Hewson, Janet K.

Parker, M.G.

Weitzman, P.D.J.

Elsevier - Publisher Connector 

Volume 43, number 1 FEBS LETTERS July 1974 
PREPARATION OF DIHYDROLIPOAMIDE BY ELECTROLYTIC REDUCTION 
P. D. J. WEITZMAN, Janet K. HEWSON and M. G. PARKER 
Department of Biochemistry, University of Leicester, 
Leicester, England 
Received 28 March 1974 
1. Introduction 
Lipoic acid plays a central role in the action of the 
cy-oxoacid ehydrogenase enzyme complexes. Enzyme- 
bound in the form of lipoamide it participates in a 
cyclic series of reactions comprising reductive acyla- 
tion, acyl transfer to coenzyme A and reoxidation 
by NAD’. The individual enzymes are also active with 
externally-supplied, unbound, lipoamide and studies 
of these reactions ometimes require the use of the 
reduced form, dihydrolipoamide, in which the disul- 
phide bond of lipoamide has been reduced to the 
dithiol form. Reduction is generally achieved by treat- 
ment with sodium borohydride followed by extraction 
into benzene or chloroform and subsequent removal 
of the solvent by distillation or evaporation i  vacua 
[l-3]. 
In the course of investigations on the regulation of 
a bacterial cr-oxoglutarate d hydrogenase [4] we re- 
quired dihydrolipoamide to study the dihydrolipoa- 
mide dehydrogenase component of the complex and 
devised asimple electrolytic procedure for its ;;oduc- 
tion from lipoamide. This communication presents the 
details and advantages of the procedure. 
2. Experimental 
The technique mployed follows that previously 
used for the electroreduction of disulphide bonds in 
proteins [5]. The apparatus used is illustrated in fig. 
The cathode compartment, A  is a small rimless 
beaker (about 40 ml capacity) fitted with a side-arm 
and contains a pool of mercury (Analar grade) the 
surface of which can be stirred with a small magnetic 
1. 
North-Holland fiblishing Company - Amsterdam 
Fig. 1. Apparatus for electrolytic reduction. See text for 
explanation. 
‘flea’. A piece of platinum wire is fused into the side 
of the beaker and makes contact with the mercury. 
The solution to be electrolysed sits over the mercury 
pool and electrical contact between this solution and 
the anode compartment, B (a platinum electrode dip- 
ping into saturated KC 1 solution) is effected through 
the side-arm which contains 2% agar gel in saturated 
KCl. The cathode compartment isfitted with a stop- 
per through which two syringe needles pass; these 
allow nitrogen to be flushed through the solution 
being electrolysed. The source of the potential is a dry 
101 
Volume 43. number 1 FEBS LETTERS July 1974 
battery connected to the two electrodes via a variable 
potentiometer; both the, potential and the current 
may be monitored with suitable meters. 
A 20 mM solutiokof oxidised lipoamide (Sigma) 
in ethanol was first prepared and diluted to 1 mM 
with 0.1 M Tris-HCl, pH 8.0. Ten ml of this solution 
were introduced into the electrolysis cell over the mer- 
cury pool, stirred continuously and de-oxygenated 
for a few minutes by a stream of O2 -free nitrogen. 
A potential of -3 V was then applied to the mercu- 
ry pool cathode and the progressive decrease in cur- 
rent was followed. At intervals, 0.05 ml portions were 
withdrawn and added to a spectrophotometer cuvette 
containing 0.93 ml of 0.1 M Tris-HCl, pH 8.0 and 
0.02 ml of 10 mM 5,5’-dithiobis-(2-nitrobenzoate); 
after mixing the absorbance at 412 nm was measured. 
On the basis of a molar absorbance of 13 600 
1 . mole-’ . cm- ’ for the yellow thionitrobenzoate 
ion [6] the thiol content and state of reduction of 
the lipoamide were determined. 
Polarographic measurements on oxidised and redu- 
ced lipoamide were made with a Beckman Electroscan 
30 recording polarograph using a dropping mercury 
cathode and a saturated calomel reference anode. 
3. Results and discussion 
Typical progress curves for the electroreduction of 
lipoamide are shown in fig. 2. After 30 min of electro- 
-2 
I I ’ -0 
0 20 40 60 
ELECTROLYSIS TIME (min) 
Fig. 2. Time course of the electrolytic reduction of lipoamide. 
-0.4e 
0.2 0.4 0.6 0.6 1.0 
POTENTIAL (4) 
Fig. 3. Polarographic waves of oxidised and electroreduced 
lipoamide. Measurements were made on 40 rM lipoamide in 
0.1 M Tris-HCl, pH 8.0. A, oxidised lipoamide; B, dihydro- 
lipoamide. Current compensation of 0.25 PA/V was applied. 
lysis, both the current and thiol content reached limit- 
ing values corresponding to total reduction of the 
lipoamide disulphide bond to two thiol groups. Re- 
duction was also accompanied by a complete change 
of polarographic activity (fig. 3). Whereas the oxidised 
lipoamide gave rise to a cathodic reduction wave due 
to the disulphide bond, the dihydrolipoamide showed 
no cathodic wave but only an anodic oxidation wave 
due to its thiol groups. Titration withN-ethylmalei- 
mide resulted in progressive destruction of this anodic 
wave and gave an end-point again corresponding to 
two thiol groups per molecule of dihydrolipoamide. 
It is important to note that although the reduction 
is carried out over a pool of mercury, no interference 
with the dihydrolipoamide is thereby created. The 
fact that reactions with 5,5’-dithiobis-(2-nitrobenzoa- 
te) and N-ethylmaleimide indicated quantitative reduc- 
tion to the dithiol confirms that no mercury mercap- 
tide formation occurs. Moreover, the activity of the 
dihydrolipoamide with dihydrolipoamide dehydroge- 
102 
Voolilme 43, number 1 FEBS LETTERS July 1974 
nase [4] indicates that only the disulphide bond is 
attacked uring electrolysis. 
The polarographic activity of lipoic acid and its 
reduced form was demonstrated some years ago 
[7-93 but, to our knowledge, the electroreducibility 
which it suggests has not been utilized for the prepara- 
tion of dihydrolipoamide. The electrolytic method 
offers the advantages of being simple, inexpensive, 
rapid and quantitative. No excess reducing agent needs 
to be removed at the end of the reduction and the 
dihydrolipoamide is usable without further treatment 
or purification. These features make the method of 
particular value in investigations of lipoamide-linked 
enzyme systems. 
Acknowledgement 
We thank the Science Research Council for support 
(Grants B/SR/7443 and B/RG/45952) and for a Stu- 
dentship to M.G.P. 
References 
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103 


Preparation of dihydrolipoamide by electrolytic reduction 

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Preparation of dihydrolipoamide by electrolytic reduction

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