP:D-hexose 6-phosphotransferase; EC

Hexokinase (HK) catalyzes the phosphorylation of glucose. In mammals, four distinct enzymes-types I to IV hexokinases-have been identified. The enzyme is found in most cells, but there is tissue specificity for the particular type of hexokinase. Thus skeletal muscle has type II, adipose tissue has both types I and II, and the liver has all four types of hexokinases. However, the type IV hexokinase (glucokinase) is the one that predominates in the liver (Human Biochemistry; Orten, J.M. and Neuhaus, O.W.; 10th edition, p. 226, 1982, Mosby, St. Louis).

Two hexokinases, designated P-I and P-II, have been isolated from yeast. These are separate, noninterconvertible enzymes (Womack, F.C.; Welch, M.K.; Nielsen, J.; and Colowick, S.P., Arch. Biochem. Biophys., 158, 451, 1973). The yeast hexokinase has a molecular weight of 100,000 and the optimum pH for its catalytic activity is 7.5.

Hexokinase is used to determine glucose, fructose, mannose and ATP.

That amount of enzyme which causes the phosphorylation of one micromole of D-glucose per minute at 25C and pH 7.6.

This assay is based upon the phosphorylation of D-glucose by HK and the subsequent reduction of NADP by glucose-6-phosphate dehydrogenase.

  1. 0.1 M Triethanolamine (TEA) buffer, pH 7.6. Dissolve 1.86 g TEA-HCl in 80 ml redistilled water. Adjust to pH 7.6 with 1 M NaOH and adjust volume to 100 ml with water.
  2. 10% Glucose: Dissolve 1 g glucose in 10 ml TEA buffer. Prepare fresh daily.
  3. 0.1 M MgCl2: Dissolve 2.03 g MgCl2&6H2O in 100 ml redistilled water.
  4. 80 mM ATP: Dissolve 50 mg ATP-Na2 in 1 ml redistilled water.
  5. 12.7 mM NADP: Dissolve 10 mg NADP-Na2 in 1 ml redistilled water.
  6. G-6-PDH from yeast: 1 mg/ml.
  7. 1% Albumin: 1 g BSA/100 ml Triethanolamine buffer.
  8. Hexokinase (enzyme) solution: For freeze-dried enzyme, dissolve 10 mg/1 ml redistilled water, and dilute to 1:1250 with cold albumin solution. For enzyme suspension, dilute in ratio suspension: solution of 1:5000 with cold albumin solution. Perform these steps just prior to measurement. Volume activity should be 0.3 U/ml.

  1. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 340 nm and 25C.
  2. Into a cuvette pipette the following reagents:
    TEA buffer 1.28 ml
    Glucose 1.20 ml
    MgCl2 0.20 ml
    ATP 0.10 ml
    NADP 0.20 ml
    G-6-PDH 0.005 ml
    Incubate in spectrophotometer at 25C for 5 min.
  3. Record blank rate at 340 nm.
  4. Initiate the reaction by adding 0.2 ml of the enzyme solution to the cuvette. Record the absorbance at 340 nm for 5 min.
  5. Calculate (delta)E340nm/min

Activity (U/mg) = (ΔE340nm/min)(Total Vol.)(Enz. Diln.)
(6.22)(Enz. Vol.)(mg Enz./ml)

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