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Triose Phosphate Isomerase (TPI)

(D-Glyceraldehyde-3-phosphate ketol isomerase; EC

Triose phosphate isomerase (TPI) catalyzes the following reaction in carbohydrate metabolism:

D-Glyceraldehyde-3-P <------>Dihydroxyacetone Phosphate

The enzyme is widely distributed. TPI from rabbit muscle has a molecular weight of 43,000 (Burton, P.M. and Waley, S.G., Biochem. J., 100, 702, 1966). Two isozymes of TPI have been isolated and purified from the human placenta (Yuan, P.M., Dewan, R.N., Zaun, M., Thompson, R.E. and Gracy, R.W., Arch. Biochem. Biophys., 198, No. 1, 42, 1979). Triose phosphate isomerase can be used in coupled enzyme reactions for the determination of metabolites in biological fluids.

The amount of enzyme which will convert one micromole of D- glyceraldehyde-3-phosphate to dihydroxyacetone phosphate per minute at 25ºC and pH 7.6.

The method of assay is based on the following reactions (Methods of Enzymatic Analysis, Bergmeyer, H.U. ed Vol 1, 515, 1974, Academic Press, New York).

  1. 0.3 M Triethanolamine HCl, pH 7.6.
  2. 0.02 M DL-Glyceraldehyde-3-P, free acid. Dilute 0.1 ml to 1.5 ml with distilled water.
  3. 0.008 M NADH disodium salt (5 mg/ml) in buffer.
  4. Glycerol-3-phosphate dehydrogenase (G3PDH). Dissolve in buffer to give a concentration of 50 U/ml. Prepare fresh prior to assay.
  5. Triose phosphate isomerase (enzyme) solution. Dissolve in buffer to give a concentration of 0.1-0.2 U/ml. Must be prepared fresh prior to assay.

  1. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 340 nm and 25ºC.
  2. In a cuvette, pipette the following reagents:
    Triethanolamine buffer 2.20 ml
    DL-Glyceraldehyde-3-P 0.50 ml
    NADH 0.10 ml
    Glycerol-3-P-dehydrogenase 0.10 ml
  3. Incubate cuvette in spectrophotometer at 25ºC for 5 minutes to attain temperature equilibration. Record absorbance at 340 nm (blank).
  4. Initiate the reaction by adding 0.1 triose phosphate isomerase (enzyme) solution to the cuvette. Record decrease in absorbance at 240 nm for 5-8 minutes.
  5. Calculate µE340 nm/min

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

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