Lipoamide Dehydrogenase (LD)
(NADH: lipoamide oxidoreductase; EC 126.96.36.199)
Lipoamide dehydrogenase (or diaphorase) catalyzes the
Lipoamide + NADH + H+ ------> Dihydrolipoamide + NAD+
The enzyme occurs in mammalian and microbial cells and it
catalyzes a number of reactions which involve NAD+ or
NADH. Lipoamide dehydrogenase from porcine heart contains two
polypeptide chains which are similar. It has two molecules of
tightly bound flavin adenine dinucleotide (FAD). The molecular
weight of the porcine heart enzyme is between 100,000 and
114,000 (Massey, V., Hofmann, T. and Palmer, G.,
J. Biol. Chem., 237, 3820, 1962).
||Soluble in distilled water or dilute buffer
||-20° C; -4° F
||25 U/mg protein
The amount of enzyme which catalyzes the oxidation of one micromole NADH per minute at pH 5.65 and 25°C.
The assay of lipoamide dehydrogenase is based on the method of Massey, et al (Massey, V., Gibson, Q.M. and Veeger, C., Biochem. J., 77, 341, 1960). The decrease in the absorbance at 340 nm, caused by the oxidation of NADH, is a measure of the enzyme activity. Note: DL-8,6-Thioctic Acid is the same as DL-a-Lipoic Acid and the same as DL-1,2-Dithiolane-3-pentanoic Acid.
- 1.0 M Sodium citrate buffer, pH 5.65.
- 2% Bovine serum albumin (BSA) in 0.03 M EDTA disodium salt, pH 7.0.
- 0.002 M NAD+ (1.33 mg/ml) in buffer.
- 0.0015 M NADH disodium salt (1.0 mg/ml) in buffer.
- 0.02 M DL-6,8-Thioctic acid (4.24 mg/ml), pH 7.0. Suspend 80 mg of DL-6,8-Thioctic acid in 10 ml distilled water. Adjust pH to 12.0 with 2 M NaOH. Stir for 15 minutes, maintaining pH at 12.0. Adjust pH to 7.0 with citrate buffer. Add distilled water to bring the final concentration to 0.02 M Thioctic acid. Prepare fresh daily.
- Lipoamide dehydrogenase (enzyme). Dissolve and dilute in citrate buffer to a final concentration of 0.1-0.2 U/ml. Prepare fresh prior to assay.
- Set spectrophotometer (equipped with strip chart recorder and temperature
control) at 340 nm and 25°C.
- Into a cuvette pipette the following reagents in the amounts
Citrate buffer 2.5 ml
BSA-EDTA 0.10 ml
NAD+ 0.10 ml
NADH 0.10 ml
DL-6,8-Thioctic Acid 0.10 ml
Incubate in the
spectrophotometer at 25°C for 5 minutes. Record absorbance at 340 nm (blank).
- Initiate the reaction by adding 0.1 ml of enzyme solution to the cuvette.
Record the decrease in absorbance at 340 nm for 5-8 minutes.
- Calculate E340 nm/min