Aldehyde Dehydrogenase (ALDH)
(Aldehyde: NAD+ (P+) oxi1doreductase; EC 22.214.171.124)
Aldehyde dehydrogenase from yeast catalyzes the following reaction:
RCHO + NAD/NADP+ + H2O -----> RCOOH + NADH/NADPH+ + H+
The yeast enzyme requires potassium ions and thiols
(glutathione, 2-mercaptoethanol, cysteine) for its
activity. It is inhibited by traces of heavy metals,
particularly copper. Similar enzymes, but with different
requirements for their catalytic activities, have been
purified from equine liver (Feldman, R.I. and Weiner, H;
J. Biol Chem., 247, 260, 1972) and from human erythrocytes
(Inoue, K., Nishimukai, H. and Yamasawa, K.;
Biochim. Biophys. Acta, 569, 117, 1979). The enzyme from yeast
has a molecular weight of 200,000. (Steinman, C.R. and Jakoby,
W.B., J. Biol. Chem., 242, 5019, 1967).
The amount of enzyme which will oxidize one micromole of acetaldehyde to acetic acid per minute at 25°C and pH 8.0 in the presence of potassium ions, thiols and §-NAD+.
The increase in the absorbance at 340 nm, caused by the reduction of NAD+ to NADH, is a measure of the catalytic activity of aldehyde dehydrogenase. The assay method has been described (Bostian and Betts, Biochem. J., 173, 787, 1978).
- 1.0 M Tris-HCl buffer, pH 8.0
- 3.0 M KCl in distilled water
- 0.005 M Acetaldehyde. Prepare fresh from stock 2.0 M Acetaldehyde.
- 0.33 M 2-Mercaptoethanol. Prepared fresh.
- 0.02 M §-NAD+ (15 mg/ml). Prepared fresh in distilled water.
- Aldehyde dehydrogenase (enzyme) solution. Prepare in 0.1 M Tris-HCl, pH 8.0 containing 0.02% bovine serum albumin, to give a final concentration of 0.1-0.2 U/ml. Must be prepared fresh prior to assay.
- Set spectrophotometer (equipped with strip chart recorder and temperature
control) at 340 nm and 25°C.
- In a cuvette, pipette the following reagents in the amounts indicated:
Distilled water 2.20 ml KCl 0.10 ml
Tris-HCl buffer 0.30 ml a-2-Mercaptoethanol 0.10 ml
Acetaldehyde 0.10 ml §-NAD+ 0.10 ml
Incubate cuvette in the spectrophotometer at 25°C for 5 minutes to attain
temperature equilibration. Record absorbance at 340 nm (blank).
- Initiate reaction by adding 0.1 ml enzyme solution to the cuvette. Record
the increase in absorbance at 340 nm for 5 min.
- Calculate E340nm/min
|Activity (U/mg) =
||(ΔE340nm/min)(Total Vol.)(Enz. Diln.)
|(6.22)(Enz. Vol.)(mg Enz./ml)